3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF's blood vessel forming potential prior to clinical implantation.

Formation of Adipose Stromal Vascular Fraction Cell-Laden Spheroids Using a Three-Dimensional Bioprinter and Superhydrophobic Surfaces.

The therapeutic infusion of adipose-derived stromal vascular fraction (SVF) cells for the treatment of multiple diseases, has progressed to numerous human clinical trials; however, the often poor retention of the cells following implantation remains a common drawback of direct cell injection. One solution to cellular retention at the injection site has been the use of biogels to encapsulate cells within a microenvironment before and upon implantation. The current study utilized three-dimensional bioprinting technology to evaluate the ability to form SVF cell-laden spheroids with collagen I as a gel-forming biomatrix. A superhydrophobic surface was created to maintain the bioprinted structures in a spheroid shape. A hydrophilic disc was printed onto the hydrophobic surface to immobilize the spheroids during the gelation process. Conditions for the automated rapid formation of SVF cell-laden spheroids were explored, including time/pressure relationships for spheroid extrusion during bioprinting. The formed spheroids maintain SVF viability in both static culture and dynamic spinner culture. Spheroids also undergo a time-dependent contraction with the retention of angiogenic sprout phenotype over the 14-day culture period. The use of a biphilic surface exhibiting both superhydrophobicity to maintain the spheroid shape and a hydrophilicity to immobilize the spheroid during gel formation produces SVF cell-laden spheroids that can be immediately transplanted for therapeutic applications.

Oligoclonal antibody targeting ghrelin increases energy expenditure and reduces food intake in fasted mice.

Ghrelin, an enteric peptide hormone linked to the pathophysiology of obesity has been a therapeutic target of great interest over the past decade. Many research efforts have focused on the antagonism of ghrelin's endogenous receptor GHSR1a, which is found along ascending vagal afferent fibers, as well as in the arcuate nucleus of the hypothalamus. Additionally, peptidic inhibitors of ghrelin O-acyltransferase, the enzyme responsible for the paracrine activation of ghrelin, have recently been studied. Our research has taken an alternative immunological approach, studying both active and passive vaccination as a means to sequester ghrelin in the periphery, with the original discovery in rat of decreased feed efficiency and adiposity, as well as increased metabolic activity. Using our previous hapten designs as a stepping-stone, three monoclonal antibodies (JG2, JG3, and JG4) were procured against ghrelin and tested in vivo. While mAb JG4 had the highest affinity for ghrelin, it failed to attenuate the orexigenic effects of food deprivation on energy metabolism or food intake in mice. However, animals that were administered a combination of JG3:JG4 (termed a doublet) or JG2:JG3:JG4 (termed a triplet) demonstrated higher heat dispersion and rate of respiration (higher CO(2) emission and O(2) consumption) during a 24 h fast refeed. Mice administered the triplet cocktail of JG2:JG3:JG4 also demonstrated decreased food intake upon refeeding as compared to control animals. Recently, Lu and colleagues reported that a passive approach using a single, high affinity N-terminally directed monoclonal antibody did not abrogate the effects of endogenous ghrelin. Our current report corroborates this finding, yet, refutes that a monoclonal antibody approach cannot be efficacious. Rather, we find that a multiple monoclonal antibody (oligoclonal) approach can reproduce the underlying logic to previously reported efficacies using active vaccinations.

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization.

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K(+) channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase--a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.

A multivalent probe for AI-2 quorum-sensing receptors.

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum-sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum-sensing signaling molecule.

A vaccine strategy that induces protective immunity against heroin.

Heroin addiction is a wide-reaching problem with a spectrum of damaging social consequences. A vaccine capable of blocking heroin's effects could provide a long-lasting and sustainable adjunct to heroin addiction therapy. Heroin, however, presents a particularly challenging immunotherapeutic target, as it is metabolized to multiple psychoactive molecules. To reconcile this dilemma, we examined the idea of a singular vaccine with the potential to display multiple drug-like antigens; thus two haptens were synthesized, one heroin-like and another morphine-like in chemical structure. A key feature in this approach is that immunopresentation with the heroin-like hapten is thought to be immunochemically dynamic such that multiple haptens are simultaneously presented to the immune system. We demonstrate the significance of this approach through the extremely rapid generation of robust polyclonal antibody titers with remarkable specificity. Importantly, both the antinociceptive effects of heroin and acquisition of heroin self-administration were blocked in rats vaccinated using the heroin-like hapten.

Design, synthesis, and biological activities of closantel analogues: structural promiscuity and its impact on Onchocerca volvulus.

Onchocerciasis, or river blindness, is a neglected tropical disease that affects more than 37 million people worldwide, primarily in Africa and Central and South America. We have disclosed evidence that the larval-stage-specific chitinase, OvCHT1, may be a potential biological target for affecting nematode development. On the basis of screening efforts, closantel, a known anthelmintic drug, was discovered as a potent and highly specific OvCHT1 inhibitor. Originally, closantel's anthelmintic mode of action was believed to rely solely on its role as a proton ionophore; thus, the impact of each of its biological activities on O. volvulus L3 molting was investigated. Structure-activity relationship studies on an active closantel fragment are detailed, and remarkably, by use of a simple salicylanilide scaffold, compounds acting only as protonophores or chitinase inhibitors were identified. From these data, unexpected synergistic protonophore and chitinase inhibition activities have also been found to be critical for molting in O. volvulus L3 larvae.

Synthesis and molecular modeling provide insight into a Pseudomonas aeruginosa quorum sensing conundrum.

The triphenyl amide/ester 12 was originally reported to be a potent mimic of the natural 3-oxo-dodecanoyl homoserine lactone quorum sensing molecule in Pseudomonas aeruginosa. However, explicit synthesis/chemical characterization was lacking, and a later report providing protein crystallographic data inferred 12 to be incorrect, with 9 now being the surmised structure. Because of these inconsistencies and our interest in quorum sensing molecules utilized by gram-negative bacteria, we found it necessary to synthesize 9 and 12 to test for agonistic activity in a P. aeruginosa reporter assay. Despite distinct regiochemical differences, both 9 and 12 were found to have comparable EC(50) values. To reconcile these unanticipated findings, modeling studies were conducted, and both compounds were revealed to have comparable properties for binding to the LasR receptor.

A cross-over inhibitor of the botulinum neurotoxin light chain B: a natural product implicating an exosite mechanism of action.

Clostridium botulinum produces the most lethal toxins known to man, as such they are high risk terrorist threats, and alarmingly there is no approved therapeutic. We report the first cross-over small molecule inhibitor of these neurotoxins and propose a mechanism by which it may impart its inhibitory activity.

Developing a vaccine against multiple psychoactive targets: a case study of heroin.

Heroin addiction is a wide-reaching problem with a spectrum of damaging social consequences. Currently approved heroin addiction medications include drugs that bind at the same receptors (e.g. opioid receptors) occupied by heroin and/or its metabolites in the brain, but undesired side effects of these treatments, maintenance dependence and relapse to drug taking remains problematic. A vaccine capable of blocking heroin's effects could provide an economical, long-lasting and sustainable adjunct to heroin addiction therapy without the side effects associated with available treatment options. Heroin, however, presents a particularly challenging vaccine target as it is metabolized to multiple psychoactive molecules of differing lipophilicity, with differing abilities to cross the blood brain barrier. In this review, we discuss the opiate scaffolding and hapten design considerations to confer immunogenicity as well as the specificity of the immune response towards structurally similar opiates. In addition, we detail different strategies employed in the design of immunoconjugates for a vaccine-based therapy for heroin addiction treatment.

Identification of a Natural Product Antagonist against the Botulinum Neurotoxin Light Chain Protease.

Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960's, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a K(i) of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.