Search

The regulation of reactive oxygen species (ROS) in red blood cells (RBCs) is crucial for maintaining functionality and lifespan. Indeed, dysregulated ROS occurs in haematological diseases such as sickle cell disease and β-thalassaemia. In order to combat this, RBCs possess high levels of protective antioxidant enzymes. We aimed to further boost RBC antioxidant capacity by overexpressing peroxiredoxin (Prxs) and glutathione peroxidase (GPxs) enzymes. Multiple antioxidant enzyme cDNAs were individually overexpressed in expanding immortalised erythroblasts using lentivirus, including Prx isoforms 1, 2, and 6 and GPx isoforms 1 and 4. Enhancing Prx protein expression proved straightforward, but GPx overexpression required modifications. For GPx4, these modifications included adding a SECIS element in the 3'UTR, the removal of a mitochondrial-targeting sequence, and removing putative ubiquitination sites. Culture-derived reticulocytes exhibiting enhanced levels of Prx and GPx antioxidant proteins were successfully engineered, demonstrating a novel approach to improve RBC resilience to oxidative stress. Further work is needed to explore the activity of these proteins and their impact on RBC metabolism, but this strategy shows promise for improving RBC function in physiological and pathological contexts and during storage for transfusion. Enhancing the antioxidant capacity of reticulocytes has exciting promise for developing culture-derived RBCs with enhanced resistance to oxidative damage and offers new therapeutic interventions in diseases with elevated oxidative stress. [ABSTRACT FROM AUTHOR]

After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging, we show that protocells loaded with R848 cargo (which targets TLR7 and TLR8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic. [ABSTRACT FROM AUTHOR]

Background: Neutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development. Methods: We optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors. Results: We obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis. Conclusion: Ex vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils. [ABSTRACT FROM AUTHOR]

Summary: Despite significant global morbidity associated with respiratory infection, there is a paucity of data examining the association between severity of non‐SARS‐CoV‐2 respiratory infection and blood group. We analysed a prospective cohort of adults hospitalised in Bristol, UK, from 1 August 2020 to 31 July 2022, including patients with acute respiratory infection (pneumonia [n = 1934] and non‐pneumonic lower respiratory tract infection [NP‐LRTI] [n = 1184]), a negative SARS‐CoV‐2 test and known blood group status. The likelihood of cardiovascular complication, survival and hospital admission length was assessed using regression models with group O and RhD‐negative status as reference groups. Group A and RhD‐positive were over‐represented in both pneumonia and NP‐LRTI compared to a first‐time donor population (p < 0.05 in all); contrastingly, group O was under‐represented. ABO group did not influence cardiovascular complication risk; however, RhD‐positive patients with pneumonia had a reduced odds ratio (OR) for cardiovascular complications (OR = 0.77 [95% CI = 0.59–0.98]). Compared to group O, group A individuals with NP‐LRTI were more likely to be discharged within 60 days (hazard ratio [HR] = 1.17 [95% CI = 1.03–1.33]), while group B with pneumonia was less likely (HR = 0.8 [95% CI = 0.66–0.96]). This analysis provides some evidence that blood group status may influence clinical outcome following respiratory infection, with group A having increased risk of hospitalisation and RhD‐positive patients having reduced cardiovascular complications. [ABSTRACT FROM AUTHOR]

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well‐established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole‐cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross‐polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery. [ABSTRACT FROM AUTHOR]

The bicarbonate/chloride exchanger 1 (AE1, Band 3) is abundantly expressed in the red blood cell membrane, where it is involved in gas exchange and functions as a major site of cytoskeletal attachment to the erythrocyte membrane. A truncated kidney isoform (kAE1) is highly expressed in type A intercalated cells of the distal tubules, where it is vital for urinary acidification. Recently, kAE1 has emerged as a novel physiologically significant protein in the kidney glomerulus. This minireview will discuss the known interactions of kAE1 in the podocytes and the possible mechanisms whereby this important multispanning membrane protein may contribute to the function of the glomerular filtration barrier and prevent proteinuria. Key words: AE1, Band 3, podocytes, type A intercalated cells, erythrocytes. L'echangeur bicarbonate/chlorure 1 (AE1, Band 3) est abondamment exprime; dans la membrane des globules rouges ou il est implique; dans les exchanges gazeux et constitue le principal site d'attachement du cytosquelette a la membrane des erythrocytes. Une isoforme renale tronquee (kAE1) est fortement exprimee chez les cellules intercalaires de type A des tubules distaux, ou elle joue un role essentiel dans l'acidification urinaire. Reecemment, kAE1 est apparue comme etant une proteine physiologiquement significative dans le glomerule renal. Cette minisyntheese presentera les interactions connues de kAE1 chez les podocytes ainsi que les mecanismes possibles par lesquels cette importante proteine a traversees multiples peut contribuer au fonctionnement de la barriere de filtration glomeerulaire et prevenir la proteinurie. Mots-cles: AE1, Band 3, podocytes, cellules intercalaires de type A, erythrocytes. [Traduit par la Redaction], AE1 in erythrocytes and distal kidney tubules Anion exchanger 1 (AE1, also known as Band 3; SLC4A1) is a multispanning transmembrane protein of the SLC4A bicarbonate transporter superfamily and exists [...]

Rhesus glycoprotein homologs RhAG, RhBG, and RhCG comprise a recently identified branch of the Mep/Amt ammonia transporter family. Animal studies have shown that RhBG and RhCG are present in the kidney distal tubules. Studies in mouse and rat tissue suggest a basolateral localization for RhBG in cells of the distal tubules including the a-intercalated cells ([alpha]-IC), but no localization of RhBG has been reported in human tissue. To date RhCG localization has been described as exclusively apical plasma membrane in mouse and rat kidney, or apical and basolateral in humans, and some mouse and rat tissue studies. We raised novel antibodies to RhBG and RhCG to examine their localization in the human kidney. Madin-Darby canine kidney (MDCKI) cell lines stably expressing human green fluorescent protein-tagged RhBG or RhCG and human tissue lysates were used to demonstrate the specificity of these antibodies for detecting RhBG and RhCG. Using immunoperoxidase staining and antigen liberation techniques, both apical and basolateral RhCG localization was observed in the majority of the cells of the distal convoluted tubule and IC of the connecting tubule and collecting duct. Confocal microscopic imaging of normal human kidney cryosections showed that RhCG staining was predominantly localized to the apical membrane in these cells with some basolateral and intracellular staining evident. A proportion of RhCG staining labeled kAE1-positive cells, confirming that RhCG is localized to the [alpha]-IC cells. Surprisingly, no RhBG protein was detectable in the human kidney by Western blot analysis of tissue lysates, or by immunohistochemistry or confocal microscopy of tissue sections. The same antibodies, however, could detect RhBG in rat tissue. We conclude that under normal conditions, RhCG is the major putative ammonia transporter expressed in the human kidney and RhBG is not expressed at detectable levels. Rh glycoprotein; MDCK; distal tubule; acid-base homeostasis

The human electrogenic renal Na-HC[O.sub.3] cotransporter (NBCel-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HC[O.sub.3] from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCel-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal [Na.sup.+] affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semi-quantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; [Na.sup.+]-HC[O.sub.3] cotransporter 1