No description available.

No description available.

No description available.

No description available.

Background: Despite the availability of a yearly vaccine and antivirals, the incidence of influenza infections remains high. The genome of the influenza virus can mutate rapidly, therefore novel influenza strains that may be resistant to the current vaccine or antivirals frequently enter the population. Because of the long production time necessary to produce a vaccine, new antivirals must be created to combat early stages of influenza outbreaks. The most effective antivirals will target a highly conserved and essential stage of virus replication. The influenza RNA-dependent RNA polymerase is a heterotrimeric complex composed of three subunits: PA, PB1, and PB2. The three components of the polymerase interact through well-defined domains and are essential for viral replication. Previously, influenza replication has been inhibited using a small synthetic peptide that mimics the interaction domain between PA and PB1 and inhibits the formation of the heterotrimeric complex. Problem and Hypothesis: Although the peptide could inhibit influenza replication, synthetic peptides are costly to produce and are not a viable option for large-scale production. This problem can often be overcome by attaching the peptide to a highly soluble carrier protein. We hypothesize that influenza replication can be inhibited by attaching a peptide, that mimics the binding domain between the PA and PB1 subunits, to a human elastin-like polypeptide (ELP) carrier protein. Methods and Results: The peptide and a nuclear localization sequence was genetically linked to a maltose binding protein (MBP) or ELP carrier protein. The MBP construct was purified by affinity chromatography using FPLC. A high yield of the ELP construct was obtained using inverse transition cycling, a method unique to ELPs because of their temperature-dependent solubility. The ELP construct was designed to be soluble at physiological temperature to limit cellular toxicity due to protein aggregation. The cytotoxicity of the ELP construct was assessed by monitoring the growth of A549 cells, a human lung epithelial cell line. The ELP construct did not have any adverse effects on A549 cell growth. Both constructs could localize to cell nuclei using their respective nuclear localization sequences and could also interact with the PA subunit, demonstrating their potential to inhibit influenza replication. Despite this, only the MBP construct was able to inhibit the replication of influenza. The MBP construct could inhibit the replication of both the H1N1 and H3N2 subtypes of influenza, indicating the recombinant protein had cross-strain activity. Conclusion: Linking a small peptide to carrier protein can result in high protein yields, however a carrier protein must be chosen that will maintain the peptides’ therapeutic activity. In this study, a small anti-influenza peptide inhibited influenza replication when attached to an MBP carrier protein, however was not able to inhibit influenza replication when attached to an ELP carrier protein. Although the peptide was ineffective when attached to this particular ELP carrier protein, different ELP proteins of various lengths and compositions may still be effective carrier proteins for an antiviral peptide. / Thesis / Master of Science (MSc) / The influenza virus causes seasonal outbreaks each year and can have life-threatening symptoms in the young and elderly. In addition, it can rapidly mutate through antigenic drift; therefore, a new vaccine is required each year. Pandemic influenza strains can enter the population when the virus undergoes genetic reassortment by antigenic shift. However, it can take a significant amount of time to formulate a vaccine against pandemic influenzas, which means antiviral drugs are often used as the first line of defense. New antivirals to treat influenza must be developed because resistance to the current influenza antivirals has steadily increased. In this work, we developed an antiviral peptide to disrupt a critical interaction in the influenza RNA-dependent RNA polymerase and inhibit virus replication. This peptide was previously conjugated to an E. coli MBP carrier protein, which would likely not be compatible in vivo. This thesis focused on attaching the antiviral peptide to an elastin-like polypeptide protein, which mimics human tropoelastin, and should be non-immunogenic in humans.

Yes / Alopecia areata (AA) is thought to be an autoimmune process. In other autoimmune diseases, the innate immune system and Toll‐like receptors (TLRs) can play a significant role. Expression of TLR7, TLR9 and associated inducible genes was evaluated by quantitative PCR in peripheral blood mononuclear cells (PBMCs) from 10 healthy individuals and 19 AA patients, categorized according to disease duration, activity and hair loss extent. Microdissected scalp biopsies from five patients and four controls were also assessed by quantitative PCR and immunohistology. TLR9 was significantly upregulated 2.37 fold in AA PBMCs. Notably, TLR9 was most significantly upregulated in patients with active AA, as shown by a positive hair pull test, compared to stable AA patients. In hair follicle bulbs from AA patients, IFNG and TLR7 exhibited statistically significant 3.85 and 2.70 fold increases in mRNA, respectively. Immunohistology revealed TLR7 present in lesional follicles, while TLR9 positive cells were primarily observed peri‐bulbar to AA affected hair follicles. The increased expression of TLR7 and TLR9 suggest components of the innate immune system may be active in AA pathogenesis. / National Alopecia Areata Foundation; Canadian Dermatology Foundation; Michael Smith Foundation for Health Research, Grant/Award Number: CI‐SCH‐00480(06‐1); Canadian Institutes of Health Research, Grant/Award Number: MOP‐167368 and MSH‐192593‐140450

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...

8 Abstract Introduction: Celiac disease is indentified as the loss of oral tolerance to gluten, it is an organ-specific autoimmune disease in which both, adaptive and innate immunity participate. Monocytes are important part of immune system; they have many functions and express very diverse membrane receptors including Toll-like receptors (TLRs). TLRs are involved in the innate immune response, specifically TLR2 and TLR4 are crucial for recognition of bacterial components and TLR7 recognizes virus's ssRNA. Monocytes also produce prolaktin (PRL), which acts as a cytokine that modulates immune responses. To clarify the role of innate immunity and circulating monocytes in pathogenesis of celiac disease, we focused on changes in expression of selected Toll-like receptors (TLR2, TLR4, TLR7), prolactin, some pro- a anti-inflammatory cytokines (TNF-α, IL-6, IL-12, IL-10). We monitored the influence of the SNP - 1149 G/T on the expression of PRL mRNA. Another objective of this work was the introduction and optimization of in vitro methods for cultivation and stimulation of peripheral monocytes. Material and Methods: This pilot study includes 21 patients with celiac disease and 40 healthy controls. For determination of mRNA levels of the studied genes we isolated RNA from monocytes that were isolated by...

Introduction: Communication between neuroendocrinne and immune system is arranged by hormones and cytokines in endocrinne, paracrinne and autocrinne manner. One of the factors involved is also prolactin, a pituitary hormone and an immune cytokine. Sepsis is a system reaction to inflammation mediated by Th1 immune response, which is supported by prolactin as well. Primary protection against sepsis is mediated by innate immunity. Toll- like receptors distinguish molecules, which are connected with pathogens. Afterwards this identification of a specific pathogen toll-like receptors trigger immune reaction with the main goal of destroying this pathogen and also with the goal of renewing the balance of the organism. It is supposed that in the organism that is hardly attacked by a pathogen, the PRL, TLR2 and TLR4 gene expression is on the increase. We studied the levels of PRL, TLR2 and TLR4 mRNA production in circulating monocytes derived from septic patients. Simultaneously, the effect of PRL -1149 G/T SNP on physiological levels of PRL mRNA and its expression in the course of sepsis was evaluated. Materials and methods: As a source of monocytes, blood specimens from 43 septic patients and 40 healthy controls were used. The blood of septic patients was taken three times with some time difference and...

Introduction: Diabetes mellitus is a polygenic disease and its development is influenced to some extent by environmental factors as well. Innate immunity triggers nonspecifically first defense reactions after penetration of the pathogen into the body, while overstimulation components of innate immunity may give rise to autoimmune diseases, including diabetes type 1. The components of innate immunity are, among others, Toll-like receptors (TLRs) belonging to a group of the structures recognizing preserved molecular structures characteristic of pathogens. Toll-like receptors are abundantly expressed by monocytes which produce prolactin (PRL) having an immunostimulatory function. To clarify the role of innate immunity in the pathogenesis of diabetes, we focused on the expression of mRNA and protein expression of TLR2 and TLR4. The expression of PRL was studied only at the level of mRNA. Monocytes were separated by flow cytometry into classical (CD14++) and nonclassical (CD14+). We monitored their percentages and the degree of expression of CD14 antigen on their surface.The operational objective of this dissertation was to optimize the stimulation of monocytes for the planned study of the function of non-pituitary prolactin in vitro and determine the appropriateness of the use of healthy donors' buffy...