Investigating the immunomodulatory potential of metals present in Sudbury Particulate Matter (SPaM)

The incidence of allergic disease has risen dramatically over the last century. This increase cannot simply be attributed to genetics, and so environmental contributions to allergic disease must be considered. Airborne particulate matter is recognized as a contributing factor in the initiation and exacerbation of allergic respiratory diseases due to its ability to act as an adjuvant and irritant. Particulate matter is heterogeneous in composition, and different components contribute to its adjuvant effects, including metals. This thesis investigates whether metals contained in Sudbury Particulate Matter (SPaM) are potential immunomodulators. We investigated the ability of metals in SPaM to influence inflammation both in vivo and in vitro. We demonstrate that the metals are able to alter the inflammatory profile after physiologically relevant exposure levels, suggesting that some of the metals present in SPaM are immunomodulatory in nature and may influence allergic disease. Key WordsMaster of Science (MSc) in Biolog

Case Reports of Situs Inversus Totalis and Dextrocardia in Sprague Dawley Rats

Situs inversus totalis is a condition where there is a transposition of all internal organs from their normal anatomical location. This infrequent and rare congenital condition has been described in several species of mammals. Dextorcardia is a series of conditions associated with an abnormal congenital positioning of the heart, and is often associated with situs inversus totalis. Here we report a case of situs inversus totalis and two cases of dextrocardia identified in Sprague Dawley rats during gross necropsy evaluations at both the Health Sciences North Research Institute (HSNRI) in Canada and Finlay Institute of Vaccine Research and Production in Cuba. The intent of this report is to share our findings and aid in the accumulation of data on these rare conditions

Assessing the immunogenicity and toxicity of the AFPL1-conjugate nicotine vaccine using heterologous and homologous vaccination routes.

Despite the increased risks of cancers and cardiovascular related diseases, tobacco smoking continues to be prevalent in the population due largely in part to the addictive nature of nicotine. Nicotine vaccines are an attractive alternative to the current smoking cessation options but have yet to be successful enough in clinical trials to reach the market due to a lack of neutralizing antibodies and inconsistent results. Using AFPL1 derived from the Cuban meningococcal vaccine as an adjuvant, we have previously published promising results with an intranasally administered nicotine vaccine. In order to examine the immunogenicity and safety of this vaccine in mice we set up a pilot trial administering the vaccine either intranasally, intramuscularly or utilizing both routes simultaneously and evaluated immune responses and clinical symptoms throughout the duration of the vaccination protocol and post-mortem. These data further demonstrate the ability of the AFPL1 nicotine conjugate vaccine to be a safe and potential candidate for clinical use

First steps for integrating sex and gender considerations into basic experimental biomedical research.

International audienceIn recent decades there has been an increasing recognition of the need to account for sex and gender in biology and medicine, in order to develop a more comprehensive understanding of biological phenomena and to address gaps in medical knowledge that have arisen due to a generally masculine bias in research. We have noted that as basic experimental biomedical researchers, we face unique challenges to the incorporation of sex and gender in our work, and that these have remained largely unarticulated, misunderstood, and unaddressed in the literature. Here, we describe some of the specific challenges to the incorporation of sex and gender considerations in research involving cell cultures and laboratory animals. In our view, the mainstreaming of sex and gender considerations in basic biomedical research depends on an approach that will allow scientists to address these issues in ways that do not undermine our ability to pursue our fundamental scientific interests. To that end, we suggest a number of strategies that allow basic experimental researchers to feasibly and meaningfully take sex and gender into account in their work

Evaluating the immunogenicity of an intranasal vaccine against nicotine in mice using the Adjuvant Finlay Proteoliposome (AFPL1)

Tobacco smoking is recognized as a global pandemic resulting in 6 million deaths per year. Despite a variety of anti-smoking products available to aid with tobacco cessation, the majority of people who attempt to quit smoking relapse within 6 months due to the addictive nature of nicotine. An immunotherapy approach could offer a promising treatment option by inducing a potent selective antibody response against nicotine in order to block its distribution to the brain and its addictive effects in the central nervous system. Our nicotine vaccine candidate was administered intranasally using the Neisseria meningitidis serogroup B Adjuvant Finlay Proteoliposome 1 (AFPL1) as a part of the delivery system. This system was designed to generate a robust immune response by stimulating IL-1β production through Toll-like receptor 4 (TLR4), a potent mechanism for mucosal immunity. The vaccine induced high antibody titers in mice sera in addition to inducing mucosal antibodies. The efficacy of our vaccine was demonstrated using in vivo challenge experiments with radioactive [3H]-nicotine, followed by an analysis of nicotine distribution in the lung, liver, blood and brain. Our results were encouraging as the nicotine concentration in the brain tissue of mice vaccinated with our candidate vaccine was four times lower than in non-vaccinated controls; suggesting that the anti-nicotine antibodies were able to block nicotine from crossing the blood brain barrier. In summary, we have developed a novel nicotine vaccine for the treatment of tobacco addiction by intranasal administration and also demonstrated that the AFPL1 can be used as a potential adjuvant for this vaccine design

Enhancing the Immune Response of a Nicotine Vaccine with Synthetic Small “Non-Natural” Peptides

The addictive nature of nicotine is likely the most significant reason for the continued prevalence of tobacco smoking despite the widespread reports of its negative health effects. Nicotine vaccines are an alternative to the currently available smoking cessation treatments, which have limited efficacy. However, the nicotine hapten is non-immunogenic, and successful vaccine formulations to treat nicotine addiction require both effective adjuvants and delivery systems. The immunomodulatory properties of short, non-natural peptide sequences not found in human systems and their ability to improve vaccine efficacy continue to be reported. The aim of this study was to determine if small “non-natural peptides,” as part of a conjugate nicotine vaccine, could improve immune responses. Four peptides were synthesized via solid phase methodology, purified, and characterized. Ex vivo plasma stability studies using RP-HPLC confirmed that the peptides were not subject to proteolytic degradation. The peptides were formulated into conjugate nicotine vaccine candidates along with a bacterial derived adjuvant vaccine delivery system and chitosan as a stabilizing compound. Formulations were tested in vitro in a dendritic cell line to determine the combination that would elicit the greatest 1L-1β response using ELISAs. Three of the peptides were able to enhance the cytokine response above that induced by the adjuvant delivery system alone. In vivo vaccination studies in BALB/c mice demonstrated that the best immune response, as measured by nicotine-specific antibody levels, was elicited from the conjugate vaccine structure, which included the peptide, as well as the other components. Isotype analyses highlighted that the peptide was able to shift immune response toward being more humorally dominant. Overall, the results have implications for the use of non-natural peptides as adjuvants not only for the development of a nicotine vaccine but also for use with other addictive substances and conventional vaccination targets as well

Repeat-Dose Toxicity Study Using the AFPL1-Conjugate Nicotine Vaccine in Male Sprague Dawley Rats

Tobacco smoking is the cause of 20% of Canadian deaths per year. Nicotine vaccines present a promising alternative to traditional smoking cessation products, but to date, no vaccine has been able to move through all phases of clinical trials. We have previously demonstrated that the AFPL1-conjugate nicotine vaccine does not induce systemic or immunotoxicity in a mouse model and that a heterologous vaccination approach is more advantageous than the homologous routes to inducing mucosal and systemic anti-nicotine antibodies. The purpose of this study was to confirm the safety profile of the vaccine in a repeat-dose toxicity study. The heterologous vaccination strategy was again used, and Sprague Dawley rats were administered a dose five times greater than in our previous studies. Physiological conditions, food and water consumption, body temperature, injection site inflammation, relative weights of organs, histopathology, and blood chemistry and hematology were evaluated during the course of the vaccination period to determine the safety of the vaccine. The AFPL1-conjugate nicotine vaccine did not induce clinically relevant changes or induce symptoms that would be associated with toxicity, making it a promising candidate for future investigations

Strengthening peptide-based drug activity with novel glyconanoparticle.

The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes

Improving the Utility of a Dynorphin Peptide Analogue Using Mannosylated Glycoliposomes

Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood–brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3–3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30–40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS

Synthesis & Evaluation of Novel Mannosylated Neoglycolipids for Liposomal Delivery System Applications

Glycosylated NPs, including liposomes, are known to target various receptors involved in cellular carbohydrate transport, of which the mannoside binding receptors are attracting particular attention for their expression on various immune cells, cancers, and cells involved in maintaining central nervous system (CNS) integrity. As part of our interest in NP drug delivery, mannosylated glycoliposomal delivery systems formed from the self-assembly of amphiphilic neoglycolipids were developed, with a C12-alkyl mannopyranoside (ML-C12) being identified as a lead compoundcapable of entrapping, protecting, and improving the delivery of structurally diverse payloads. However, ML-C12 was not without limitations in both the synthesis of the glycolipids, and the physicochemical properties of the resulting glycoliposomes. Herein, the chemical syntheses of a novel series of mannosylated neoglycolipids are reported with the goal of further improving on the previous ML-C12 glyconanoparticles. The current work aimed to use a self-contingent strategy which overcomes previous synthetic limitations to produce neoglycolipids that have one exposed mannose residue, an aromatic scaffold, and two lipid tails with varied alkyl chains. The azido-ending carbohydrates and the carboxylic acid-ending lipid tails were ligated using a new one-pot modified Staudinger chemistry that differed advantageously to previous syntheses. The formation of stable neoglycoliposomes of controllable and ideal sizes (≈100–400 nm) was confirmed via dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM). Beyond chemical advantages, the present study further aimed to establish potential improvements in the biological activity of the neoglycoliposomes. Concanavalin A (Con A) agglutination studies demonstrated efficient and stable cross-linking abilities dependent on the length of the linkers and lipid tails. The efficacy of the glycoliposomes in improving cytosolic uptake was investigated using Nile Red as probe in immune and cancer cell lines. Preliminary ex vivo safety assessments showed that the mannosylated glycoliposomes are hemocompatible, and non-immunogenic. Finally, using a model peptide therapeutic, the relative entrapment capacity and plasma stability of the optimal glycoliposome delivery system was evaluated and compared to the previous neoglycoliposomes. Overall, the new lead glycoliposome showed improved biological activity over ML-C12, in addition to having several chemical benefits including the lack of stereocenters, a longer linker allowing better sugar availability, and ease of synthesis using novel one-pot modified Staudinger chemistry