Longitudinal Trend of Plasma Concentrations of Extracellular Vesicles in Patients Hospitalized for COVID-19

Plasma concentrations of extracellular vesicles (EVs) originating from cells involved in COVID-19-associated coagulopathy (CAC), their longitudinal trend and association with clinical outcomes were evaluated. Blood samples of consecutive COVID-19 patients admitted to a medical Unit were longitudinally collected within 48 h of admission, at discharge and 30 days post-discharge. EVs were analyzed using high sensitivity flow cytometry and phospholipid-dependent clotting time (PPL). The following EVs were measured: endothelium-, platelet-, leukocyte-derived, bearing tissue factor (TF)+, angiotensin-converting enzyme (ACE2)+, platelet-derived growth factor receptor-β (PDGF-β)+ and SARS-CoV-2-nucleoprotein (NP)+. 91 patients were recruited for baseline EV analysis (mean age 67 ± 14 years, 50.5% male) and 48 underwent the longitudinal evaluation. From baseline to 30-days post-discharge, we observed significantly decreased plasma concentrations of endothelium-derived EVs (E-Selectin+), endothelium-derived bearing TF (E-Selectin+ TF+), endothelium-derived bearing ACE2 (E-Selectin+ACE2+) and leukocyte-EVs bearing TF (CD45+TF+), p < 0.001, p = 0.03, p = 0.001, p = 0.001, respectively. Conversely, platelet-derived (P-Selectin+) and leukocyte-derived EVs (CD45+) increased from baseline to 30-days post-discharge (p = 0.038 and 0.032, respectively). EVs TF+, ACE2+, PDGF-β+, and SARS-CoV-2-NP+ did not significantly change during the monitoring. PPL increased from baseline to 30-days post-discharge (+ 6.3 s, p = 0.006). P-Selectin + EVs >1,054/µL were associated with thrombosis (p = 0.024), E-Selectin + EVs ≤531/µL with worsening/death (p 0.026) and 30-days P-Selectin+ and CD45 + EVs with persistent symptoms (p < 0.0001). We confirmed increased EVs originating from cells involved in CAC at admission and discharge. EVs derived from activated pericytes and expressing SARS-CoV-2-NP were also detected. 30-days post-discharge, endothelium-EVs decreased, while platelet- and leukocyte-EVs further increased, indicating that cellular activation persists long after the acute phase.

Nerve-mediated amputation-induced stem cell activation primes distant appendages for future regeneration events in axolotl

Animals exhibit extreme diversity in regenerative ability. This likely reflects different, lineage-specific selective pressures in their evolutionary histories, but how specific molecular features of regenerative programs help solve species-specific challenges has not been examined in detail. Here we discover that, in the highly-regenerative axolotl salamander, a conserved, body-wide stem cell activation response triggered in response to limb removal primes undisturbed limbs for regeneration upon subsequent amputation. This response should be particularly useful to salamanders, which frequently lose limbs in response to cannibalism. We further demonstrate the body-wide response requires both peripheral nervous system input at these distant sites and mTOR signaling. We defined gene expression changes within the nerves and nearby tissues, harboring responsive stem cells, leading to identification of candidate genetic pathways influencing distant stem cell activation following amputation. Functional experimentation confirmed a requirement for adrenergic signaling in amputation-induced activation of distant stem cells. These findings reveal a direct link between systemic cellular activation responses to local tissue damage and overall regenerative ability. Similar systemic activation responses to tissue removal have been observed in animals with widely differing regenerative abilities (e.g., planaria to mice), suggesting that it is the responses downstream of these signals, likely sculpted by differing evolutionary pressures, that ultimately distinguish regenerators from non-regenerators.

Carbohydrates from Pseudomonas aeruginosa biofilms interact with immune C-type lectins and interfere with their receptor function

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of polysaccharides, nucleic acids and proteins. We hypothesised that carbohydrates could contribute to immune recognition of Pseudomonas aeruginosa biofilms by engaging C-type lectins. Here we show binding of Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), mannose receptor (MR, CD206) and Dectin-2 to P. aeruginosa biofilms. We also demonstrate that DC-SIGN, unlike MR and Dectin-2, recognises planktonic P. aeruginosa cultures and this interaction depends on the presence of the common polysaccharide antigen. Within biofilms DC-SIGN, Dectin-2 and MR ligands appear as discrete clusters with dispersed DC-SIGN ligands also found among bacterial aggregates. DC-SIGN, MR and Dectin-2 bind to carbohydrates purified from P. aeruginosa biofilms, particularly the high molecular weight fraction (HMW; >132,000 Da), with KDs in the nM range. These HMW carbohydrates contain 74.9–80.9% mannose, display α-mannan segments, interfere with the endocytic activity of cell-associated DC-SIGN and MR and inhibit Dectin-2-mediated cellular activation. In addition, biofilm carbohydrates reduce the association of the DC-SIGN ligand Lewisx, but not fucose, to human monocyte-derived dendritic cells (moDCs), and alter moDC morphology without affecting early cytokine production in response to lipopolysaccharide or P. aeruginosa cultures. This work identifies the presence of ligands for three important C-type lectins within P. aeruginosa biofilm structures and purified biofilm carbohydrates and highlights the potential for these receptors to impact immunity to P. aeruginosa infection.

The Deglycosylated Form of 1E12, a Monoclonal Anti-PF4 IgG, Strongly Inhibits Antibody-Triggered Cellular Activation in Vaccine-Induced Thrombotic Thrombocytopenia, and Is a Potential New Treatment for Vιττ

Introduction Vaccine-induced thrombotic thrombocytopenia (VITT) is a severe complication of recombinant adenoviral vector vaccines used to prevent COVID-19, likely due to anti-platelet factor 4 (PF4) IgG antibodies. The specificity and platelet-activating activity of VITT antibodies strikingly resemble that of antibodies detected in "autoimmune" heparin-induced thrombocytopenia (HIT), but their features remain poorly characterized. In particular, a better knowledge of these antibodies should help to understand the mechanisms leading to hypercoagulability and the particular thrombotic events observed in VITT, but rarely in typical HIT. We have recently developed a chimeric IgG1 anti-PF4 antibody, 1E12, which strongly mimics "autoimmune" HIT antibodies in terms of specificity and cellular effects. Therefore, we assessed whether 1E12 could mimic VITT antibodies. We then evaluated the capability of DG-1E12, a deglycosylated form of 1E12 unable to bind FcγR, to inhibit cellular activation induced by VITT antibodies. Methods and Results Using a PF4-sensitized serotonin release assay (PF4-SRA) (Vayne C, New Engl J Med, 2021), we demonstrated that 1E12 (5 and 10 μg/mL) strongly activated platelets, with a pattern similar to that obtained with human VITT samples (n=7), i.e. in a PF4-dependent manner and without heparin. This platelet activation was inhibited by low heparin concentration (0.5 IU/mL), an effect also observed with VITT samples. Serotonin release induced by 1E12 was also fully inhibited by IV-3, a monoclonal antibody blocking FcγRIIa, or by IdeS, a bacterial protease that cleaves IgG and strongly inhibits the binding of IgG antibodies to FcγRIIa. This inhibitory effect of IV-3 and IdeS strongly supports that interactions between pathogenic anti-PF4 IgG and FcγRIIa play a central role in VITT. Incubation of 1E12 or VITT samples with isolated neutrophils (PMN) and platelets with PF4 (10 µg/mL) strongly induced DNA release and NETosis, supporting that PMN are involved in the processes leading to thrombosis in VITT. Furthermore, when whole blood from healthy donors incubated with 1E12 or VITT plasma was perfused in capillaries coated with von Willebrand Factor, numerous large platelet/leukocyte aggregates containing fibrin(ogen) were formed. To investigate whether 1E12 and VITT antibodies recognize overlapping epitopes on PF4, we then performed competitive assays with a deglycosylated form of 1E12 (DG-1E12), still able to bind PF4 but not to interact with Fcγ receptors. In PF4-SRA, pre-incubation of DG-1E12 (50 µg/mL) dramatically reduced platelet activation induced by VITT antibodies, which was fully abrogated for 9 of the 14 VITT samples tested. Additional experiments using a whole blood PF4-enhanced flow cytometry assay recently designed for VITT diagnosis (Handtke et al, Blood 2021), confirmed that DG-1E12 fully prevented platelet activation induced by VITT antibodies. Moreover, when platelets and neutrophils were pre-incubated with DG-1E12 (100 µg/mL), NETosis and thus DNA release, nuclear rounding, and DNA decondensation induced by VITT antibodies were completely inhibited. Finally, DG-1E12 (100 µg/mL) also fully abolished VITT antibody-mediated thrombus formation in whole blood in vitro under vein flow conditions. Comparatively, DG-1E12 did not inhibit ALB6, a murine monoclonal anti-CD9 antibody, which also strongly activates platelets in a FcγRIIa-dependent manner. Conclusions Our results show that 1E12 exhibits features similar to those of human VITT antibodies in terms of specificity, affinity and cellular effects, and could therefore be used as a model antibody to study the pathophysiology of VITT. Our data also demonstrate that DG-1E12 prevents blood cell activation and thrombus formation induced by VITT antibodies, likely due to the competitive effect of its Fab fragment on antibody binding to PF4. DG-1E12 may allow the development of a new drug neutralizing the pathogenic effect of autoimmune anti-PF4 antibodies, such as those associated with VITT. Disclosures Thiele: Bristol Myers Squibb: Honoraria, Other; Pfizer: Honoraria, Other; Bayer: Honoraria; Chugai Pharma: Honoraria, Other; Novo Nordisk: Other; Novartis: Honoraria; Daichii Sankyo: Other. Pouplard: Stago: Research Funding. Greinacher: Macopharma: Honoraria; Biomarin/Prosensa: Other, Research Funding; Sagent: Other, Research Funding; Rovi: Other, Research Funding; Gore inc.: Other, Research Funding; Bayer Healthcare: Other, Research Funding; Paringenix: Other, Research Funding; BMS: Honoraria, Other, Research Funding; MSD: Honoraria, Other, Research Funding; Boehringer Ingelheim: Honoraria, Other, Research Funding; Aspen: Honoraria, Other, Research Funding; Portola: Other; Ergomed: Other; Instrument Laboratory: Honoraria; Chromatec: Honoraria. Gruel: Stago: Other: symposium fees, Research Funding. Rollin: Stago: Research Funding.

Minimal Role for the Alternative Pathway in Complement Activation By HIT Immune Complexes

Background: Recent studies show that ultra-large immune complexes consisting of IgG and platelet factor 4 and heparin (P+H) potently activate complement and facilitate complement dependent activation of cellular FcgRIIA (PMID 34189574). In whole blood assays using KKO, a monoclonal anti-PF4/heparin antibody, or antibodies from patients with heparin induced thrombocytopenia (HIT), we showed that classical pathway (CP) inhibition reduced immune complex-mediated complement activation (C3c and soluble C5b-9 generation), cell surface deposition of immune complexes and cellular activation. Aims: As previous studies suggest that the alternative pathway (AP) provides significant amplification (>80%) of the CP pathway, (PMID: 15544620) we compared the effects of AP, CP, and CP/AP inhibitors by KKO and HIT immune complexes in whole blood. Methods: Inhibitors of the CP (BBK32, a borrelia protein inhibitor to C1r), AP (anti-factor B antibody (α-fB), or Factor D (fD inhibitor or fD-INH) Alexion Pharmaceuticals, Boston, MA) or combined AP/CP (C1-esterase inhibitor, C1-INH, Berinert, CSL Behring; or soluble complement receptor 1, sCR1, Alexion) were tested in hemolytic assays of CP or AP to confirm pathway specificity. To examine effects of CP or AP inhibition on complement activation by immune complexes consisting of KKO or HIT IgG, whole blood was pre-incubated with CP, AP or CP/AP inhibitors prior to addition of P+H ± KKO/HIT IgG or isotype controls. WB was incubated for 45 minutes at 37ºC followed by addition of 10mM EDTA to quench further complement activation. Complement activation products (C3c and sC5b-9) and neutrophil degranulation (MMP9) markers were measured using commercial immunoassays. Effects of complement inhibitors on cellular deposition of immune complexes was examined by flow, using previously described methods (PMID 34189574) using fluorescently labeled anti-C3c antibody (Quidel, San Diego, CA) and anti-mouse or human IgG (Biolegend, San Diego, CA). Results: Consistent with prior publications (PMID: 26808924), BBK32 showed marked reduction CP, but not AP-dependent hemolytic assays. The converse was true of AP inhibitors: α-fB and fD-INH prevented AP-dependent, but not CP-dependent hemolysis (data not shown). C1-INH and sCR1 showed activity in both CP- and AP-dependent assays. The CP or CP/AP inhibitors showed potent inhibition of C3c and sC5b-9 generation by KKO and HIT immune complexes, while AP inhibitors had no effect (Figure A for KKO C3c generation; and Table 1 for KKO/HIT C3c generation; sC5b-9 data not shown). For a given CP or CP/AP inhibitor, the concentrations leading to 50% inhibition (IC 50) were generally comparable for KKO and HIT immune complexes for C3c (Figure A and Table 1) and sC5b-9 generation (data not shown), with potency as follows: C1-INH>>BBK32>sCR1 (Table 1). On the other hand, the AP inhibitors, α-fB and fD-INH, showed no inhibitory activity in C3c (Figure A and Table 1)/sC5b-9 (data not shown) generation by KKO or HIT ULICs. As our recent studies indicate that complement activation is critical to cell surface deposition of immune complexes and cellular activation via FcgRIIA, we examined effects of complement inhibitors on IC deposition on B-cells and MMP9 release from neutrophils. CP or CP/AP inhibitors, but not AP inhibitors, reduced cell surface binding of immune complexes (Figure B) as well as MMP9 release (Figure C and Table 1). Conclusion: Together, these studies demonstrate that the AP has a minimal role in supporting complement activation by KKO/HIT ULICs. Future studies should examine CP inhibition as a therapeutic strategy for modulating the cellular activating effects of HIT antibodies. To what extent these findings apply to other immune complexes and/or CP activators requires further study. Funding Agency: NIH HL151730; α-fB antibody, fD inhibitor and sCR1 was provided by Alexion Pharmaceuticals, Boston, MA. BBK32 was provided by Dr. Brandon Garcia, East Carolina University, Greenville, NC. Figure 1 Figure 1. Disclosures Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board. OffLabel Disclosure: C1-esterase inhibitor off label for HIT

Lamin B1 Accumulation’s Effects on Autosomal Dominant Leukodystrophy (ADLD): Induction of Reactivity in the Astrocytes

Autosomal dominant leukodystrophy (ADLD) is an extremely rare and fatal neurodegenerative disease due to the overexpression of the nuclear lamina component Lamin B1. Many aspects of the pathology still remain unrevealed. This work highlights the effect of Lamin B1 accumulation on different cellular functions in an ADLD astrocytic in vitro model. Lamin B1 overexpression induces alterations in cell survival signaling pathways with GSK3β inactivation, but not the upregulation of β-catenin targets, therefore resulting in a reduction in astrocyte survival. Moreover, Lamin B1 build up affects proliferation and cell cycle progression with an increase of PPARγ and p27 and a decrease of Cyclin D1. These events are also associated to a reduction in cell viability and an induction of apoptosis. Interestingly, ADLD astrocytes trigger a tentative activation of survival pathways that are ineffective. Finally, astrocytes overexpressing Lamin B1 show increased immunoreactivity for both GFAP and vimentin together with NF-kB phosphorylation and c-Fos increase, suggesting astrocytes reactivity and substantial cellular activation. These data demonstrate that Lamin B1 accumulation is correlated to biochemical, metabolic, and morphologic remodeling, probably related to the induction of a reactive astrocytes phenotype that could be strictly associated to ADLD pathological mechanisms.

Inflammation in Asthma Pathogenesis: Role of T cells, Macrophages, Epithelial Cells and Type 2 Inflammation

: Asthma is a chronic disease with abnormal inflammatory and immunological responses. The disease initiated by antigens in subjects with genetic susceptibility. However, environmental factors play a role in the initiation and exacerbation of asthma attack. Asthma is T helper 2 (Th2)-cell-mediated disease. Recent studies indicated that asthma is not a single disease entity, but it is with multiple phenotypes and endotypes. The pathophysiological changes in asthma included a series of subsequent continuous vicious circle of cellular activation contributed to induction of chemokines and cytokines that potentiate inflammation. The heterogeneity of asthma influenced the treatment response. The asthma pathogenesis driven by varied set of cells such as eosinophils, basophils, neutrophils, mast cells, macrophages, epithelial cells and T cells. In this review the role of T cells, macrophage, and epithelial cells are discussed.

Differentially expressed genes in the femur cartilage transcriptome clarify the understanding of femoral head separation in chickens

Locomotor problems are among one of the main concerns in the current poultry industry, causing major economic losses and affecting animal welfare. The most common bone anomalies in the femur are dyschondroplasia, femoral head separation (FHS), and bacterial chondronecrosis with osteomyelitis (BCO), also known as femoral head necrosis (FHN). The present study aimed to identify differentially expressed (DE) genes in the articular cartilage (AC) of normal and FHS-affected broilers by RNA-Seq analysis. In the transcriptome analysis, 12,169 genes were expressed in the femur AC. Of those, 107 genes were DE (FDR < 0.05) between normal and affected chickens, of which 9 were downregulated and 98 were upregulated in the affected broilers. In the gene-set enrichment analysis using the DE genes, 79 biological processes (BP) were identified and were grouped into 12 superclusters. The main BP found were involved in the response to biotic stimulus, gas transport, cellular activation, carbohydrate-derived catabolism, multi-organism regulation, immune system, muscle contraction, multi-organism process, cytolysis, leukocytes and cell adhesion. In this study, the first transcriptome analysis of the broilers femur articular cartilage was performed, and a set of candidate genes (AvBD1, AvBD2, ANK1, EPX, ADA, RHAG) that could trigger changes in the broiler´s femoral growth plate was identified. Moreover, these results could be helpful to better understand FHN in chickens and possibly in humans.

NF-κB and Pancreatic Cancer; Chapter and Verse

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the world’s most lethal cancers. An increase in occurrence, coupled with, presently limited treatment options, necessitates the pursuit of new therapeutic approaches. Many human cancers, including PDAC are initiated by unresolved inflammation. The transcription factor NF-κB coordinates many signals that drive cellular activation and proliferation during immunity but also those involved in inflammation and autophagy which may instigate tumorigenesis. It is not surprising therefore, that activation of canonical and non-canonical NF-κB pathways is increasingly recognized as an important driver of pancreatic injury, progression to tumorigenesis and drug resistance. Paradoxically, NF-κB dysregulation has also been shown to inhibit pancreatic inflammation and pancreatic cancer, depending on the context. A pro-oncogenic or pro-suppressive role for individual components of the NF-κB pathway appears to be cell type, microenvironment and even stage dependent. This review provides an outline of NF-κB signaling, focusing on the role of the various NF-κB family members in the evolving inflammatory PDAC microenvironment. Finally, we discuss pharmacological control of NF-κB to curb inflammation, focussing on novel anti-cancer agents which reinstate the process of cancer cell death, the Smac mimetics and their pre-clinical and early clinical trials.