Biochemical Stimulation of Immune Cells and Measurement of Mechanical Responses Using Atomic Force Microscopy.

This manuscript details methods to ligate cell-surface receptors on live cells with precise spatiotemporal control using an atomic force microscope (AFM) to deliver ligands. This approach can be used to image cellular responses upon activating T cell receptors when the AFM is mounted on an optical microscope. Moreover, the AFM measures forces generated by the cell during the contact. Using AFM to trigger cellular responses adds an important capability to the field of mechanobiology. We describe how to incorporate anti-CD3 antibodies or other molecules onto an AFM cantilever and how to use AFM to activate T cells. © 2019 by John Wiley & Sons, Inc.

Cytoskeletal adaptivity regulates T cell receptor signaling.

The factors that govern T cell activation control the initiation and progression of adaptive immune responses. T cells recognize their cognate antigen on the surface of antigen-presenting cells (APCs) through the T cell receptor, which results in the formation of a contact region (immune synapse) between the two cells and the activation of the T cells. Activated T cells proliferate and differentiate into effector T cells that secrete cytokines, provide help to B cells, and kill target cells. We asked whether the actin cytoskeleton governs differences in signaling in effector T cells versus naïve (unstimulated) T cells. Using atomic force microscopy and quantitative confocal microscopy, we found that naïve T cells had a mechanically stiffer cortical cytoskeleton than that of effector cells, which resulted in naïve cells forming smaller immune synapses with APCs. This suggests that the cytoskeletal stiffness of the T cell before it undergoes antigen stimulation predicts its subsequent dynamic engagement with APCs and its activation potential. Cytoskeletal rigidity depended on the activity of the actin-severing enzyme cofilin through a pathway requiring the small guanosine triphosphatase RhoA and the kinases ROCK (Rho-activated kinase) and LIMK. These findings suggest that the baseline cytoskeletal state controls T cell responses and that the underlying pathway could be a therapeutic target for modulating adaptive immunity.

Spatiotemporally and mechanically controlled triggering of mast cells using atomic force microscopy.

Mast cells are thought to be sensitive to mechanical forces, for example, coughing in asthma or pressure in "physical urticarias." Conversion of mechanical forces to biochemical signals could potentially augment antigenic signaling. Studying the combined effects of mechanical and antigenic cues on mast cells and other hematopoietic cells has proven difficult. Here, we present an approach using a modified atomic force microscope cantilever to deliver antigenic signals to mast cells while simultaneously applying mechanical forces. We developed a strategy to concurrently record degranulation events by fluorescence microscopy during antigenic triggering. Finally, we also measured the mechanical forces generated by mast cells while antigen receptors are ligated. We showed that mast cells respond to antigen delivered by the atomic force microscopy cantilever with prompt degranulation and the generation of strong pushing and pulling forces. We did not discern any relationship between applied mechanical forces and the kinetics of degranulation. These experiments present a new method for dissecting the interactions of mechanical and biochemical cues in the signaling responses of immune cells.

Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes.

Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.

Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications.

We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.

Real-time GPU-based 3D Deconvolution.

Confocal microscopy is an oft-used technique in biology. Deconvolution of 3D images reduces blurring from out-of-focus light and enables quantitative analyses, but existing software for deconvolution is slow and expensive. We present a parallelized software method that runs within ImageJ and deconvolves 3D images ~100 times faster than conventional software (few seconds per image) by running on a low-cost graphics processor board (GPU). We demonstrate the utility of this software by analyzing microclusters of T cell receptors in the immunological synapse of a CD4 + T cell and dendritic cell. This software provides a low-cost and rapid way to improve the accuracy of 3D microscopic images obtained by any method.

Atomic force mechanobiology of pluripotent stem cell-derived cardiomyocytes.

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent, stem cell-derived cardiomyocytes, including contraction force, rate, duration, and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy, and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive, inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents, or as a platform for studying cardiomyocyte biology.

Discovery of a Novel Class of Bicyclo[3.1.0]hexanylpiperazines as Noncompetitive Neuropeptide Y Y1 Antagonists.

A novel class of bicyclo[3.1.0]hexanylpiperazine neuropeptide Y (NPY) Y1 antagonists has been designed and synthesized. Scatchard binding analysis showed these compounds to be noncompetitive with [(125)I]PYY binding to the Y1 receptor. The most potent member, 1-((1α,3α,5α,6ß)-6-(3-ethoxyphenyl)-3-methylbicyclo[3.1.0]hexan-6-yl)-4-phenylpiperazine (2) had an IC50 = 62 nM and displayed excellent oral bioavailability in rat (% F po = 80), as well as good brain penetration (B/P ratio = 0.61). In a spontaneous nocturnal feeding study with male Sprague-Dawley rats, 2 significantly reduced food intake during a 12 h period.

Pharmacological characterization and appetite suppressive properties of BMS-193885, a novel and selective neuropeptide Y(1) receptor antagonist.

Treatment of obesity is still a large unmet medical need. Neuropeptide Y is the most potent orexigenic peptide in the animal kingdom. Its five cloned G-protein couple receptors are all implicated in the regulation of energy homeostasis evidenced by overexpression or deletion of neuropeptide Y or its receptors. Neuropeptide Y most likely exerts its orexigenic activity via the neuropeptide Y(1) and neuropeptide Y(5) receptors, although the involvement of the neuropeptide Y(2) and neuropeptide Y(4) receptors are also gaining importance. The lack of potent, selective, and brain penetrable pharmacologic agents at these receptors made our understanding of the modulation of food intake by neuropeptide Y-ergic agents elusive. BMS-193885 (1,4-dihydro-[3-[[[[3-[4-(3-methoxyphenyl)-1-piperidinyl]propyl]amino] carbonyl]amino]phenyl]-2,6-dimethyl-3,5-pyridinedicarboxylic acid, dimethyl ester) is a potent and selective neuropeptide Y(1) receptor antagonist. BMS-193885 has 3.3 nM affinity at the neuropeptide Y(1) receptor, acting competitively at the neuropeptide Y binding site. BMS-193885 increased the K(d) of [(125)I]PeptideYY from 0.35 nM to 0.65 nM without changing the B(max) (0.16 pmol/mg of protein) in SK-N-MC cells that endogenously express the neuropeptide Y(1) receptor. It is also found to be a full antagonist with an apparent K(b) of 4.5 nM measured by reversal of forskolin (FK)-stimulated inhibition of cAMP production by neuropeptide Y. Pharmacological profiling showed that BMS-193885 has no appreciable affinity at the other neuropeptide Y receptors, and is also 200-fold less potent at the alpha(2) adrenergic receptor. Testing the compound in a panel of 70 G-protein coupled receptors and ion channels resulted in at least 200-fold or greater selectivity, with the exception of the sigma(1) receptor, where the selectivity was 100-fold. When administered intracerebroventricularly or directly into the paraventricular nucleus of the hypothalamus, it blocked neuropeptide Y-induced food intake in rats. Intraperitoneal administration of BMS-193885 (10 mg/kg) also reduced one-hour neuropeptide Y-induced food intake in satiated rats, as well as spontaneous overnight food consumption. Chronic administration of BMS-193885 (10 mg/kg) i.p. for 44 days significantly reduced food intake and the rate of body weight gain compared to vehicle treated control without developing tolerance or affecting water intake. These results provide supporting evidence that BMS-193885 reduces food intake and body weight via inhibition of the central neuropeptide Y(1) receptor. BMS-193885 has no significant effect of locomotor activity up to 20 mg/kg dose after 1 h of treatment. It also showed no activity in the elevated plus maze when tested after i.p. and i.c.v. administration, indicating that reduction of food intake is unrelated to anxious behavior. BMS-193885 has good systemic bioavailability and brain penetration, but lacks oral bioavailability. The compound had no serious cardiovascular adverse effect in rats and dogs up to 30 and 10 mg/kg dose, respectively, when dosed intravenously. These data demonstrate that BMS-193885 is a potent, selective, brain penetrant Y(1) receptor antagonist that reduces food intake and body weight in animal models of obesity both after acute and chronic administration. Taken together the data suggest that a potent and selective neuropeptide Y(1) receptor antagonist might be an efficacious treatment for obesity in humans.

Isosteric N-arylpiperazine replacements in a series of dihydropyridine NPY1 receptor antagonists.

4-Amino-N-arylpiperidines serve as effective bioisosteres for N-arylpiperazines in the series of dihydropyridine NPY1 receptor antagonists. These were prepared by a ZnCl2-mediated reductive amination reaction between elaborated primary amines, 2 or 5, and 4-arylpiperidones.

Chronobiotic activity of N-[2-(2,7-dimethoxyfluoren-9-yl)ethyl]-propanamide. Synthesis and melatonergic pharmacology of fluoren-9-ylethyl amides.

A series of fluoren-9-yl ethyl amides (2) were synthesized and evaluated for human melatonin MT(1) and MT(2) receptor binding. N-[2-(2,7-dimethoxyfluoren-9-yl)ethyl]propanamide (2b) was selected and evaluated in functional assays measuring intrinsic activity at the human MT(1) and MT(2) receptors and demonstrated full agonism at both receptors. The chronobiotic properties of 2b were demonstrated in both acute and chronic rat models where 2b produced an acute phase advance of 32 min at 1mg/kg and chronically entrained free-running rats with a mean effective dose of 0.23 mg/kg. Compound 2b is significantly less efficacious than melatonin in constricting human coronary artery.

Dihydropyridine neuropeptide Y Y1 receptor antagonists 2. bioisosteric urea replacements.

Structure-activity studies around the urea linkage in BMS-193885 (4a) identified the cyanoguanidine moiety as an effective urea replacement in a series of dihydropyridine NPY Y(1) receptor antagonists. In comparison to urea 4a (K(i)=3.3 nM), cyanoguanidine 20 (BMS-205749) displayed similar binding potency at the Y(1) receptor (K(i)=5.1 nM) and full functional antagonism (K(b)=2.6 nM) in SK-N-MC cells. Cyanoguanidine 20 also demonstrated improved permeability properties in Caco-2 cells in comparison to urea 4a (43 vs 19 nm/s).

Dihydropyridine neuropeptide Y Y(1) receptor antagonists.

Dihydropyridine 5a was found to be an inhibitor of neuropeptide Y(1) binding in a high throughput (125)I-PYY screening assay. Structure-activity studies around certain portions of the dihydropyridine chemotype identified BMS-193885 (6e) as a potent and selective Y(1) receptor antagonist. In a forskolin-stimulated c-AMP production assay using CHO cells expressing the human Y(1) receptor, 6e demonstrated full functional antagonism (K(b)=4.5 nM). Compound 6e inhibited NPY-induced feeding in satiated rats when dosed at 3.0 and 10.0 mg/kg (ip), and also decreased spontaneous overnight food consumption in rats at doses of 10 and 20 mg/kg (ip).