Fluorescence imaging of lamellipodin-mediated biomolecular condensates on solid supported lipid bilayer membranes.

Biomolecular condensates play a major role in numerous cellular processes, including several that occur on the surface of lipid bilayer membranes. There is increasing evidence that cellular membrane trafficking phenomena, including the internalization of the plasma membrane through endocytosis, are mediated by multivalent protein-protein interactions that can lead to phase separation. We have recently found that proteins involved in the clathrin-independent endocytic pathway named Fast Endophilin Mediated Endocytosis can undergo liquid-liquid phase separation (LLPS) in solution and on lipid bilayer membranes. Here, the protein solution concentrations required for phase separation to be observed are significantly smaller compared to those required for phase separation in solution. LLPS is challenging to systematically characterize in cellular systems in general, and on biological membranes in particular. Model membrane approaches are more suitable for this purpose as they allow for precise control over the nature and amount of the components present in a mixture. Here we describe a method that enables the imaging of LLPS domain formation on solid supported lipid bilayers. These allow for facile imaging, provide long-term stability, and avoid clustering of vesicles and vesicle-attached features (such as buds and tethers) in the presence of multi-valent membrane interacting proteins.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

Advances in Hierarchical Inorganic Nanostructures for Efficient Solar Energy Harvesting Systems.

The urgent need to address the global energy and environmental crisis necessitates the development of efficient solar-power harvesting systems. Among the promising candidates, hierarchical inorganic nanostructures stand out due to their exceptional attributes, including a high specific surface area, abundant active sites, and tunable optoelectronic properties. In this comprehensive review, we delve into the fundamental principles underlying various solar energy harvesting technologies, including dye-sensitized solar cells (DSSCs), photocatalytic, photoelectrocatalytic (water splitting), and photothermal (water purification) systems, providing a foundational understanding of their operation. Thereafter, the discussion is focused on recent advancements in the synthesis, design, and development of hierarchical nanostructures composed of diverse inorganic material combinations, tailored for each of these solar energy harvesting systems. We meticulously elaborate on the distinct synthesis methods and conditions employed to fine-tune the morphological features of these hierarchical nanostructures. Furthermore, this review offers profound insights into critical aspects such as electron transfer mechanisms, band gap engineering, the creation of hetero-hybrid structures to optimize interface chemistry through diverse synthesis approaches, and precise adjustments of structural features. Beyond elucidating the scientific fundamentals, this review explores the large-scale applications of the aforementioned solar harvesting systems. Additionally, it addresses the existing challenges and outlines the prospects for achieving heightened solar-energy conversion efficiency.

A Model Study to Assess Fibrillation and Product Stability to Support Peptide Drug Design.

The fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and can accelerate product development by guiding the selection of solution-stable candidates and formulations. The studies reported here investigated the effects of structural modifications on the fibrillation of a 29-residue peptide (PepA) and two sequence modified variants (PepB, PepC). The C-terminus of PepA was amidated, whereas both PepB and PepC retained the carboxylate, and Ser16 in PepA and PepB was substituted with a helix-stabilizing residue, α-aminoisobutyric acid (Aib), in PepC. In thermal denaturation studies by far-UV CD spectroscopy and fibrillation kinetic studies by fluorescence and turbidity measurements, PepA and PepB showed heat-induced conformational changes and were found to form fibrils, whereas PepC did not fibrillate and showed only minor changes in the CD signal. Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed a high degree of protection from HD exchange in mature PepA fibrils and its proteolytic fragments, indicating that most of the sequence had been incorporated into the fibril structure and occurred nearly simultaneously throughout the sequence. The effects of the net peptide charge and formulation pH on fibrillation kinetics were investigated. In real-time stability studies of two formulations of PepA at pH's 7.4 and 8.0, analytical methods detected significant changes in the stability of the formulations at different time points during the study, which were not observed during accelerated studies. Additionally, PepA samples were withdrawn from real-time stability and subjected to additional stress (40 °C, continuous shaking) to induce fibrillation; an approach that successfully amplified oligomers or prefibrillar species previously undetected in a thioflavin T assay. Taken together, these studies present an approach to differentiate and characterize fibrillation risk in structurally related peptides under accelerated and real-time conditions, providing a model for rapid, iterative structural design to optimize the stability of therapeutic peptides.

mRNA vaccines against SARS-CoV-2 induce divergent antigen-specific T-cell responses in patients with lung cancer.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is highly transmissible and evades pre-established immunity. Messenger RNA (mRNA) vaccination against ancestral strain spike protein can induce intact T-cell immunity against the Omicron variant, but efficacy of booster vaccination in patients with late-stage lung cancer on immune-modulating agents including anti-programmed cell death protein 1(PD-1)/programmed death-ligand 1 (PD-L1) has not yet been elucidated. METHODS: We assessed T-cell responses using a modified activation-induced marker assay, coupled with high-dimension flow cytometry analyses. Peripheral blood mononuclear cells (PBMCs) were stimulated with various viral peptides and antigen-specific T-cell responses were evaluated using flow cytometry. RESULTS: Booster vaccines induced CD8+ T-cell response against the ancestral SARS-CoV-2 strain and Omicron variant in both non-cancer subjects and patients with lung cancer, but only a marginal induction was detected for CD4+ T cells. Importantly, antigen-specific T cells from patients with lung cancer showed distinct subpopulation dynamics with varying degrees of differentiation compared with non-cancer subjects, with evidence of dysfunction. Notably, female-biased T-cell responses were observed. CONCLUSION: We conclude that patients with lung cancer on immunotherapy show a substantial qualitative deviation from non-cancer subjects in their T-cell response to mRNA vaccines, highlighting the need for heightened protective measures for patients with cancer to minimize the risk of breakthrough infection with the Omicron and other future variants.

Neuromuscular disease genetics in under-represented populations: increasing data diversity.

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% 'solved' and ∼13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.

Electrochemical treatment of aquaculture wastewater effluent and optimization of the parameters using response surface methodology.

The electrocoagulation (EC) and electrooxidation (EO) processes are employed widely as treatment processes for industrial, agricultural, and domestic wastewater. In the present study, EC, EO, and a combination of EC + EO were evaluated as methods of removing pollutants from shrimp aquaculture wastewater. Process parameters for electrochemical processes, including current density, pH, and operation time were studied, and response surface methodology was employed to determine the optimum condition for the treatment. The effectiveness of the combined EC + EO process was assessed by measuring the reduction of targeted pollutants, including dissolved inorganic nitrogen species, total dissolved nitrogen (TDN), phosphate, and soluble chemical oxygen demand (sCOD). Using EC + EO process, more than 87% reduction was achieved for inorganic nitrogen, TDN, and phosphate, while 76.2% reduction was achieved for sCOD. These results demonstrated that the combined EC + EO process provided better treatment performance in removing the pollutants from shrimp wastewater. The kinetic results suggested that the effects of pH, current density, and operation time were significant on the degradation process when using iron and aluminum electrodes. Comparatively, iron electrodes were effective at reducing the half-life (t1/2) of each of the pollutants in the samples. The application of the optimized process parameters on shrimp wastewater could be used for large-scale treatment in aquaculture.

Hyperphosphorylated PTEN exerts oncogenic properties.

PTEN is a multifaceted tumor suppressor that is highly sensitive to alterations in expression or function. The PTEN C-tail domain, which is rich in phosphorylation sites, has been implicated in PTEN stability, localization, catalytic activity, and protein interactions, but its role in tumorigenesis remains unclear. To address this, we utilized several mouse strains with nonlethal C-tail mutations. Mice homozygous for a deletion that includes S370, S380, T382 and T383 contain low PTEN levels and hyperactive AKT but are not tumor prone. Analysis of mice containing nonphosphorylatable or phosphomimetic versions of S380, a residue hyperphosphorylated in human gastric cancers, reveal that PTEN stability and ability to inhibit PI3K-AKT depends on dynamic phosphorylation-dephosphorylation of this residue. While phosphomimetic S380 drives neoplastic growth in prostate by promoting nuclear accumulation of ß-catenin, nonphosphorylatable S380 is not tumorigenic. These data suggest that C-tail hyperphosphorylation creates oncogenic PTEN and is a potential target for anti-cancer therapy.

A Randomized Comparative Study of Functional and Radiological Outcome of Tension Band Wiring for Patella Fractures Using SS Wire Versus Fiberwire.

Background: Patellar fractures account for 1% of all skeletal injuries. Tension band wiring using SS wire has been the most commonly practiced procedure. Although this has shown good results, many patients experience hardware related problems like pain, irritation and prominence which necessitate it's removal. Recent studies have highlighted braided sutures as a possible alternative to SS wire. The purpose of this study is to evaluate the functional and radiological outcomes and complications of TBW using SS wire versus FiberWire (a reinforced braided polyblend suture) for the treatment of displaced transverse patellar fractures. Methods: A randomized comparative study was carried out at a tertiary care center from November 2019 to May 2021. 32 patients were randomized into two equal groups, one treated with TBW using FiberWire and the other with SS wire. Patients were followed up for a period of 20 weeks and evaluated for functional outcome using the Bostman scoring scale, radiological union, complications and hardware removal rates. Results: The mean duration for radiological union was 12.85 weeks using FiberWire and 12.75 weeks using SS wire. The mean knee range of motion was 118.57° in the FiberWire group and 117.18° in the SS wire group. Functional scores in the FiberWire and SS wire groups were 24 (good) and 26 (good) respectively measured using the Bostman scoring scale at end of 20 weeks. Complications like hardware prominence, soft tissue irritation and hardware removal rates were significantly higher in the SS wire group with a p value of 0.023. Conclusion: SS wire is biomechanically stronger than FiberWire when used for TBW. Both implants produce comparable results with respect to union rate, ROM and functional outcome, however, FiberWire causes fewer hardware complications like prominence and pain and hence alleviates the need for a second surgical procedure for implant removal. Thus, surgical treatment of transverse and inferior pole of patella fractures with TBW using FiberWire is a better alternative to SS wire considering early rehabilitation and lesser complication rates.

Selective suppression of de novo SARS-CoV-2 vaccine antibody responses in patients with cancer on B cell-targeted therapy.

We assessed vaccine-induced antibody responses to the SARS-CoV-2 ancestral virus and Omicron variant before and after booster immunization in 57 patients with B cell malignancies. Over one-third of vaccinated patients at the pre-booster time point were seronegative, and these patients were predominantly on active cancer therapies such as anti-CD20 monoclonal antibody. While booster immunization was able to induce detectable antibodies in a small fraction of seronegative patients, the overall booster benefit was disproportionately evident in patients already seropositive and not receiving active therapy. While ancestral virus- and Omicron variant-reactive antibody levels among individual patients were largely concordant, neutralizing antibodies against Omicron tended to be reduced. Interestingly, in all patients, including those unable to generate detectable antibodies against SARS-CoV-2 spike, we observed comparable levels of EBV- and influenza-reactive antibodies, demonstrating that B cell-targeting therapies primarily impair de novo but not preexisting antibody levels. These findings support rationale for vaccination before cancer treatment.

Formulation Development of Modafinil Sprinkle Dosage Form: Study on the Effect of Food on Dosage.

Sprinkles, drug-containing pellets or granules that can be mixed with soft food before administration, may be administered either directly in the mouth, or mixed with a spoonful of cold or room temperature soft foods. In this study, attempts were made to develop a new oral sprinkle formulation for modafinil in granular form. In order to increase the oral bioavailability and taste masking to food taken along with the drug, modafinil-hydroxypropyl-beta-cyclodextrin complexes were prepared and evaluated. A molar ratio of 1:5 was optimized. In vitro drug-release studies showed a maximum drug release rate of 93%. In-use analysis study also showed that the drug release was more than 90% within 45 minutes. The formulation was found to be stable for 3 months under accelerated conditions.

Selective targeting of GARP-LTGFß axis in the tumor microenvironment augments PD-1 blockade via enhancing CD8+ T cell antitumor immunity.

BACKGROUND: Immune checkpoint blockade (ICB) has revolutionized cancer immunotherapy. However, most patients with cancer fail to respond clinically. One potential reason is the accumulation of immunosuppressive transforming growth factor ß (TGFß) in the tumor microenvironment (TME). TGFß drives cancer immune evasion in part by inducing regulatory T cells (Tregs) and limiting CD8+ T cell function. Glycoprotein-A repetitions predominant (GARP) is a cell surface docking receptor for activating latent TGFß1, TGFß2 and TGFß3, with its expression restricted predominantly to effector Tregs, cancer cells, and platelets. METHODS: We investigated the role of GARP in human patients with cancer by analyzing existing large databases. In addition, we generated and humanized an anti-GARP monoclonal antibody and evaluated its antitumor efficacy and underlying mechanisms of action in murine models of cancer. RESULTS: We demonstrate that GARP overexpression in human cancers correlates with a tolerogenic TME and poor clinical response to ICB, suggesting GARP blockade may improve cancer immunotherapy. We report on a unique anti-human GARP antibody (named PIIO-1) that specifically binds the ligand-interacting domain of all latent TGFß isoforms. PIIO-1 lacks recognition of GARP-TGFß complex on platelets. Using human LRRC32 (encoding GARP) knock-in mice, we find that PIIO-1 does not cause thrombocytopenia; is preferentially distributed in the TME; and exhibits therapeutic efficacy against GARP+ and GARP- cancers, alone or in combination with anti-PD-1 antibody. Mechanistically, PIIO-1 treatment reduces canonical TGFß signaling in tumor-infiltrating immune cells, prevents T cell exhaustion, and enhances CD8+ T cell migration into the TME in a C-X-C motif chemokine receptor 3 (CXCR3)-dependent manner. CONCLUSION: GARP contributes to multiple aspects of immune resistance in cancer. Anti-human GARP antibody PIIO-1 is an efficacious and safe strategy to block GARP-mediated LTGFß activation, enhance CD8+ T cell trafficking and functionality in the tumor, and overcome primary resistance to anti-PD-1 ICB. PIIO-1 therefore warrants clinical development as a novel cancer immunotherapeutic.

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional.

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

Senescent cells limit p53 activity via multiple mechanisms to remain viable.

Super-enhancers regulate genes with important functions in processes that are cell type-specific or define cell identity. Mouse embryonic fibroblasts establish 40 senescence-associated super-enhancers regardless of how they become senescent, with 50 activated genes located in the vicinity of these enhancers. Here we show, through gene knockdown and analysis of three core biological properties of senescent cells that a relatively large number of senescence-associated super-enhancer-regulated genes promote survival of senescent mouse embryonic fibroblasts. Of these, Mdm2, Rnase4, and Ang act by suppressing p53-mediated apoptosis through various mechanisms that are also engaged in response to DNA damage. MDM2 and RNASE4 transcription is also elevated in human senescent fibroblasts to restrain p53 and promote survival. These insights identify key survival mechanisms of senescent cells and provide molecular entry points for the development of targeted therapeutics that eliminate senescent cells at sites of pathology.

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

p21 produces a bioactive secretome that places stressed cells under immunosurveillance.

Immune cells identify and destroy damaged cells to prevent them from causing cancer or other pathologies by mechanisms that remain poorly understood. Here, we report that the cell-cycle inhibitor p21 places cells under immunosurveillance to establish a biological timer mechanism that controls cell fate. p21 activates retinoblastoma protein (Rb)­dependent transcription at select gene promoters to generate a complex bioactive secretome, termed p21-activated secretory phenotype (PASP). The PASP includes the chemokine CXCL14, which promptly attracts macrophages. These macrophages disengage if cells normalize p21 within 4 days, but if p21 induction persists, they polarize toward an M1 phenotype and lymphocytes mount a cytotoxic T cell response to eliminate target cells, including preneoplastic cells. Thus, p21 concurrently induces proliferative arrest and immunosurveillance of cells under duress.

TREATMENT WITH SOLUBLE CD24 ATTENUATES COVID-19-ASSOCIATED SYSTEMIC IMMUNOPATHOLOGY.

BACKGROUND: SARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy. METHODS: We studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. FINDINGS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity. INTERPRETATION: Our data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19. FUNDING: NIH.

Immunological Insights Into the Therapeutic Roles of CD24Fc Against Severe COVID-19

BACKGROUNDSARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy. METHODSWe studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. FINDINGSTwenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity. INTERPRETATIONOur data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19. FUNDINGNIH

Development of EndoScreen Chip, a Microfluidic Pre-Endoscopy Triage Test for Esophageal Adenocarcinoma.

The current endoscopy and biopsy diagnosis of esophageal adenocarcinoma (EAC) and its premalignant condition Barrett's esophagus (BE) is not cost-effective. To enable EAC screening and patient triaging for endoscopy, we developed a microfluidic lectin immunoassay, the EndoScreen Chip, which allows sensitive multiplex serum biomarker measurements. Here, we report the proof-of-concept deployment for the EAC biomarker Jacalin lectin binding complement C9 (JAC-C9), which we previously discovered and validated by mass spectrometry. A monoclonal C9 antibody (m26 3C9) was generated and validated in microplate ELISA, and then deployed for JAC-C9 measurement on EndoScreen Chip. Cohort evaluation (n = 46) confirmed the expected elevation of serum JAC-C9 in EAC, along with elevated total serum C9 level. Next, we asked if the small panel of serum biomarkers improves detection of EAC in this cohort when used in conjunction with patient risk factors (age, body mass index and heartburn history). Using logistic regression modeling, we found that serum C9 and JAC-C9 significantly improved EAC prediction from AUROC of 0.838 to 0.931, with JAC-C9 strongly predictive of EAC (vs. BE OR = 4.6, 95% CI: 1.6-15.6, p = 0.014; vs. Healthy OR = 4.1, 95% CI: 1.2-13.7, p = 0.024). This proof-of-concept study confirms the microfluidic EndoScreen Chip technology and supports the potential utility of blood biomarkers in improving triaging for diagnostic endoscopy. Future work will expand the number of markers on EndoScreen Chip from our list of validated EAC biomarkers.