A backpack-based myeloid cell therapy for multiple sclerosis.

Multiple sclerosis (MS) is an incurable autoimmune disease and is currently treated by systemic immunosuppressants with off-target side effects. Although aberrant myeloid function is often observed in MS plaques in the central nervous system (CNS), the role of myeloid cells in therapeutic intervention is currently overlooked. Here, we developed a myeloid cell-based strategy to reduce the disease burden in experimental autoimmune encephalomyelitis (EAE), a mouse model of progressive MS. We developed monocyte-adhered microparticles ("backpacks") for activating myeloid cell phenotype to an anti-inflammatory state through localized interleukin-4 and dexamethasone signals. We demonstrate that backpack-laden monocytes infiltrated into the inflamed CNS and modulated both the local and systemic immune responses. Within the CNS, backpack-carrying monocytes regulated both the infiltrating and tissue-resident myeloid cell compartments in the spinal cord for functions related to antigen presentation and reactive species production. Treatment with backpack-monocytes also decreased the level of systemic pro-inflammatory cytokines. Additionally, backpack-laden monocytes induced modulatory effects on TH1 and TH17 populations in the spinal cord and blood, demonstrating cross talk between the myeloid and lymphoid arms of disease. Backpack-carrying monocytes conferred therapeutic benefit in EAE mice, as quantified by improved motor function. The use of backpack-laden monocytes offers an antigen-free, biomaterial-based approach to precisely tune cell phenotype in vivo, demonstrating the utility of myeloid cells as a therapeutic modality and target.

A single combination gene therapy treats multiple age-related diseases.

Comorbidity is common as age increases, and currently prescribed treatments often ignore the interconnectedness of the involved age-related diseases. The presence of any one such disease usually increases the risk of having others, and new approaches will be more effective at increasing an individual's health span by taking this systems-level view into account. In this study, we developed gene therapies based on 3 longevity associated genes (fibroblast growth factor 21 [FGF21], αKlotho, soluble form of mouse transforming growth factor-ß receptor 2 [sTGFßR2]) delivered using adeno-associated viruses and explored their ability to mitigate 4 age-related diseases: obesity, type II diabetes, heart failure, and renal failure. Individually and combinatorially, we applied these therapies to disease-specific mouse models and found that this set of diverse pathologies could be effectively treated and in some cases, even reversed with a single dose. We observed a 58% increase in heart function in ascending aortic constriction ensuing heart failure, a 38% reduction in α-smooth muscle actin (αSMA) expression, and a 75% reduction in renal medullary atrophy in mice subjected to unilateral ureteral obstruction and a complete reversal of obesity and diabetes phenotypes in mice fed a constant high-fat diet. Crucially, we discovered that a single formulation combining 2 separate therapies into 1 was able to treat all 4 diseases. These results emphasize the promise of gene therapy for treating diverse age-related ailments and demonstrate the potential of combination gene therapy that may improve health span and longevity by addressing multiple diseases at once.

A facile approach to enhance antigen response for personalized cancer vaccination.

Existing strategies to enhance peptide immunogenicity for cancer vaccination generally require direct peptide alteration, which, beyond practical issues, may impact peptide presentation and result in vaccine variability. Here, we report a simple adsorption approach using polyethyleneimine (PEI) in a mesoporous silica microrod (MSR) vaccine to enhance antigen immunogenicity. The MSR-PEI vaccine significantly enhanced host dendritic cell activation and T-cell response over the existing MSR vaccine and bolus vaccine formulations. Impressively, a single injection of the MSR-PEI vaccine using an E7 peptide completely eradicated large, established TC-1 tumours in about 80% of mice and generated immunological memory. When immunized with a pool of B16F10 or CT26 neoantigens, the MSR-PEI vaccine eradicated established lung metastases, controlled tumour growth and synergized with anti-CTLA4 therapy. Our findings from three independent tumour models suggest that the MSR-PEI vaccine approach may serve as a facile and powerful multi-antigen platform to enable robust personalized cancer vaccination.

DNA origami vaccine (DoriVac) nanoparticles improve both humoral and cellular immune responses to infectious diseases.

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine platform, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific HR2 peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular immune responses in human cells. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities. The programmability of this platform underscores its potential utility in addressing future pandemics.

Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination.

Multivalent presentation of ligands often enhances receptor activation and downstream signalling. DNA origami offers a precise nanoscale spacing of ligands, a potentially useful feature for therapeutic nanoparticles. Here we use a square-block DNA origami platform to explore the importance of the spacing of CpG oligonucleotides. CpG engages Toll-like receptors and therefore acts to activate dendritic cells. Through in vitro cell culture studies and in vivo tumour treatment models, we demonstrate that square blocks induce Th1 immune polarization when CpG is spaced at 3.5 nm. We observe that this DNA origami vaccine enhances DC activation, antigen cross-presentation, CD8 T-cell activation, Th1-polarized CD4 activation and natural-killer-cell activation. The vaccine also effectively synergizes with anti-PD-L1 for improved cancer immunotherapy in melanoma and lymphoma models and induces long-term T-cell memory. Our results suggest that DNA origami may serve as a platform for controlling adjuvant spacing and co-delivering antigens in vaccines.

Biomaterial vaccines capturing pathogen-associated molecular patterns protect against bacterial infections and septic shock.

Most bacterial vaccines work for a subset of bacterial strains or require the modification of the antigen or isolation of the pathogen before vaccine development. Here we report injectable biomaterial vaccines that trigger potent humoral and T-cell responses to bacterial antigens by recruiting, reprogramming and releasing dendritic cells. The vaccines are assembled from regulatorily approved products and consist of a scaffold with absorbed granulocyte-macrophage colony-stimulating factor and CpG-rich oligonucleotides incorporating superparamagnetic microbeads coated with the broad-spectrum opsonin Fc-mannose-binding lectin for the magnetic capture of pathogen-associated molecular patterns from inactivated bacterial-cell-wall lysates. The vaccines protect mice against skin infection with methicillin-resistant Staphylococcus aureus, mice and pigs against septic shock from a lethal Escherichia coli challenge and, when loaded with pathogen-associated molecular patterns isolated from infected animals, uninfected animals against a challenge with different E. coli serotypes. The strong immunogenicity and low incidence of adverse events, a modular manufacturing process, and the use of components compatible with current good manufacturing practice could make this vaccine technology suitable for responding to bacterial pandemics and biothreats.

Nitric oxide regulates pulmonary vascular smooth muscle cell expression of the inducible cAMP early repressor gene.

Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca(2+)) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca(2+) channel inhibitors, 2-ABP, and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca(2+) signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca(2+) in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.

Lineage barcoding in mice with homing CRISPR.

Classic approaches to mapping the developmental history of cells in vivo have relied on techniques that require complex interventions and often capture only a single trajectory or moment in time. We have previously described a developmental barcoding system to address these issues using synthetically induced mutations to record information about each cell's lineage in its genome. This system uses MARC1 mouse lines, which have multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Here, we detail two MARC1 lines that are available from a public repository. We describe strategies for using MARC1 mice and experimental design considerations. We provide a protocol for barcode retrieval and sequencing as well as the analysis of the sequencing data. This protocol generates barcodes based on synthetically induced mutations in mice to enable lineage analysis.

A Modular Biomaterial Scaffold-Based Vaccine Elicits Durable Adaptive Immunity to Subunit SARS-CoV-2 Antigens.

The coronavirus disease 2019 (COVID-19) pandemic demonstrates the importance of generating safe and efficacious vaccines that can be rapidly deployed against emerging pathogens. Subunit vaccines are considered among the safest, but proteins used in these typically lack strong immunogenicity, leading to poor immune responses. Here, a biomaterial COVID-19 vaccine based on a mesoporous silica rods (MSRs) platform is described. MSRs loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF), the toll-like receptor 4 (TLR-4) agonist monophosphoryl lipid A (MPLA), and SARS-CoV-2 viral protein antigens slowly release their cargo and form subcutaneous scaffolds that locally recruit and activate antigen-presenting cells (APCs) for the generation of adaptive immunity. MSR-based vaccines generate robust and durable cellular and humoral responses against SARS-CoV-2 antigens, including the poorly immunogenic receptor binding domain (RBD) of the spike (S) protein. Persistent antibodies over the course of 8 months are found in all vaccine configurations tested and robust in vitro viral neutralization is observed both in a prime-boost and a single-dose regimen. These vaccines can be fully formulated ahead of time or stored lyophilized and reconstituted with an antigen mixture moments before injection, which can facilitate its rapid deployment against emerging SARS-CoV-2 variants or new pathogens. Together, the data show a promising COVID-19 vaccine candidate and a generally adaptable vaccine platform against infectious pathogens.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues. We used four of the hits to program hPSCs into neurons, fibroblasts, oligodendrocytes and vascular endothelial-like cells that have molecular and functional similarity to primary cells. Our cell-autonomous approach enabled parallel programming of hPSCs into multiple cell types simultaneously. We also demonstrated orthogonal programming by including oligodendrocyte-inducible hPSCs with unmodified hPSCs to generate cerebral organoids, which expedited in situ myelination. Large-scale combinatorial screening of the Human TFome will complement other strategies for cell engineering based on developmental biology and computational systems biology.

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Cardiomyocyte-specific overexpression of nitric oxide synthase 3 prevents myocardial dysfunction in murine models of septic shock.

Myocardial dysfunction contributes to the high mortality of patients with endotoxemia. Although nitric oxide (NO) has been implicated in the pathogenesis of septic cardiovascular dysfunction, the role of myocardial NO synthase 3 (NOS3) remains incompletely defined. Here we show that mice with cardiomyocyte-specific NOS3 overexpression (NOS3TG) are protected from myocardial dysfunction and death associated with endotoxemia. Endotoxin induced more marked impairment of Ca(2+) transients and cellular contraction in wild-type than in NOS3TG cardiomyocytes, in part, because of greater total sarcoplasmic reticulum Ca(2+) load and myofilament sensitivity to Ca(2+) in the latter during endotoxemia. Endotoxin increased reactive oxygen species production in wild-type but not NOS3TG hearts, in part, because of increased xanthine oxidase activity. Inhibition of NOS by N(G)-nitro-l-arginine-methyl ester restored the ability of endotoxin to increase reactive oxygen species production and xanthine oxidase activity in NOS3TG hearts to the levels measured in endotoxin-challenged wild-type hearts. Allopurinol, a xanthine oxidase inhibitor, attenuated endotoxin-induced reactive oxygen species accumulation and myocardial dysfunction in wild-type mice. The protective effects of cardiomyocyte NOS3 on myocardial function and survival were further confirmed in a murine model of polymicrobial sepsis. These results suggest that increased myocardial NO levels attenuate endotoxin-induced reactive oxygen species production and increase total sarcoplasmic reticulum Ca(2+) load and myofilament sensitivity to Ca(2+), thereby reducing myocardial dysfunction and mortality in murine models of septic shock.

Platelet decoys inhibit thrombosis and prevent metastatic tumor formation in preclinical models.

Platelets are crucial for normal hemostasis; however, their hyperactivation also contributes to many potentially lethal pathologies including myocardial infarction, stroke, and cancer. We hypothesized that modified platelets lacking their aggregation and activation capacity could act as reversible inhibitors of platelet activation cascades. Here, we describe the development of detergent-extracted human modified platelets (platelet decoys) that retained platelet binding functions but were incapable of functional activation and aggregation. Platelet decoys inhibited aggregation and adhesion of platelets on thrombogenic surfaces in vitro, which could be immediately reversed by the addition of normal platelets; in vivo in a rabbit model, pretreatment with platelet decoys inhibited arterial injury-induced thromboembolism. Decoys also interfered with platelet-mediated human breast cancer cell aggregation, and their presence decreased cancer cell arrest and extravasation in a microfluidic human microvasculature on a chip. In a mouse model of metastasis, simultaneous injection of the platelet decoys with tumor cells inhibited metastatic tumor growth. Thus, our results suggest that platelet decoys might represent an effective strategy for obtaining antithrombotic and antimetastatic effects.

Soluble guanylate cyclase-alpha1 deficiency selectively inhibits the pulmonary vasodilator response to nitric oxide and increases the pulmonary vascular remodeling response to chronic hypoxia.

BACKGROUND: Nitric oxide (NO) activates soluble guanylate cyclase (sGC), a heterodimer composed of alpha- and beta-subunits, to produce cGMP. NO reduces pulmonary vascular remodeling, but the role of sGC in vascular responses to acute and chronic hypoxia remains incompletely elucidated. We therefore studied pulmonary vascular responses to acute and chronic hypoxia in wild-type (WT) mice and mice with a nonfunctional alpha1-subunit (sGCalpha1-/-). METHODS AND RESULTS: sGCalpha1-/- mice had significantly reduced lung sGC activity and vasodilator-stimulated phosphoprotein phosphorylation. Right ventricular systolic pressure did not differ between genotypes at baseline and increased similarly in WT (22+/-2 to 34+/-2 mm Hg) and sGCalpha1-/- (23+/-2 to 34+/-1 mm Hg) mice in response to acute hypoxia. Inhaled NO (40 ppm) blunted the increase in right ventricular systolic pressure in WT mice (22+/-2 to 24+/-2 mm Hg, P<0.01 versus hypoxia without NO) but not in sGCalpha1-/- mice (22+/-1 to 33+/-1 mm Hg) and was accompanied by a significant rise in lung cGMP content only in WT mice. In contrast, the NO-donor sodium nitroprusside (1.5 mg/kg) decreased systemic blood pressure similarly in awake WT and sGCalpha1-/- mice as measured by telemetry (-37+/-2 versus -42+/-4 mm Hg). After 3 weeks of hypoxia, the increases in right ventricular systolic pressure, right ventricular hypertrophy, and muscularization of intra-acinar pulmonary vessels were 43%, 135%, and 46% greater, respectively, in sGCalpha1-/- than in WT mice (P<0.01). Increased remodeling in sGCalpha1-/- mice was associated with an increased frequency of 5'-bromo-deoxyuridine-positive vessels after 1 and 3 weeks (P<0.01 versus WT). CONCLUSIONS: Deficiency of sGCalpha1 does not alter hypoxic pulmonary vasoconstriction. sGCalpha1 is essential for NO-mediated pulmonary vasodilation and limits chronic hypoxia-induced pulmonary vascular remodeling.

Disruption of nitric oxide synthase 3 protects against the cardiac injury, dysfunction, and mortality induced by doxorubicin.

BACKGROUND: Flavoprotein reductases are involved in the generation of reactive oxygen species by doxorubicin. The objective of the present study was to determine whether or not one flavoprotein reductase, endothelial nitric oxide synthase (nitric oxide synthase 3 [NOS3]), contributes to the cardiac dysfunction and injury seen after the administration of doxorubicin. METHODS AND RESULTS: A single dose of doxorubicin (20 mg/kg) was administered to wild-type (WT) mice, NOS3-deficient mice (NOS3-/-), and mice with cardiomyocyte-specific overexpression of NOS3 (NOS3-TG). Cardiac function was assessed after 5 days with the use of echocardiography. Doxorubicin decreased left ventricular fractional shortening from 57+/-2% to 47+/-1% (P<0.001) in WT mice. Compared with WT mice, fractional shortening was greater in NOS3-/- and less in NOS3-TG after doxorubicin (55+/-1% and 35+/-2%; P<0.001 for both). Cardiac tissue was harvested from additional mice at 24 hours after doxorubicin administration for measurement of cell death and reactive oxygen species production. Doxorubicin induced cardiac cell death and reactive oxygen species production in WT mice, effects that were attenuated in NOS3-/- and were more marked in NOS3-TG mice. Finally, WT and NOS3-/- mice were treated with a lower dose of doxorubicin (4 mg/kg) administered weekly over 5 weeks. Sixteen weeks after beginning doxorubicin treatment, fractional shortening was greater in NOS3-/- than in WT mice (45+/-2% versus 28+/-1%; P<0.001), and mortality was reduced (7% versus 60%; P<0.001). CONCLUSIONS: These findings implicate NOS3 as a key mediator in the development of left ventricular dysfunction after administration of doxorubicin.

Inhaled agonists of soluble guanylate cyclase induce selective pulmonary vasodilation.

RATIONALE: Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed. OBJECTIVES: We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired. METHODS: In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg. MEASUREMENTS AND MAIN RESULTS: Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ. CONCLUSIONS: Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

Developmental barcoding of whole mouse via homing CRISPR.

In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.

Liposomal Delivery Enhances Immune Activation by STING Agonists for Cancer Immunotherapy.

Overcoming the immunosuppressive tumor microenvironment (TME) is critical to realizing the potential of cancer immunotherapy strategies. Agonists of stimulator of interferon genes (STING), a cytosolic immune adaptor protein, have been shown to induce potent anti-tumor activity when delivered into the TME. However, the anionic properties of STING agonists make them poorly membrane permeable, and limit their ability to engage STING in the cytosol of responding cells. In this study, cationic liposomes with varying surface polyethylene glycol (PEG) levels were used to encapsulate cGAMP to facilitate its cytosolic delivery. In vitro studies with antigen-presenting cells (APCs) revealed that liposomal formulations substantially improved the cellular uptake of cGAMP and pro-inflammatory gene induction relative to free drug. Liposomal encapsulation allowed cGAMP delivery to metastatic melanoma tumors in the lung, leading to anti-tumor activity, whereas free drug produced no effect at the same dose. Injection of liposomal cGAMP into orthotopic melanoma tumors showed retention of cGAMP at the tumor site and co-localization with tumor-associated APCs. Liposomal delivery induced regression of injected tumors and produced immunological memory that protected previously treated mice from rechallenge with tumor cells. These results show that liposomal delivery improves STING agonist activity, and could improve their utility in clinical oncology.

A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function.

Accurate assessment of blood haemostasis is essential for the management of patients who use extracorporeal devices, receive anticoagulation therapy or experience coagulopathies. However, current monitoring devices do not measure effects of haemodynamic forces that contribute significantly to platelet function and thrombus formation. Here we describe a microfluidic device that mimics a network of stenosed arteriolar vessels, permitting evaluation of blood clotting within small sample volumes under pathophysiological flow. By applying a clotting time analysis based on a phenomenological mathematical model of thrombus formation, coagulation and platelet function can be accurately measured in vitro in patient blood samples. When the device is integrated into an extracorporeal circuit in pig endotoxemia or heparin therapy models, it produces real-time readouts of alterations in coagulation ex vivo that are more reliable than standard clotting assays. Thus, this disposable device may be useful for personalized diagnostics and for real-time surveillance of antithrombotic therapy in clinic.

A Broad-Spectrum Infection Diagnostic that Detects Pathogen-Associated Molecular Patterns (PAMPs) in Whole Blood.

BACKGROUND: Blood cultures, and molecular diagnostic tests that directly detect pathogen DNA in blood, fail to detect bloodstream infections in most infected patients. Thus, there is a need for a rapid test that can diagnose the presence of infection to triage patients, guide therapy, and decrease the incidence of sepsis. METHODS: An Enzyme-Linked Lectin-Sorbent Assay (ELLecSA) that uses magnetic microbeads coated with an engineered version of the human opsonin, Mannose Binding Lectin, containing the Fc immunoglobulin domain linked to its carbohydrate recognition domain (FcMBL) was developed to quantify pathogen-associated molecular patterns (PAMPs) in whole blood. This assay was tested in rats and pigs to explore whether it can detect infections and monitor disease progression, and in prospectively enrolled, emergency room patients with suspected sepsis. These results were also compared with data obtained from non-infected patients with or without traumatic injuries. RESULTS: The FcMBL ELLecSA was able to detect PAMPS present on, or released by, 85% of clinical isolates representing 47 of 55 different pathogen species, including the most common causes of sepsis. The PAMP assay rapidly (<1h) detected the presence of active infection in animals, even when blood cultures were negative and bacteriocidal antibiotics were administered. In patients with suspected sepsis, the FcMBL ELLecSA detected infection in 55 of 67 patients with high sensitivity (>81%), specificity (>89%), and diagnostic accuracy of 0·87. It also distinguished infection from trauma-related inflammation in the same patient cohorts with a higher specificity than the clinical sepsis biomarker, C-reactive Protein. CONCLUSION: The FcMBL ELLecSA-based PAMP assay offers a rapid, simple, sensitive and specific method for diagnosing infections, even when blood cultures are negative and antibiotic therapy has been initiated. It may help to triage patients with suspected systemic infections, and serve as a companion diagnostic to guide administration of emerging dialysis-like sepsis therapies.