Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's disease.

OBJECTIVES: Inflammatory cytokines that signal through the Janus kinases-signal transducer and activator of transcription (JAK-STAT) pathway, especially interferons (IFNs), are implicated in Sjögren's disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signalling and the effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been fully investigated. METHODS: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single-cell (sc) RNA sequencing (RNAseq), immunofluorescence (IF) microscopy and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. RESULTS: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (eg, focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell type-specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFN-ß, which were normalised by JAKi without cytotoxicity. CONCLUSIONS: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalises this aberrant signalling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a phase Ib/IIa randomised controlled trial to treat SjD with tofacitinib was initiated.

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils.

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

Resilience of hospital and allied infrastructure during pandemic and post pandemic periods for maternal health care of pregnant women and infants in Tamil Nadu, India - A counterfactual analysis.

COVID-19 has impacted the healthcare system across the globe. The study will span three pandemic waves in 2020, 2021, and 2022. The goal is to learn how the pandemic affects antenatal care (ANC) and emergency delivery care for pregnant women in Tamil Nadu, India, and how medical services respond. The study employs counterfactual analysis to evaluate the causal impact of the pandemic. A feedforward in combination with a simple auto-regressive neural network (AR-Net) is used to predict the daily number of calls for ambulance services (CAS). Three categories of the daily CAS count between January 2016 and December 2022 are utilised. The total CAS includes all types of medical emergencies; the second group pertains to planned ANC for high-risk pregnant women and the third group comprises CAS from pregnant women for medical emergencies. The second wave's infection and mortality rates were up to six times higher than the first. The phases in wave-II, post-wave-II, wave-III, and post-wave-III experienced a significant increase in both total IFT (inter-facility transfer) and total non-IFT calls covering all emergencies relative to the counterfactual, as evidenced by reported effect sizes of 1 and a range of 0.65 to 0.85, respectively. This highlights overwhelmed health services. In Tamil Nadu, neither emergency prenatal care nor planned prenatal care was affected by the pandemic. In contrast, the increase in actual emergency-related IFT calls during wave-II, post-wave-II, wave-III, and post-wave-III was 62%, 160%, 141%, and 165%, respectively, relative to the counterfactual. During the same time periods, the mean daily CAS related to prenatal care increased by 47%, 51%, 38%, and 38%, respectively, compared to pre-pandemic levels. The expansion of ambulance services and increased awareness of these services during wave II and the ensuing phases of Covid-19 pandemic have enhanced emergency care delivery for all, including obstetric and neonatal cohorts.

Inhibition of JAK-STAT pathway corrects salivary gland inflammation and interferon driven immune activation in Sjögren's Disease.

Objectives: Inflammatory cytokines that signal through the JAK- STAT pathway, especially interferons (IFNs), are implicated in Sjögren's Disease (SjD). Although inhibition of JAKs is effective in other autoimmune diseases, a systematic investigation of IFN-JAK-STAT signaling and effect of JAK inhibitor (JAKi) therapy in SjD-affected human tissues has not been reported. Methods: Human minor salivary glands (MSGs) and peripheral blood mononuclear cells (PBMCs) were investigated using bulk or single cell (sc) RNA sequencing (RNAseq), immunofluorescence microscopy (IF), and flow cytometry. Ex vivo culture assays on PBMCs and primary salivary gland epithelial cell (pSGEC) lines were performed to model changes in target tissues before and after JAKi. Results: RNAseq and IF showed activated JAK-STAT pathway in SjD MSGs. Elevated IFN-stimulated gene (ISGs) expression associated with clinical variables (e.g., focus scores, anti-SSA positivity). scRNAseq of MSGs exhibited cell-type specific upregulation of JAK-STAT and ISGs; PBMCs showed similar trends, including markedly upregulated ISGs in monocytes. Ex vivo studies showed elevated basal pSTAT levels in SjD MSGs and PBMCs that were corrected with JAKi. SjD-derived pSGECs exhibited higher basal ISG expressions and exaggerated responses to IFNß, which were normalized by JAKi without cytotoxicity. Conclusions: SjD patients' tissues exhibit increased expression of ISGs and activation of the JAK-STAT pathway in a cell type-dependent manner. JAKi normalizes this aberrant signaling at the tissue level and in PBMCs, suggesting a putative viable therapy for SjD, targeting both glandular and extraglandular symptoms. Predicated on these data, a Phase Ib/IIa randomized controlled trial to treat SjD with tofacitinib was initiated.

Transcription inhibition suppresses nuclear blebbing and rupture independently of nuclear rigidity.

Chromatin plays an essential role in the nuclear mechanical response and determining nuclear shape, which maintain nuclear compartmentalization and function. However, major genomic functions, such as transcription activity, might also impact cell nuclear shape via blebbing and rupture through their effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with several RNA polymerase II inhibitors in wild-type cells and perturbed cells that presented increased nuclear blebbing. Transcription inhibition suppressed nuclear blebbing for several cell types, nuclear perturbations and transcription inhibitors. Furthermore, transcription inhibition suppressed nuclear bleb formation, bleb stabilization and bleb-based nuclear ruptures. Interestingly, transcription inhibition did not alter the histone H3 lysine 9 (H3K9) modification state, nuclear rigidity, and actin compression and contraction, which typically control nuclear blebbing. Polymer simulations suggested that RNA polymerase II motor activity within chromatin could drive chromatin motions that deform the nuclear periphery. Our data provide evidence that transcription inhibition suppresses nuclear blebbing and rupture, in a manner separate and distinct from chromatin rigidity.

How cells wrap around virus-like particles using extracellular filamentous protein structures.

Nanoparticles, such as viruses, can enter cells via endocytosis. During endocytosis, the cell surface wraps around the nanoparticle to effectively eat it. Prior focus has been on how nanoparticle size and shape impacts endocytosis. However, inspired by the noted presence of extracellular vimentin affecting viral and bacteria uptake, as well as the structure of coronaviruses, we construct a computational model in which both the cell-like construct and the virus-like construct contain filamentous protein structures protruding from their surfaces. We then study the impact of these additional degrees of freedom on viral wrapping. We find that cells with an optimal density of filamentous extracellular components (ECCs) are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus. At the optimal density, the cell surface folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. We also find that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. However, changing other mechanical parameters, such as the stretching stiffness of filamentous ECCs or virus spikes, can drive crumple-like formation of the cell surface. We conclude with the implications of our study on the evolutionary pressures of virus-like particles, with a particular focus on the cellular microenvironment that may include filamentous ECCs.

How cells wrap around virus-like particles using extracellular filamentous protein structures.

Nanoparticles, such as viruses, can enter cells via endocytosis. During endocytosis, the cell surface wraps around the nanoparticle to effectively eat it. Prior focus has been on how nanoparticle size and shape impacts endocytosis. However, inspired by the noted presence of extracellular vimentin affecting viral and bacteria uptake, as well as the structure of coronaviruses, we construct a computational model in which both the cell-like construct and the virus-like construct contain filamentous protein structures protruding from their surfaces. We then study the impact of these additional degrees of freedom on viral wrapping. We find that cells with an optimal density of filamentous extracellular components (ECCs) are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus. At the optimal density, the cell surface folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. We also find that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. However, changing other mechanical parameters, such as the stretching stiffness of filamentous ECCs or virus spikes, can drive crumple-like formation of the cell surface. We conclude with the implications of our study on the evolutionary pressures of virus-like particles, with a particular focus on the cellular microenvironment that may include filamentous ECCs.

Adsorptive removal of Cd2+ ions using dolochar at an industrial-scale process optimization by response surface methodology.

In this work, performance of laboratory-synthesized dolochar has been investigated for adsorption of Cd2+ ions in a large-scale process with the application of Aspen Adsorption. Moreover, the optimum values of the operating parameters (namely, flow rate, bed height, and inlet metal ion concentration) that would result into maximum amount of cadmium ion adsorption (high exhaustion capacity) in minimum time (less exhaustion time) for a fixed mass of dolochar have been calculated via the application of response surface methodology. It was found that, at optimum values of bed height (3.48 m), flow rate (76.31 m3/day), and inlet concentration (10 ppm), the optimized value of exhaustion capacity and exhaustion time for cadmium ion adsorption in dolochar packed bed is equal to 1.85 mg/g and 11.39 h, respectively. The validity of these simulation experiments can be proven by the fact that the obtained exhaustion capacity of dolochar packed bed always remained in close proximity of the experimentally obtained value of adsorption capacity of the dolochar in batch process mode (equal to 2.1 mg/g).

Freeze v/s air-dried alginate-pectin gel beads modified with sodium dodecyl sulphate for enhanced removal of copper ions.

This work presents a novel polymer-based adsorbent, Sodium Dodecyl Sulphate modified alginate-pectin gel beads (APS221) prepared via controlled freeze drying & air drying, for the removal of copper ions from the aqueous solution. This work also critically discusses the role played by various components and their concentrations in the success of APS221. Addition of pectin to alginate resulted into approximately 150 % increase in the metal removal performance of the adsorbent while addition of SDS into alginate-pectin complex enhanced the performance by 14 % approximately, taking the maximum adsorption capacity of final complex APS221 to 111.11 mg/g. Our characterization studies revealed that the adsorption happened predominantly by complexation and ion-exchange mechanisms, and hence despite having a higher surface area, freeze-dried variant showed lesser adsorption capacity than air-dried variant as there was a loss of ion-exchange sites resulting from breakage of crosslinking bonds due to chain elongation. The adsorption process was found to follow Langmuir isotherm and pseudo-second order kinetics with a good fit of experimental data. Further, operating parameters have been optimized via RSM to, simultaneously, maximize the utilization of the adsorbent and minimize the cost of the process. Stability studies showed that APS221 beads could be used up to eight cycles.

GZMK+CD8+ T cells Target a Specific Acinar Cell Type in Sjögren's Disease.

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2- seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

Surgical Repair of Achilles Tendon Rupture by Turn-O-Plasty: A Case Report.

Achilles tendon rupture has been a difficult problem for surgeons, especially in older patients, since tendon strength and flexibility are significantly diminished compared to young people. The Achilles tendon endures the highest tensile stresses in the body while running, leaping, and skipping, with tensile loads up to 10 times body weight. There are many treatment options for Achilles tendon repair, including open surgery, percutaneous repair, and ultrasound therapy. Open repair has the danger of scar dehiscence owing to poor skin conditions. In contrast, small invasive operations have the risk of sural nerve damage and a higher possibility of re-rupture. The gold standard method or approach is still under question. Plantar flexion in the ankle is primarily a function of the Achilles tendon; hence, post-operative plantar flexion is a significant determinant of the desired result. We present the case of a 57-year-old male farmer suffering from a left Achilles tendon rupture due to trivial trauma. This rupture consisted of a significant defect, present in the watershed area with signs of tendinosis at the insertion of the tendon. The patient was managed surgically by turn-o-plasty and the degenerated insertion site was augmented with the help of a suture disc. This case report focuses on surgical management by turn-o-plasty for significant defects in the Achilles tendon by using a suture disc to augment the defect.

Changes in cardiorespiratory function and fatigue following 12 weeks of exercise training in women with systemic lupus erythematosus: a pilot study.

OBJECTIVE: In patients with systemic lupus erythematosus (SLE), fatigue is a debilitating symptom with poorly understood pathophysiology. Cardiorespiratory dysfunction has been hypothesised as a contributor to SLE-fatigue. The purpose of this exploratory study was to examine changes in cardiorespiratory function, following an exercise training programme in women with SLE, together with patient reported outcomes and other pathophysiological measures that may underlie SLE-fatigue. METHODS: Sixteen women with SLE and fatigue (Fatigue Severity Scale (FSS) ≥3) were enrolled in a supervised aerobic exercise training programme of vigorous intensity. The primary outcome was time to reach anaerobic threshold (AT-Time) during a cardiopulmonary exercise test (CPET). Secondary outcomes included changes in the 10-minute walk test (10MWT), FSS scores and the Patient Reported Outcomes Measurement Information System (PROMIS-57) survey. Mitochondrial function was assessed by the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) metabolic potential ratio. RESULTS: Following 12 weeks of exercise training, AT-Time increased by 93±82 (mean±SD) s (p<0.001), 10MWT increased by 84±66 m (p<0.001) and peak oxygen uptake (VO2) increased by 1.4±2.0 mL/kg/min (p=0.013). There were improvements in FSS score (-1.4±1.0, p<0.0001) and in most of the PROMIS-57 domains. The decrease in FSS scores correlated with an increase in the OCR/ECAR ratio (Pearson's correlation r=-0.59, p=0.03). A subset of subjects (9/15) had significant reduction in their Interferon Stimulated Genes (ISG) (p=0.007) accompanied by a significant increase in the OCR/ECAR ratio (p=0.013). CONCLUSIONS: Cardiorespiratory function was improved in concomitance with reductions in fatigue following a 12-week aerobic exercise programme. The reduction in fatigue scores correlated with improvements in mitochondrial function.

Quantification of Neutrophils Undergoing NET Formation and Distinguishing Mechanisms of Neutrophil Cell Death by Use of a High-Throughput Method.

Neutrophils, the most abundant white blood cell type in humans, play a crucial role in innate host defenses. Recent studies have revealed additional key roles in the pathogenesis of cancer and autoimmune diseases through multiple mechanisms including the formation of neutrophil extracellular traps (NETs). Further research to expand the understanding of neutrophils' role in health and diseases is limited by lack of techniques to quantify neutrophils undergoing NET formation in an objective, reproducible, and efficient manner. In this chapter, we describe an automated high-throughput method to quantify NETting neutrophils in real time using a two-color, live-content imaging platform, the IncuCyte™S3 (Essen BioScience, Inc.) system, coupled to membrane integrity-dependent dual-dye approach to image intracellular and extracellular DNA. Based on characteristic differences in nuclear morphology and membrane integrity, this method may also be used to distinguish between different types of neutrophil cell death. This platform can help to assess neutrophil physiology and to develop and test therapeutic targets.

Peroxisome proliferator activated receptor-γ agonist pioglitazone improves vascular and metabolic dysfunction in systemic lupus erythematosus.

OBJECTIVES: Premature cardiovascular events in systemic lupus erythematosus (SLE) contribute to morbidity and mortality, with no effective preventive strategies described to date. Immune dysregulation and metabolic disturbances appear to play prominent roles in the induction of vascular disease in SLE. The peroxisome proliferator activated receptor-gamma agonist pioglitazone (PGZ suppresses vascular damage and immune dysregulation in murine lupus and improves endothelial dysfunction in other inflammatory diseases. We hypothesised that PGZ could improve vascular dysfunction and cardiometabolic parameters in SLE. METHODS: Eighty SLE subjects with mild to severe disease activity were randomised to a sequence of PGZ followed by placebo for 3 months, or vice versa, in a double-blind, cross-over design with a 2-month wash-out period. Primary endpoints were parameters of endothelial function and arterial inflammation, measured by multimodal assessments. Additional outcome measures of disease activity, neutrophil dysregulation, metabolic disturbances and gene expression studies were performed. RESULTS: Seventy-two subjects completed the study. PGZ was associated with a significant reduction in Cardio-Ankle Vascular Index (a measure of arterial stiffness) compared with placebo. Various metabolic parameters improved with PGZ, including insulin resistance and lipoprotein profiles. Circulating neutrophil extracellular trap levels also significantly decreased with PGZ compared with placebo. Most adverse events experienced while on PGZ were mild and resolved with reduction in PGZ dose. CONCLUSION: PGZ was well tolerated and induced significant improvement in vascular stiffness and cardiometabolic parameters in SLE. The results suggest that PGZ should be further explored as a modulator of cardiovascular disease risk in SLE. TRIAL REGISTRATION NUMBER: NCT02338999.

Single-Cell Analysis Reveals the Range of Transcriptional States of Circulating Human Neutrophils.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.

The Importance of Prompt Management of Morel-Lavallée Lesions.

The Morel-Lavallée lesion (MLL) is a closed soft-tissue injury that is frequently associated with high-intensity trauma. The thigh, hip, and pelvic regions are the most typically affected regions. It is critical to recognize and treat an MLL as soon as possible because it is often neglected or its identification is delayed because of other distracting injuries in a polytrauma patient. Bacterial colonization of these closed soft-tissue wounds can result in an increased risk of perioperative and postoperative infection. Magnetic resonance imaging has recently been used to define and grade these lesions. To reduce the dangers of these situations, clinical suspicion and on-the-spot identification of these lesions are essential. Here, we report an operated case of fracture shaft femur associated with MLLs and discuss the diagnostic and surgical approaches.

Joint-Predominant Rheumatic Complications of Immune Checkpoint Inhibitor Therapy in Patients with Thymic Epithelial Tumors.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced cancers. However, activation of the immune system can occasionally cause life-threatening toxicity involving critical organs. Induction of immune-mediated toxicity is a significant concern for patients with thymic epithelial tumors (TETs) due to defects in immune tolerance. An increased risk of skeletal and cardiac muscle inflammation following treatment with ICIs is well recognized in patients with advanced TETs. However, uncommon musculoskeletal and rheumatic complications can also occur. The cases presented in this report highlight the spectrum of presentation of immune-mediated, joint-predominant musculoskeletal adverse events in patients with advanced TETs treated with ICIs, including polymyalgia rheumatica-like illness and inflammatory arthritis.

Vimentin Intermediate Filaments Mediate Cell Morphology on Viscoelastic Substrates.

The ability of cells to take and change shape is a fundamental feature underlying development, wound repair, and tissue maintenance. Central to this process is physical and signaling interactions between the three cytoskeletal polymeric networks: F-actin, microtubules, and intermediate filaments (IFs). Vimentin is an IF protein that is essential to the mechanical resilience of cells and regulates cross-talk among the cytoskeleton, but its role in how cells sense and respond to the surrounding extracellular matrix is largely unclear. To investigate vimentin's role in substrate sensing, we designed polyacrylamide hydrogels that mimic the elastic and viscoelastic nature of in vivo tissues. Using wild-type and vimentin-null mouse embryonic fibroblasts, we show that vimentin enhances cell spreading on viscoelastic substrates, even though it has little effect in the limit of purely elastic substrates. Our results provide compelling evidence that vimentin modulates how cells sense and respond to their environment and thus plays a key role in cell mechanosensing.

Extracellular Vimentin as a Target Against SARS-CoV-2 Host Cell Invasion.

Infection of human cells by pathogens, including SARS-CoV-2, typically proceeds by cell surface binding to a crucial receptor. The primary receptor for SARS-CoV-2 is the angiotensin-converting enzyme 2 (ACE2), yet new studies reveal the importance of additional extracellular co-receptors that mediate binding and host cell invasion by SARS-CoV-2. Vimentin is an intermediate filament protein that is increasingly recognized as being present on the extracellular surface of a subset of cell types, where it can bind to and facilitate pathogens' cellular uptake. Biophysical and cell infection studies are done to determine whether vimentin might bind SARS-CoV-2 and facilitate its uptake. Dynamic light scattering shows that vimentin binds to pseudovirus coated with the SARS-CoV-2 spike protein, and antibodies against vimentin block in vitro SARS-CoV-2 pseudovirus infection of ACE2-expressing cells. The results are consistent with a model in which extracellular vimentin acts as a co-receptor for SARS-CoV-2 spike protein with a binding affinity less than that of the spike protein with ACE2. Extracellular vimentin may thus serve as a critical component of the SARS-CoV-2 spike protein-ACE2 complex in mediating SARS-CoV-2 cell entry, and vimentin-targeting agents may yield new therapeutic strategies for preventing and slowing SARS-CoV-2 infection.