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Restitution slope is principally determined by steady-state action potential duration.
AIMS: The steepness of the action potential duration (APD) restitution curve and local tissue refractoriness are both thought to play important roles in arrhythmogenesis. Despite this, there has been little recognition of the apparent association between steady-state APD and the slope of the restitution curve. The objective of this study was to test the hypothesis that restitution slope is determined by APD and to examine the relationship between restitution slope, refractoriness and susceptibility to VF. METHODS AND RESULTS: Experiments were conducted in isolated hearts and ventricular myocytes from adult guinea pigs and rabbits. Restitution curves were measured under control conditions and following intervention to prolong (clofilium, veratridine, bretylium, low [Ca]e, chronic transverse aortic constriction) or shorten (catecholamines, rapid pacing) ventricular APD. Despite markedly differing mechanisms of action, all interventions that prolonged the action potential led to a steepening of the restitution curve (and vice versa). Normalizing the restitution curve as a % of steady-state APD abolished the difference in restitution curves with all interventions. Effects on restitution were preserved when APD was modulated by current injection in myocytes pre-treated with the calcium chelator BAPTA-AM - to abolish the intracellular calcium transient. The non-linear relation between APD and the rate of repolarization of the action potential is shown to underpin the common influence of APD on the slope of the restitution curve. Susceptibility to VF was found to parallel changes in APD/refractoriness, rather than restitution slope. CONCLUSION(S): Steady-state APD is the principal determinant of the slope of the ventricular electrical restitution curve. In the absence of post-repolarization refractoriness, factors that prolong the action potential would be expected to steepen the restitution curve. However, concomitant changes in tissue refractoriness act to reduce susceptibility to sustained VF. Dependence on steady-state APD may contribute to the failure of restitution slope to predict sudden cardiac death.
Endoscopic transsphenoidal drainage of an aggressive petrous apex cholesterol granuloma: unusual complications and lessons learnt.
OBJECTIVES: This case report describes the endoscopic transsphenoidal management of a cholesterol granuloma situated in a technically challenging part of the petrous apex, and the associated peri- and post-operative complications that arose. The literature on diagnosis and management of petrous apex cholesterol granulomas is reviewed. METHOD AND RESULTS: Surgical intervention was attempted on three occasions, each time via an endoscopic, transsphenoidal approach with image guidance. The procedure was abandoned on the first occasion as there was a significant risk to the carotid artery; only a small drainage ostium was created because of the proximity of the carotid artery. The second attempt, complicated by copious bleeding from the clival venous plexus, was arrested prematurely. Successful drainage was achieved at the third attempt, but recovery was complicated by tension pneumocephalus. CONCLUSION: The transnasal route is less invasive than a lateral labyrinthine or cochlear approach, and spares cochlear and vestibular function. However, this approach is not without risk. It is important to consider the natural anatomical variance of vasculature when planning surgical intervention for a lesion situated in a technically challenging part of the petrous apex. Additional magnetic resonance venography is recommended to circumnavigate the venous plexus, thereby avoiding an unexpected breach.
Clinical practice recommendations for the diagnosis and management of X-linked hypophosphataemia.
X-linked hypophosphataemia (XLH) is a rare metabolic bone disorder caused by pathogenic variants in the PHEX gene, which is predominantly expressed in osteoblasts, osteocytes and odontoblasts. XLH is characterized by increased synthesis of the bone-derived phosphaturic hormone fibroblast growth factor 23 (FGF23), which results in renal phosphate wasting with consecutive hypophosphataemia, rickets, osteomalacia, disproportionate short stature, oral manifestations, pseudofractures, craniosynostosis, enthesopathies and osteoarthritis. Patients with XLH should be provided with multidisciplinary care organized by a metabolic bone expert. Historically, these patients were treated with frequent doses of oral phosphate supplements and active vitamin D, which was of limited efficiency and associated with adverse effects. However, the management of XLH has evolved in the past few years owing to the availability of burosumab, a fully humanized monoclonal antibody that neutralizes circulating FGF23. Here, we provide updated clinical practice recommendations for the diagnosis and management of XLH to improve outcomes and quality of life in these patients.
Fibroblasts andFibroblast-like Synoviocytes
Recent advances in single-cell transcriptomic analysis have demonstrated unexpected diversity of fibroblast phenotypes within the synovium during embryogenesis, adulthood, and disease, with discrete markers and functions, offering dramatic new understanding of disease mechanisms and the potential to transform therapeutic approaches to disease. Fibroblasts are programmed epigenetically to determine the unique structure and function of different organs and tissues. However, these unique features might contribute to organ-specific disease. Tissue fibroblasts may be recruited from a number of sources and cell types including the bone marrow, blood, and local stromal cells and act as organ-specific innate immune sentinel cells. Under inflammatory conditions, fibroblasts become key immune system players by recruiting and modulating the behavior and survival of infiltrating immune cells. Fibroblasts can be programmed epigenetically through exposure to inflammatory and environmental stress such that they inappropriately prolong inflammation, which becomes persistent. Within the synovium, persistent abnormal behavior of fibroblasts results in continued damage to vital joint structures such as cartilage and bone, which, if untreated, will result in deformity and functional impairment.
X-Linked Hypophosphatemia Management in Children: An International Working Group Clinical Practice Guideline.
BACKGROUND: An International Working Group (IWG) developed new guidelines on the diagnosis, evaluation, management, and monitoring of X-linked hypophosphatemia (XLH) in children. Over the past 5 years, important advances have occurred in our understanding of the presentation, complications and treatment of XLH. METHODS: A group of 50 international experts in XLH from Canada, the United States, Europe, Asia and South America, along with methodology experts and patient partners, held 18 teleconference meetings in 2023-2024. These meetings addressed key issues regarding diagnosing, evaluating, managing, and monitoring XLH in children. Two systematic reviews were conducted to examine the impact of burosumab compared to conventional therapy (phosphate salts and active vitamin D) or no therapy, and to assess the impact of conventional therapy versus no therapy on patient-important outcomes. The certainty of evidence was evaluated using the GRADE methodology. Additionally, narrative reviews were completed on XLH diagnosis and the role of genetic testing, and an expert clinical practice survey informed the monitoring recommendations. OUTCOMES: An approach to establishing the diagnosis of XLH is presented. GRADEd recommendations were developed on treatment strategies for XLH in children. Monitoring recommendations, GRADEd as weak with very low certainty, were based on clinical practice survey of the IWG experts. The guidelines also addressed dental complications and proposed potential strategies to mitigate them. CONCLUSION: These clinical practice guidelines provide an update of the current evidence on the diagnosis and management of XLH and provide a comprehensive guidance for multidisciplinary healthcare professionals involved in the care of children with XLH.
Neutrophil nucleus: shaping the past and the future.
Neutrophils are innate immune cells that are key to protecting the host against infection and maintaining body homeostasis. However, if dysregulated, they can contribute to disease, such as in cancer or chronic autoinflammatory disorders. Recent studies have highlighted the heterogeneity in the neutrophil compartment and identified the presence of immature neutrophils and their precursors in these pathologies. Therefore, understanding neutrophil maturity and the mechanisms through which they contribute to disease is critical. Neutrophils were first characterized morphologically by Ehrlich in 1879 using microscopy, and since then, different technologies have been used to assess neutrophil maturity. The advances in the imaging field, including state-of-the-art microscopy and machine learning algorithms for image analysis, reinforce the use of neutrophil nuclear morphology as a fundamental marker of maturity, applicable for objective classification in clinical diagnostics. New emerging approaches, such as the capture of changes in chromatin topology, will provide mechanistic links between the nuclear shape, chromatin organization, and transcriptional regulation during neutrophil maturation.
Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation.
At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.
A blood atlas of COVID-19 defines hallmarks of disease severity and specificity
SummaryTreatment of severe COVID-19 is currently limited by clinical heterogeneity and incomplete understanding of potentially druggable immune mediators of disease. To advance this, we present a comprehensive multi-omic blood atlas in patients with varying COVID-19 severity and compare with influenza, sepsis and healthy volunteers. We identify immune signatures and correlates of host response. Hallmarks of disease severity revealed cells, their inflammatory mediators and networks as potential therapeutic targets, including progenitor cells and specific myeloid and lymphocyte subsets, features of the immune repertoire, acute phase response, metabolism and coagulation. Persisting immune activation involving AP-1/p38MAPK was a specific feature of COVID-19. The plasma proteome enabled sub-phenotyping into patient clusters, predictive of severity and outcome. Tensor and matrix decomposition of the overall dataset revealed feature groupings linked with disease severity and specificity. Our systems-based integrative approach and blood atlas will inform future drug development, clinical trial design and personalised medicine approaches for COVID-19.
MHC-related protein 1-restricted recognition of cancer via a semi-invariant TCR-α chain.
The T cell antigen presentation platform MR1 consists of 6 allomorphs in humans that differ by no more than 5 amino acids. The principal function of this highly conserved molecule involves presenting microbial metabolites to the abundant mucosal-associated invariant T (MAIT) cell subset. Recent developments suggest that the role of MR1 extends to presenting antigens from cancer cells, a function dependent on the K43 residue in the MR1 antigen binding cleft. Here, we successfully cultured cancer-activated, MR1-restricted T cells from multiple donors and confirmed that they recognized a wide range of cancer types expressing the most common MR1*01 and/or MR1*02 allomorphs (over 95% of the population), while remaining inert to healthy cells including healthy B cells and monocytes. Curiously, in all but one donor these T cells were found to incorporate a conserved TCR-α chain motif, CAXYGGSQGNLIF (where X represents 3-5 amino acids), because of pairing between 10 different TRAV genes and the TRAJ42 gene segment. This semi-invariance in the TCR-α chain is reminiscent of MAIT cells and suggests recognition of a conserved antigen bound to K43.
Proteinases and Matrix Degradation
Extra-cellular matrix (ECM) plays critical roles in the normal development and function of the organism by interacting with cells and supporting tissue and organ architectures. The cell-ECM interaction regulates various fundamental cellular functions including growth, differentiation, apoptosis, and migration. The turnover and remodeling of ECM are transient and under strict control in physiologic conditions, and excessive degradation of ECM components by proteinases causes tissue destruction in many pathologic conditions. Thus regulatory mechanisms of ECM degradation are important to understand to reveal pathogenesis of these diseases. In rheumatoid arthritis and osteoarthritis, activity of ECM-degrading proteinases is elevated, causing destruction of joint tissue including cartilage and bone. It is getting clearer that these unbalanced ECM metabolisms are not only due to upregulation of proteinase genes but also other factors. This chapter provides up-to-date information about ECM-degrading proteinases and their regulations in rheumatoid arthritis and osteoarthritis.
MAIT cells protect against sterile lung injury.
Mucosal-associated invariant T (MAIT) cells, the most abundant unconventional T cells in the lung, can exhibit a wide range of functional responses to different triggers via their T cell receptor (TCR) and/or cytokines. Their role, especially in sterile lung injury, is unknown. Using single-cell RNA sequencing (scRNA-seq), spectral analysis, and adoptive transfer in a bleomycin-induced sterile lung injury, we found that bleomycin activates murine pulmonary MAIT cells and is associated with a protective role against bleomycin-induced lung injury. MAIT cells drive the accumulation of type 1 conventional dendritic cells (cDC1s), limiting tissue damage in a DNGR-1-dependent manner. Human scRNA-seq data revealed that MAIT cells were activated, with increased cDC populations in idiopathic pulmonary fibrosis patients. Thus, MAIT cells enhance defense against sterile lung injury by fostering cDC1-driven anti-fibrotic pathways.
Activation-induced thrombospondin-4 works with thrombospondin-1 to build cytotoxic supramolecular attack particles.
Cytotoxic attack particles released by CTLs and NK cells include diverse phospholipid membrane and glycoprotein encapsulated entities that contribute to target cell killing. Supramolecular attack particles (SMAPs) are one type of particle characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a proteinaceous shell including thrombospondin (TSP)-1. TSP-4 was also detected in bulk analysis of SMAPs released by CTLs; however, it has not been investigated whether TSP-4 contributes to distinct SMAP types or the same SMAP type as TSP-1 and, if in the same type of SMAP, whether TSP-4 and TSP-1 cooperate or compete. Here, we observed that TSP-4 expression increased upon CD8+ T cell activation while, surprisingly, TSP-1 was down-regulated. Correlative Light and Electron Microscopy and Stimulated Emission Depletion microscopy localized TSP-4 and TSP-1 in SMAP-containing multicore granules. Superresolution dSTORM revealed that TSP-4 and TSP-1 are usually enriched in the same SMAPs while particles with single-positive shells are rare. Retention Using Selective Hooks assays showed that TSP-4 localizes to the lytic granules faster than TSP-1 and promotes its accumulation therein. TSP-4 contributed to direct CTL-mediated killing, as previously shown for TSP-1. TSP-4 and TSP-1 were both required for latent SMAP-mediated cell killing, in which released SMAPs kill targets after removal of the CTLs. Of note, we found that chronic lymphocytic leukemia (CLL) cell culture supernatants suppressed expression of TSP-4 in CTL and latent SMAP-mediated killing. These results identify TSP-4 as a functionally important component of SMAPs and suggest that SMAPs may be targeted for immune suppression by CLL.
Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies.
When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling.