Kruti R. Patel

Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay

inTrOducTiOn Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are both widely used as diagnostic tools in medicine and as quality control measures in various industries; they are also used as analytical tools in biomedical research for the detection and quantification of specific antigens or antibodies in a given sample. These two procedures share similar basic principles and are derived from the radioimmunoassay (RIA). RIA was first described by Berson and Yalow (Yalow and Berson, 1960), for which Yalow was awarded the Nobel Prize in 1977, to measure endogenous plasma insulin. RIA was then developed into a novel technique to detect and measure biological molecules present in very small quantities, paving the way for the analysis and detection of countless other biological molecules, including hormones, peptides, and proteins. Because of the safety concern regarding its use of radioactivity, RIA assays were modified by replacing the radioisotope with an enzyme, thus creating the modern-day EIA and ELISA.

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists

Human precision-cut lung slices (hPCLSs) provide a unique ex vivo model for translational research. However, the limited and unpredictable availability of human lung tissue greatly impedes their use. Here, we demonstrate that cryopreservation of hPCLSs facilitates banking of live human lung tissue for routine use. Our results show that cryopreservation had little effect on overall cell viability and vital functions of immune cells, including phagocytes and T lymphocytes. In addition, airway contraction and relaxation in response to specific agonists and antagonists, respectively, were unchanged after cryopreservation. At the subcellular level, cryopreserved hPCLSs maintained Ca(2+)-dependent regulatory mechanisms for the control of airway smooth muscle cell contractility. To exemplify the use of cryopreserved hPCLSs in smooth muscle research, we provide evidence that bitter-taste receptor (TAS2R) agonists relax airways by blocking Ca(2+) oscillations in airway smooth muscle cells. In conclusion, the banking of cryopreserved hPCLSs provides a robust bioassay for translational research of lung physiology and disease.

A human microglia-like cellular model for assessing the effects of neurodegenerative disease gene variants

A microglial-like cellular model can be used to identify functional consequences of genetic variants associated with neurodegenerative disease. The secret life of microglia In a new study, Ryan et al. characterized a cellular model of monocytes differentiated into a microglial-like state. They then demonstrated the effectiveness of this human in vitro cell model system for identifying functional consequences of genetic variation associated with neurodegenerative disease risk by performing a cis-expression quantitative trait locus (cis-eQTL) study. The authors identified six neurodegenerative disease–associated loci with genotype-driven gene expression differences in their cell model system. Two of these cis-eQTLs were not seen in ex vivo human monocytes, emphasizing the importance of examining the cell type of interest when investigating functional consequences of genetic variants. Microglia are emerging as a key cell type in neurodegenerative diseases, yet human microglia are challenging to study in vitro. We developed an in vitro cell model system composed of human monocyte-derived microglia-like (MDMi) cells that recapitulated key aspects of microglia phenotype and function. We then used this model system to perform an expression quantitative trait locus (eQTL) study examining 94 genes from loci associated with Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. We found six loci (CD33, PILRB, NUP160, LRRK2, RGS1, and METTL21B) in which the risk haplotype drives the association with both disease susceptibility and altered expression of a nearby gene (cis-eQTL). In the PILRB and LRRK2 loci, the cis-eQTL was found in the MDMi cells but not in human peripheral blood monocytes, suggesting that differentiation of monocytes into microglia-like cells led to the acquisition of a cellular state that could reveal the functional consequences of certain genetic variants. We further validated the effect of risk haplotypes at the protein level for PILRB and CD33, and we confirmed that the CD33 risk haplotype altered phagocytosis by the MDMi cells. We propose that increased LRRK2 gene expression by MDMi cells could be a functional outcome of rs76904798, a single-nucleotide polymorphism in the LRKK2 locus that is associated with Parkinson’s disease.

Targeting thrombomodulin to circulating red blood cells augments its protective effects in models of endotoxemia and ischemia-reperfusion injury

Endothelial thrombomodulin (TM) regulates coagulation and inflammation via several mechanisms, including production of activated protein C (APC). Recombinant APC and soluble fragments of TM (sTM) have been tested in settings associated with insufficiency of the endogenous TM/APC pathway, such as sepsis. We previously designed a fusion protein of TM [single‐chain variable fragment antibody (scFv)/TM] targeted to red blood cells (RBCs) to improve pharmacokinetics and antithrombotic effects without increasing bleeding. Here, scFv/ TM was studied in mouse models of systemic inflammation and ischemia‐reperfusion injury. Injected concomitantly with or before endotoxin, scFv/TM provided more potent protection against liver injury and release of pathological mediators than sTM, showing similar efficacy at up to 50‐fold lower doses. scFv/TM provided protection when injected after endotoxin, whereas sTM did not, and augmented APC production by thrombin ~50‐fold more than sTM. However, scFv/TM injected after endotoxin did not reduce thrombin/antithrombin complexes; nor did antibodies that block APC anticoagulant activity suppress the prophylactic anti‐inflammatory effect of scFv/TM. Therefore, similar to endogenous TM, RBC‐anchored scFv/TM activates several protective pathways. Finally, scFv/TM was more effective at reducing cerebral infarct volume and alleviated neurological deficits than sTM after cerebral ischemia/reperfusion injury. These results indicate that RBC‐targeted scFv/TM exerts multifaceted cytoprotective effects and may find utility in systemic and focal inflammatory and ischemic disorders.—Carnemolla, R., Villa, C.H., Greineder, C. F., Zaitseva, S., Patel, K.R., Kowalska, M.A., Atochin, D. N., Cines, D.B., Siegel, D.L., Esmon, C. T., Muzykantov, V. R. Targetingthrombomodulin to circulatingred blood cells augments its protective effects in models of endotoxemia and ischemia‐reperfusion injury FASEB J. 31, 761–770 (2017). http://www.fasebj.org

A new approach for the study of lung smooth muscle phenotypes and its application in a murine model of allergic airway inflammation.

Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained ex vivo. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.

Quantitative analysis of thrombomodulin-mediated conversion of protein C to APC: Translation from in vitro to in vivo

Thrombomodulin-bound thrombin cleaves protein C (PC) zymogen in blood plasma producing activated protein C (APC), which exerts anti-coagulant, anti-inflammatory, anti-apoptotic and CNS-protective effects. Recombinant APC and thrombomodulin (TM) are both in clinical studies for management of acute conditions including sepsis. Methods that permit accurate measurement of APC in plasma are needed for clinical monitoring and mechanistic studies in animal models. However, the two existing methods require either long incubation periods with substrate, resulting in high background or they also recognize protein C inhibitor (PCI) complexed with APC (APC:PCI), which convolutes analysis of the amount of APC generated. Here we describe a robust quantitative in vivo assay that measures APC generation at both low levels of human protein C seen in chronic inflammatory disease and at physiological levels that shows a >99% fit with in vitro data.

Platelet Endothelial Cell Adhesion Molecule Targeted Oxidant-Resistant Mutant Thrombomodulin Fusion Protein with Enhanced Potency In Vitro and In Vivo

Thrombomodulin (TM) is a glycoprotein normally present in the membrane of endothelial cells that binds thrombin and changes its substrate specificity to produce activated protein C (APC) that has antithrombotic and anti-inflammatory features. To compensate for loss of endogenous TM in pathology, we have fused recombinant TM with single chain variable fragment (scFv) of an antibody to mouse platelet endothelial cell adhesion molecule-1 (PECAM). This fusion, anti-PECAM scFv/TM, anchors on the endothelium, stimulates APC production, and provides therapeutic benefits superior to sTM in animal models of acute thrombosis and inflammation. However, in conditions of oxidative stress typical of vascular inflammation, TM is inactivated via oxidation of the methionine 388 (M388) residue. Capitalizing on the reports that M388L mutation renders TM resistant to oxidative inactivation, in this study we designed a mutant anti-PECAM scFv/TM M388L. This mutant has the same APC-producing capacity and binding to target cells, yet, in contrast to wild-type fusion, it retains APC-producing activity in an oxidizing environment in vitro and in vivo. Therefore, oxidant resistant mutant anti-PECAM scFv/TM M388L is a preferable targeted biotherapeutic to compensate for loss of antithrombotic and anti-inflammatory TM functions in the context of vascular oxidative stress.

Mast cell-derived neurotrophin 4 mediates allergen-induced airway hyperinnervation in early life.

Asthma often progresses from early episodes of insults. How early-life events connect to long-term airway dysfunction remains poorly understood. We demonstrated previously that increased neurotrophin 4 (NT4) levels following early-life allergen exposure cause persistent changes in airway smooth muscle (ASM) innervation and airway hyper-reactivity (AHR) in mice. Herein, we identify pulmonary mast cells as a key source of aberrant NT4 expression following early insults. NT4 is selectively expressed by ASM and mast cells in mice, nonhuman primates, and humans. We show in mice that mast cell-derived NT4 is dispensable for ASM innervation during development. However, upon insults, mast cells expand in number and degranulate to release NT4 and thus become the major source of NT4 under pathological condition. Adoptive transfer of wild-type mast cells, but not NT4−/− mast cells restores ASM hyperinnervation and AHR in KitW-sh/W-sh mice following early-life insults. Notably, an infant nonhuman primate model of asthma also exhibits ASM hyperinnervation associated with the expansion and degranulation of mast cells. Together, these findings identify an essential role of mast cells in mediating ASM hyperinnervation following early-life insults by producing NT4. This role may be evolutionarily conserved in linking early insults to long-term airway dysfunction.

Foxo1 Promotes Th9 Cell Differentiation and Airway Allergy

T helper 9 (Th9) cells are effector CD4+ T cells that are characterized by the production of interleukin-9 (IL-9) and have been associated with allergic responses. Here, we found that the expression of the transcription factor forkhead box O1 (Foxo1) was induced in Th9 and Foxo1 plays a crucial role in the differentiation of Th9 cells. Pharmacological inhibition of Foxo1 or genetic disruption of Foxo1 in CD4+ T cells caused a reduction in IL-9 expression while upregulating IL-17A and IFNγ production. Furthermore, chromatin immunoprecipitation (ChIP) followed by luciferase assays revealed direct binding of Foxo1 to both the Il9 and Irf4 promoters and induces their transactivation. Lastly, adoptive transfer of Th9 cells into lungs induced asthma-like symptoms that were ameliorated by Foxo1 inhibitor, AS1842856. Together, our findings demonstrate a novel regulator of Th9 cells with a direct implication in allergic inflammation.

Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion

Pulmonary neuroendocrine cells (PNECs) are the only innervated airway epithelial cells. To what extent neural innervation regulates PNEC secretion and function is unknown. Here, we discover that neurotrophin 4 (NT4) plays an essential role in mucus overproduction after early life allergen exposure by orchestrating PNEC innervation and secretion of GABA. We found that PNECs were the only cellular source of GABA in airways. In addition, PNECs expressed NT4 as a target‐derived mechanism underlying PNEC innervation during development. Early life allergen exposure elevated the level of NT4 and caused PNEC hyperinnervation and nodose neuron hyperactivity. Associated with aberrant PNEC innervation, the authors discovered that GABA hypersecretion was required for the induction of mucin Muc5ac expression. In contrast, NT4−/− mice were protected from allergen‐induced mucus overproduction and changes along the nerve–PNEC axis without any defects in inflammation. Last, GABA installation restored mucus overproduction in NT4−/− mice after early life allergen exposure. Together, our findings provide the first evidence for NT4‐dependent neural regulation of PNEC secretion of GABA in a neonatal disease model. Targeting the nerve–PNEC axis may be a valid treatment strategy for mucus overproduction in airway diseases, such as childhood asthma.—Barrios, J., Patel, K. R., Aven, L., Achey, R., Minns, M. S., Lee, Y., Trinkaus‐Randall, V. E., Ai, X. Early life allergen‐induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion. FASEB J. 31, 4117–4128 (2017). www.fasebj.org—Barrios, Juliana, Patel, Kruti R., Aven, Linh, Achey, Rebecca, Minns, Martin S., Lee, Yoonjoo, Trinkaus‐Randall, Vickery E., Ai, Xingbin Early life allergen‐induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion. FASEB J. 31, 4117–4128 (2017)