Low-Cost Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells

Biopharmaceuticals made using current production systems are prohibitively expensive and are not affordable for a large majority of the global population. The cost of protein drugs ($140 billion in 2013) exceeds GDP of \u3e75$2 per day cannot afford any protein drugs. This is because of their production in prohibitively expensive fermenters, purification, cold storage/transportation, short shelf life and sterile delivery methods. Simpler methods of delivery such as oral dosing could obviate much of the expense: however, oral delivery of protein drugs has been elusive for decades because of their degradation in the digestive system, inability to cross the gut epithelium and delivery to target cells/tissues

Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells

Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood–brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer\u27s disease, diabetes, hypertension, Gaucher\u27s or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery

Arrhythmogenic Right Ventricular Cardiomyopathy: Is Coxsackie the Criminal?

Introduction: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy characterized by right ventricular dysfunction, which can precipitate sudden cardiac death in young adults. Case Presentation: A 22-year-old Hispanic male with PMH of hypertriglyceridemia and exertional syncopal episodes was brought to the ED after experiencing a sudden cardiac arrest while on the treadmill. On arrival, the patient was intubated and placed on a defibrillator which detected Vfib with torsade de pointes. On arrival to ED, initial ECG revealed 1-2 mm ST depressions in leads II, III, and aVF and incomplete RBBB. Chest XR showed water-bottle-shaped cardiac silhouette. Coronary angiography demonstrated patent coronary arteries. Subsequently, 2D echo was performed which showed severe enlargement of RV with EF of 30-35% and RV free wall akinesis. The only pertinent positives were Coxsackie A and B antibodies with titers as high as 1:1600 in Coxsackie A Ab. The patient met the 2010 Task Force Criteria for ARVC including 1 major criterion by 2D echo and 2 major criteria by ECG. The first major criteria met were regional RV akinesia and increased RV dimensions in end-diastole of PLAX RVOT \u3e32mm and/or PSAX RVOT \u3e36 mm. The second major criteria met were inverted T-waves in V1, V2, and V3 in the absence of complete RBBB. Lastly, the third major criterion met was the presence of VT of left bundle branch morphology with superior axis. Conclusion: This case highlights the uncertainty behind the pathogenesis of ARVC and the role that cardiotropic viruses such as Coxsackie play in the pathophysiology of this disease

Oral Delivery of Bioencapsulated Exendin-4 Expressed in Chloroplasts Lowers Blood Glucose Level in Mice and Stimulates Insulin Secretion in Beta-TC6 Cells

Glucagon like peptide (GLP-1) increases insulin secretion but is rapidly degraded (half-life: 2 min in circulation). GLP-1 analog, Exenatide (Byetta) has a longer half life (3.3–4 hrs) with potent insulinotropic effects but requires cold storage, daily abdominal injections with short shelf life. Because diabetic patients take \u3e60,000 injections in their life time, alternative delivery methods are highly desired. Exenatide is ideal for oral delivery because insulinotropism is glucose dependent, with reduced risk of hypoglycemia even at higher doses. Therefore, exendin-4 (EX4) was expressed as a cholera toxin B subunit (CTB)-fusion protein in tobacco chloroplasts to facilitate bioencapsulation within plant cells and transmucosal delivery in the gut via GM1 receptors present in the intestinal epithelium. The transgene integration was confirmed by PCR and Southern blot analysis. Expression level of CTB-EX4 reached up to 14.3% of total leaf protein (TLP). Lyophilization of leaf material increased therapeutic protein concentration by 12–24 fold, extended their shelf life up to 15 months when stored at room temperature and eliminated microbes present in fresh leaves. The pentameric structure, disulfide bonds and functionality of CTB-EX4 were well preserved in lyophilized materials. Chloroplast derived CTB-EX4 showed increased insulin secretion similar to the commercial EX4 in beta-TC6, a mouse pancreatic cell line. Even when 5,000-fold excess dose of CTB-EX4 was orally delivered, it stimulated insulin secretion similar to the intraperitoneal injection of commercial EX4 but didn’t cause hypoglycemia in mice. Oral delivery of the bioencapsulated EX4 should eliminate injections, increase patient compliance/convenience and significantly lower their cost

Taking The Load Off: Device Lead-Induced Severe Tricuspid Regurgitation and Right-Sided Heart Failure Treated With Transcatheter Aortic Valve Implantation (TAVI)

Background: Interference of device leads with closure of the tricuspid leaflets can cause severe tricuspid regurgitation (TR) and right-sided heart failure, as can heart failure with reduced ejection fraction (HFrEF) and left sided valve disease. We report a case treated with transcatheter aortic valve implantation (TAVI) with improved TR. Case: A 78-year-old male with coronary artery disease status post remote myocardial infarction and coronary bypass, HFrEF with left ventricular ejection fraction 35-40%, and inducible ventricular tachycardia status post AICD placement presented with worsening dyspnea on exertion, easy fatigue, lower extremity edema, anasarca and abdominal bloating. Transthoracic echocardiography demonstrated severe aortic stenosis with secondary pulmonary hypertension, a right ventricular (RV) systolic pressure of 50 mmHg, severe bi-atrial enlargement, and severe TR in the region of his device lead. Transesophageal echocardiography confirmed mechanical interference by the lead with closure of the tricuspid septal leaflet. Symptoms were refractory to diuretics. He was a poor candidate for lead extraction given fibrotic changes. Tricuspid valve (TV) replacement was high risk given prior bypass surgery with patent grafts. TAVI was performed to reduce pulmonary artery pressures, with concomitant guideline directed medical therapy (GDMT) for HFrEF to reduce tricuspid regurgitation from RV pressure overload from left sided heart disease. Repeat echo showed a reduced RV systolic pressure of 42 mmHg, with his TR improved to moderate to severe. Conclusion: While diuretics are the mainstay of therapy, medical therapies for management of severe TR are limited. Attention should be focused on the underlying etiology of the TR and classification as primary (abnormal valve leaflets-including device lead induced) or secondary (normal valve leaflets). Our patient had mixed involvement. Management was directed accordingly with TAVI and GDMT for HFrEF, with plans for the higher risk surgical TV repair or replacement should symptoms persist

Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts

Oral Delivery of Human Biopharmaceuticals, Autoantigens and Vaccine Antigens Bioencapsulated in Plant Cells

Among 12 billion injections administered annually, unsafe delivery leads to \u3e20 million infections and \u3e100 million reactions. In an emerging new concept, freeze-dried plant cells (lettuce) expressing vaccine antigens/biopharmaceuticals are protected in the stomach from acids/enzymes but are released to the immune or blood circulatory system when plant cell walls are digested by microbes that colonize the gut. Vaccine antigens bioencapsulated in plant cells upon oral delivery after priming, conferred both mucosal and systemic immunity and protection against bacterial, viral or protozoan pathogens or toxin challenge. Oral delivery of autoantigens was effective against complications of type 1diabetes and hemophilia, by developing tolerance. Oral delivery of proinsulin or exendin-4 expressed in plant cells regulated blood glucose levels similar to injections. Therefore, this new platform offers a low cost alternative to deliver different therapeutic proteins to combat infectious or inherited diseases by eliminating inactivated pathogens, expensive purification, cold storage/transportation and sterile injections

Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species During Biotic and Abiotic Stress

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a novel signaling mechanism, especially for participation of chloroplast proteins (e.g. transcription factors) in retrograde signaling, thereby offering new opportunities to regulate pathways outside chloroplasts

Retrospective Study Examining Obesity Hypoventilation Syndrome in COVID+ Patients

Purpose: Coronavirus disease 2019 (COVID-19) has affected millions of people all over the world with worse proven outcomes in those with certain comorbid conditions, such as diabetes, cardiovascular disease, and pulmonary complications. The Rio Grande Valley located in South Texas with a largely Hispanic population has been hit especially hard during this pandemic with over 3,200 virus-related deaths. This region’s high population of diabetic and obese patients is likely correlated with the especially high mortality rate. While it is understood the impact that obesity has on worsening health outcomes, further research is needed to better understand whether more adverse COVID-19 outcomes are correlated with an underdiagnosis of Obesity Hypoventilation Syndrome (OHS) amongst obese patients. Patients and Methods: Using an observational database from Valley Baptist Medical Center (VBMC) in Harlingen, TX, we gathered a list of COVID+ patients admitted between March 19, 2020 and September 25, 2020. COVID-19 was diagnosed based on World Health Organization (WHO) guidance. The official database is still a work-in-progress, as we are still working on manual data-entry for co-existing conditions and lab values for these patients. Once the database is completed, evaluation guidelines as listed in the American Journal of Respiratory and Critical Care Medicine will be used as a screening method to identify OHS in COVID+ patients. COVID-19 outcomes including hospitalization length, ICU transfer/admission, intubation count, in-hospital death will then be evaluated. Results: Of the 1114 patients with COVID-19+ included in our database, we have completed chart review on 112 patients. Once the database is completed, statistical analysis will be performed using Python to see if there is a higher percentage of adverse COVID-19+ outcomes in OHS-suspected patients compared to obese patients who don’t meet the criteria for OHS. Further analysis will also be done to compare these outcomes to the remaining admitted COVID+ patients. Conclusion: Database still in progress and no conclusion can be drawn at this time