Definitions and measures of long COVID fatigue in adults: a scoping review protocol.

OBJECTIVE: The aim of this scoping review is to investigate how fatigue is defined and measured in adults with long COVID. INTRODUCTION: Following COVID-19 infection, 10% to 20% of individuals experience persisting symptoms for a minimum of 3 months; this is commonly known as long COVID. Fatigue is one of the most prevalent symptoms of long COVID, but there is currently no consistently applied definition of long COVID fatigue. To advance our understanding of long COVID fatigue, we must first identify the current definitions and measures being used to describe and mesure this condition. INCLUSION CRITERIA: This review will consider published and unpublished studies involving adults (≥18 years) that define and/or measure long COVID fatigue. Papers using quantitative or qualitative designs will be included. Conference abstracts, editorials, and opinion papers will be excluded. METHODS: Published studies from January 2020 onwards will be searched for across MEDLINE (Ovid), CINAHL (EBSCOhost), Embase (Ovid), Scopus, PsycINFO (Ovid), Web of Science Core Collection, Epistemonikos, and Cochrane Central Register of Controlled Trials (CENTRAL). Dimensions, Overton, and ProQuest Dissertations and Theses will be searched for unpublished literature. Eligible records will be de-duplicated, and 2 independent reviewers will carry out title, abstract, and full-text screening. A data extraction tool will be pilot tested on a small number of papers, then modified as necessary, with any modifications detailed in the scoping review. Findings will be presented in tables and charts, supported by a narrative summary. REVIEW REGISTRATION: Open Science Framework https://osf.io/hnf8z.

Determining the efficacy of disinfectants at nucleic acid degradation.

AIMS: Nucleic acids, particularly antibiotic resistance genes, are commonly found on surfaces within healthcare environments, with levels not reducing following cleaning. Within the UK, there are no regulations for testing disinfectants against nucleic acids. METHODS AND RESULTS: A series of commonplace in vitro methods were used to determine disinfectant-induced physical and functional damage to various nucleic acids; RNA (10 µg), genomic DNA (2 µg), and plasmids (1 µg). Using these methods, the optimal residence time (10 minutes) and working concentration (10%) were determined for a new disinfectant. Furthermore, comparison of disinfectants with different active ingredients including lactic acid (LA), sodium hydroxide (NaOH), chloroxylenol (PCMX), and quaternary ammonium compounds (QACs), were compared to controls. All disinfectants showed greater degradation by gel electrophoresis of genomic DNA and RNA than of purified plasmids. Functional analysis using quantitative polymerase chain reaction (qPCR) and polymerase chain reaction (PCR) demonstrated that no disinfectant tested (apart from control) could damage DNA to the level where PCR amplification was not possible, and only the NaOH reagent could achieve this for RNA. CONCLUSIONS: The set of methods described herein provides a platform for future standardization and potential regulation regarding monitoring cleaning solutions for their activity against nucleic acids.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

Pro-resolving and anti-arthritic properties of the MC1 selective agonist PL8177.

Background: Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. Methods: PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177. Results: PL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1ß, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint. Conclusion: These results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. Significance: Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

In vivo potency assay for the screening of bioactive molecules on cartilage formation.

Cartilage regeneration is a priority in medicine for the treatment of osteoarthritis and isolated cartilage defects. Several molecules with potential for cartilage regeneration are under investigation. Unfortunately, in vitro chondrogenesis assays do not always predict the stability of the newly formed cartilage in vivo. Therefore, there is a need for a stringent, quantifiable assay to assess in vivo the capacity of molecules to promote the stable formation of cartilage that is resistant to calcification and endochondral bone formation. We developed an ectopic cartilage formation assay (ECFA) that enables one to assess the capacity of bioactive molecules to support cartilage formation in vivo using cartilage organoids. The ECFA predicted good clinical outcomes when used as a quality control for efficacy of chondrocyte preparations before implantation in patients with cartilage defects. In this assay, articular chondrocytes from human donors or animals are injected either intramuscularly or subcutaneously in nude mice. As early as 2 weeks later, cartilage organoids can be retrieved. The size of the implants and their degree of differentiation can be assessed by histomorphometry, immunostainings of molecular markers and real-time PCR. Mineralization can be assessed by micro-computed tomography or by staining. The effects of molecules on cartilage formation can be tested following the systemic administration of the molecule in mice previously injected with chondrocytes, or after co-injection of chondrocytes with cell lines overexpressing and secreting the protein of interest. Here we describe the ECFA procedure, including steps for harvesting human and bovine articular cartilage, isolating primary chondrocytes, preparing overexpression cell lines, injecting the cells intramuscularly and retrieving the implants. This assay can be performed by technicians and researchers with appropriate animal training within 3 weeks.

Eosinophilic sialodochitis: An emerging atopic condition.

OBJECTIVE: To report the clinical characteristics of the largest single centre cohort of patients with eosinophilic sialodochitis. METHODS: Analysis of data relating to 37 patients seen in a dedicated multidisciplinary clinic was performed. Demographic, clinical, haematological, cytological, histological and radiological features were collated. Response to trials of allergy treatment was assessed. RESULTS: Thirty-seven patients (30 female, seven male) were identified, 42% of whom were of Afro-Caribbean origin, with a mean age of 50.4 years (range 28-80 years). Mean symptom duration at presentation was 10 years (range 2-33 years). Parotid and submandibular gland involvement was equally reported. The most commonly reported symptoms were swelling (97%), itching of the overlying skin (92%), salivary gland discomfort (84%) and "string-like" mucus discharge from salivary duct orifices (76%). Twenty-three patients (62%) demonstrated atopic disease and serum IgE level elevated in 57%. All 37 patients had eosinophils present in aspirated duct contents samples while raised peripheral eosinophil count was seen in 41%. Anecdotal symptom improvement was reported with antihistamine, antileukotriene or steroid treatment. CONCLUSION: Eosinophilic sialodochitis should be considered in any patient presenting with recurrent salivary gland swelling. Further studies are needed to evaluate treatments directed at a likely allergic pathogenesis.

WNT3A-loaded exosomes enable cartilage repair.

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

Calcium calmodulin kinase II activity is required for cartilage homeostasis in osteoarthritis.

WNT ligands can activate several signalling cascades of pivotal importance during development and regenerative processes. Their de-regulation has been associated with the onset of different diseases. Here we investigated the role of the WNT/Calcium Calmodulin Kinase II (CaMKII) pathway in osteoarthritis. We identified Heme Oxygenase I (HMOX1) and Sox-9 as specific markers of the WNT/CaMKII signalling in articular chondrocytes through a microarray analysis. We showed that the expression of the activated form of CaMKII, phospho-CaMKII, was increased in human and murine osteoarthritis and the expression of HMOX1 was accordingly reduced, demonstrating the activation of the pathway during disease progression. To elucidate its function, we administered the CaMKII inhibitor KN93 to mice in which osteoarthritis was induced by resection of the anterior horn of the medial meniscus and of the medial collateral ligament in the knee joint. Pharmacological blockade of CaMKII exacerbated cartilage damage and bone remodelling. Finally, we showed that CaMKII inhibition in articular chondrocytes upregulated the expression of matrix remodelling enzymes alone and in combination with Interleukin 1. These results suggest an important homeostatic role of the WNT/CaMKII signalling in osteoarthritis which could be exploited in the future for therapeutic purposes.

ROR2 blockade as a therapy for osteoarthritis.

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase-like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T1/2 cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade-induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis.

Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis.

Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that agrin is a potent chondrogenic factor and that a single intraarticular administration of agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of ß-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of agrin for joint surface regeneration.

Abnormal clot microstructure formed in blood containing HIT-like antibodies.

INTRODUCTION: Thrombosis is a severe and frequent complication of heparin-induced thrombocytopenia (HIT). However, there is currently no knowledge of the effects of HIT-like antibodies on the resulting microstructure of the formed clot, despite such information being linked to thrombotic events. We evaluate the effect of the addition of pathogenic HIT-like antibodies to blood on the resulting microstructure of the formed clot. MATERIALS AND METHODS: Pathogenic HIT-like antibodies (KKO) and control antibodies (RTO) were added to samples of whole blood containing Unfractionated Heparin and Platelet Factor 4. The formed clot microstructure was investigated by rheological measurements (fractal dimension; df) and scanning electron microscopy (SEM), and platelet activation was measured by flow cytometry. RESULTS AND CONCLUSIONS: Our results revealed striking effects of KKO on clot microstructure. A significant difference in df was found between samples containing KKO (df = 1.80) versus RTO (df = 1.74; p < 0.0001). This increase in df was often associated with an increase in activated platelets. SEM images of the clots formed with KKO showed a network consisting of a highly branched and compact arrangement of thin fibrin fibres, typically found in thrombotic disease. This is the first study to identify significant changes in clot microstructure formed in blood containing HIT-like antibodies. These observed alterations in clot microstructure can be potentially exploited as a much-needed biomarker for the detection, management and monitoring of HIT-associated thrombosis.

Targeting Extracellular Vesicles to the Arthritic Joint Using a Damaged Cartilage-Specific Antibody.

The targeted delivery of therapies to diseased tissues offers a safe opportunity to achieve optimal efficacy while limiting systemic exposure. These considerations apply to many disease indications but are especially relevant for rheumatoid arthritis (RA), as RA is a systemic autoimmune disease which affects multiple joints. We have identified an antibody that is specific to damaged arthritic cartilage (anti-ROS-CII) that can be used to deliver treatments specifically to arthritic joints, yielding augmented efficacy in experimental arthritis. In the current study, we demonstrate that scaffolds enriched with bioactive payloads can be delivered precisely to an inflamed joint and achieve superior efficacy outcomes consistent with the pharmacological properties of these payloads. As a scaffold, we have used extracellular vesicles (EVs) prepared from human neutrophils (PMNs), which possess intrinsic anti-inflammatory properties and the ability to penetrate inflamed arthritic cartilage. EV fortified with anti-ROS-CII (EV/anti-ROS-CII) retained anti-ROS-CII specificity and bound exclusively to the damaged cartilage. Following systemic administration, EV/anti-ROS-CII (a) exhibited the ability to localize specifically in the arthritic joint in vivo and (b) was able to specifically target single (viral IL-10 or anti-TNF) or combined (viral IL-10 and anti-TNF) anti-inflammatory treatments to the arthritic joint, which accelerated attenuation of clinical and synovial inflammation. Overall, this study demonstrates the attainability of targeting a pro-resolving biological scaffold to the arthritic joint. The potential of targeting scaffolds such as EV, nanoparticles, or a combination thereof alongside combined therapeutics is paramount for designing systemically administered broad-spectrum of anti-inflammatory treatments.

Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting.

BACKGROUND: One of the main challenges for extrusion 3D bioprinting is the identification of non-synthetic bioinks with suitable rheological properties and biocompatibility. Our aim was to optimize and compare the printability of crystal, fibril and blend formulations of novel pulp derived nanocellulose bioinks and assess biocompatibility with human nasoseptal chondrocytes. METHODS: The printability of crystalline, fibrillated and blend formulations of nanocellulose was determined by assessing resolution (grid-line assay), post-printing shape fidelity and rheology (elasticity, viscosity and shear thinning characteristics) and compared these to pure alginate bioinks. The optimized nanocellulose-alginate bioink was bioprinted with human nasoseptal chondrocytes to determine cytotoxicity, metabolic activity and bioprinted construct topography. RESULTS: All nanocellulose-alginate bioink combinations demonstrated a high degree of shear thinning with reversible stress softening behavior which contributed to post-printing shape fidelity. The unique blend of crystal and fibril nanocellulose bioink exhibited nano- as well as micro-roughness for cellular survival and differentiation, as well as maintaining the most stable construct volume in culture. Human nasoseptal chondrocytes demonstrated high metabolic activity post printing and adopted a rounded chondrogenic phenotype after prolonged culture. CONCLUSIONS: This study highlights the favorable rheological, swelling and biocompatibility properties of nanocellulose-alginate bioinks for extrusion-based bioprinting.

Artificial shear stress effects on leukocytes at a biomaterial interface.

Medical devices, such as ventricular assist devices (VADs), introduce both foreign materials and artificial shear stress to the circulatory system. The effects these have on leukocytes and the immune response are not well understood. Understanding how these two elements combine to affect leukocytes may reveal why some patients are susceptible to recurrent device-related infections and provide insight into the development of pump thrombosis. Biomaterials-DLC: diamond-like carbon-coated stainless steel; Sap: single-crystal sapphire; and Ti: titanium alloy (Ti6 Al4 V) were attached to the parallel plates of a rheometer. Whole human blood was left between the two discs for 5 minutes at +37°C with or without the application of shear stress (0 s-1 or 1000 s-1 ). Blood was removed and used for complete blood cell counts, flow cytometry (leukocyte activation, cell death, microparticle generation, phagocytic ability, and reactive oxygen species [ROS] production), and the production of pro-inflammatory cytokines. L-selectin expression on monocytes was decreased when blood was exposed to the biomaterials both with and without shear. Applying shear stress to blood on a Sap and Ti surface led to activation of neutrophils shown as decreased L-selectin expression. Sap and Ti blunted the LPS-stimulated macrophage migration inhibitory factor (MIF) production, most notably when sheared on Ti. The biomaterials used here have been shown to activate leukocytes in a static environment. The introduction of shear appears to exacerbate this activation. Interestingly, a widely accepted biocompatible material (Ti) utilized in many different types of devices has the capacity for immune cell activation and inhibition of MIF secretion when combined with shear stress. These findings contribute to our understanding of the contribution of biomaterials and shear stress to recurrent infections and vulnerability to sepsis in some VAD patients as well as pump thrombosis.

WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

OBJECTIVE: Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. METHODS: Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. RESULTS: WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. CONCLUSIONS: In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

Shall I go digital?

UNLABELLED: Dental radiographic imaging is slowly transferring to digital format. The decision to invest in this new technology should be based on a good understanding of the different types of digital imaging available within the dental field. This article outlines its use in general dental practice, highlighting the pros and cons of the various systems both for intra-oral and extra-oral radiography. CLINICAL RELEVANCE: An understanding of the mechanisms of digital imaging and their associated potential problems are required by any clinician moving to film-less imaging.

Spontaneous coronary artery dissection in men presenting with acute coronary syndrome, successfully managed by intravascular ultrasound-guided percutaneous coronary intervention.

Spontaneous coronary artery dissection is a rare cause of acute coronary syndrome. It is most commonly seen in young women, without atherosclerosis, in the peripartum period. Management options include conservative medical treatment, percutaneous coronary intervention or a surgical approach depending on the presentation, extent of dissection and luminal stenosis. We describe three unusual cases of spontaneous coronary artery dissection occurring in young/middle-aged men-the first in association with heavy mechanical work, the second in association with high levels of anxiety and emotional distress and the third in association with intense physical exercise. In each case we report the use of coronary angiography and intravascular ultrasound in the diagnosis of the condition, and their successful management using percutaneous coronary intervention.

Inequalities in premature mortality in Britain: observational study from 1921 to 2007.

OBJECTIVE: To report on the extent of inequality in premature mortality as measured between geographical areas in Britain. DESIGN: Observational study of routinely collected mortality data and public records. Population subdivided by age, sex, and geographical area (parliamentary constituencies from 1991 to2007, pre-1974 local authorities over a longer time span). SETTING: Great Britain. PARTICIPANTS: Entire population aged under 75 from 1990 to 2007, and entire population aged under 65 in the periods 1921-39, 1950-3, 1959-63, 1969-73, and 1981-2007. MAIN OUTCOME MEASURE: Relative index of inequality (RII) and ratios of inequality in age-sex standardised mortality ratios under ages 75 and 65. The relative index of inequality is the relative rate of mortality for the hypothetically worst-off compared with the hypothetically best-off person in the population, assuming a linear association between socioeconomic position and risk of mortality. The ratio of inequality is the ratio of the standardised mortality ratio of the most deprived 10% to the least deprived 10%. RESULTS: When measured by the relative index of inequality, geographical inequalities in age-sex standardised rates of mortality below age 75 have increased every two years from 1990-1 to 2006-7 without exception. Over this period the relative index of inequality increased from 1.61 (95% confidence interval 1.52 to 1.69) in 1990-1 to 2.14 (2.02 to 2.27) in 2006-7. Simple ratios indicated a brief period around 2001 when a small reduction in inequality was recorded, but this was quickly reversed and inequalities up to the age of 75 have now reached the highest levels reported since at least 1990. Similarly, inequalities in mortality ratios under the age of 65 improved slightly in the early years of this century but the latest figures surpass the most extreme previously reported. Comparison of crudely age-sex standardised rates for those below age 65 from historical records showed that geographical inequalities in mortality are higher in the most recent decade than in any similar time period for which records are available since at least 1921. CONCLUSIONS: Inequalities in premature mortality between areas of Britain continued to rise steadily during the first decade of the 21st century. The last time in the long economic record that inequalities were almost as high was in the lead up to the economic crash of 1929 and the economic depression of the 1930s. The economic crash of 2008 might precede even greater inequalities in mortality between areas in Britain.