ABSTRACT The aim of my thesis is to further investigate the mechanisms underlying inflammatory muscle pain. Despite numerous studies investigating the mechanisms of inflammatory hyperalgesia, little is known of the mechanisms underlying inflammatory muscle hyperalgesia. Using rats as experimental animals, I investigated inflammatory hyperalgesia in muscle and compared it to that of inflamed cutaneous tissue. I injected carrageenan, a plant-origin polysaccharide, into leg muscle and into the hind paw of rats, and measured the behavioural response, as well as cytokine changes, in both plasma and inflamed tissue. Carrageenan induced inflammatory hyperalgesia but the cytokine cascade was not the same in muscle and cutaneous tissue. At no time following carrageenan injection was muscle tumour necrosis factor alpha (TNF-&) concentration elevated above that of muscle injected with saline. TNF-& is a key inflammatory mediator in cutaneous tissue, but apparently not in muscle. Interleukin-1) (IL-1)) and interleukin-6 concentrations also were different during muscle inflammation compared to those of cutaneous inflammation. IL-1) and IL- 6 concentrations, following carrageenan injection, were elevated later in muscle compared to in cutaneous tissue. IL-1) is a potent sensitizer of nociceptors in cutaneous tissue, and also may play an important role in sustaining muscle pain, but it is unlikely to be an initiator of the inflammatory muscle hyperalgesia. In the course of comparing muscle hyperalgesia and cutaneous hyperalgesia, I aimed to identify whether these differences in cytokine concentrations were unique to muscle tissue or if similar differences in cytokine concentrations existed between the hind paw and other cutaneous sites. To explore an alternative cutaneous tissue site, I injected carrageenan into the rat tail and measured the behavioural response, changes in cytokine concentrations and histological changes. Elevations of pro-inflammatory cytokines occurred concurrently with the infiltration of leukocytes into the inflamed tail tissue with the thermal and mechanical hyperalgesia similar to that found in the hind paw. Different mechanisms therefore appear to underlie muscle and cutaneous inflammatory hyperalgesia, regardless of the site used to investigate cutaneous inflammation. One of the consequences of the poor understanding of muscle pain is the lack of a reliable regimen for treating human muscle pain, including delayedonset muscle soreness (DOMS). DOMS, which has a partial inflammatory pathogenesis, is not relieved by non-selective cyclo-oxygenase inhibitors. This phenomenon may be that prostaglandins are not produced peripherally or centrally, when muscle tissue is damaged. I investigated the effect of inhibiting cyclo-oxygenase-2, the isoform released during inflammation, on DOMS in human volunteers. I found that rofecoxib, a cyclo-oxygenase-2 inhibitor, did not attenuate DOMS and nor did tramadol, a central-acting analgesic. The neurochemical pathway underlying DOMS therefore appears to be distinct from the pathways which underlie pain and hyperalgesia in other syndromes. Future research should include investigations into the central mechanisms of muscle pain and blocking the action of IL-1) and CINC-1 both peripherally and centrally may prove a beneficial target for the treatment of clinical muscle pain.

No description available.

The promoter region of interferon regulatory factor 5 (IRF5) contains the rs2004640 T or G single nucleotide polymorphism (SNP) and a CGGGG indel. Both of these polymorphisms have been implicated as genetic risk factors for several autoimmune diseases, including systemic lupus erythematosus, whose pathology involves altered apoptosis and cytokine signaling. The polymorphisms' overall effect is to increase IRF5 levels. IRF5 is a transcription factor of several cytokines, including interferon, and is pro-apoptotic. Thus an alteration of cytokine levels and apoptosis signaling due to high IRF5 levels is the proposed source of autoimmune risk. Each of IRF5's four first exons (1A, 1B, 1C, 1D) has its own promoter and responds to specific stimuli. rs2004640 is a T or G polymorphism; T is the risk allele. The SNP creates a sequence-specific recognition site for the spliceosome, making exon 1B spliceable. Analysis of the 1B promoter showed putative p53 binding site. IRF5 and p53 are pro-apoptotic transcription factors, and the p53 site may provide a positive feedback loop. Apoptosis levels were altered in cells with the rs2004640 risk T/T allele when treated with DNA damaging agents (extrinsic apoptosis), but not when activating death receptors (intrinsic apoptosis). The 1B promoter was the only one to activate expression after inducing DNA damage in a luciferase reporter assay, and this activation was abolished after mutating the p53 site. The exon 1A promoter contains either three or four copies (4X) of CGGGG; the 4X variant is the risk allele. The 1A promoter is constitutively active and is responsive to the Toll-like receptor 7 agonist imiquimod. RNA folding analysis revealed a hairpin encompassing exon 1B. Mutational analysis showed that the hairpin shape decreased translation five-fold in a luciferase reporter assay. Cells with the CGGGG or rs2004640 risk allele exhibited higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR in cell lines with either risk polymorphism demonstrated decreased usage of exons 1C or 1D, although no other correlated splicing events were observed. Also, several mRNA splice variants of IRF5 were sequenced. The risk polymorphisms altered cytokine signaling as well. Expression of interferon, Toll-like receptor, and B cell receptor pathways were affected by a risk haplotype which includes the rs2004640 SNP. The CGGGG polymorphism decreased the levels of CC-chemokine receptor 7. Specific transcription factor binding sites define promoter activity and thus first exon usage and transcription levels. Translation levels are affected by mRNA folding. Overall, the rs2004640 SNP and the CGGGG indel cause high levels of IRF5. High IRF5 expression causes altered cytokine and apoptosis signaling, and may bias the immune system toward autoimmunity.

Despite being a common problem, there are many gaps in the understanding of mild traumatic brain injury (mTBI). Its pathophysiology is unclear, diagnostic criteria are variable, and the associated symptomatology is non-specific. As a result, there are challenges associated with precise mTBI diagnosis and treatment. The dissertation seeks to identify distinctive features, both clinical and pathophysiological, exclusively associated with mTBI. In addition, the neuroinflammatory component of mTBI is explored in detail in the context of inflammatory cytokines’ potential use as prognostic biomarkers and development of a targeted treatment. Three studies were conducted to explore mTBI. We conducted a retrospective chart review to identify the clinical presentation exclusively associated with mTBI that sets it apart from other similar conditions. This was accomplished through symptomatology comparison between the patients with head injuries that meet the ACRM (1993) criteria for mTBI diagnosis vs. those who do not. The results of this study showed that 20.5% of patients with chronic post-concussive symptoms do not meet the ACRM (1993) criteria of mTBI despite sustaining a head injury. In addition, no symptom specific differences were found between the two populations. A detailed systematic review and meta-analysis were also conducted to identify the common inflammatory cytokines associated with mTBI and to explore their potential use as prognostic biomarkers. The results show significantly elevated blood IL-6, IL-1RA, IFN-γ (at <24 hrs.) and MCP-1/CCL2 (within a week) levels in patients with mTBI compared to healthy controls in majority of the included studies. A meta-analysis was further conducted that supported these findings by showing significantly elevated IL-6, MCP-1/CCL2, and IL-1β levels in patients with mTBI in the acute stages (<7 days). In addition, elevated IL-6, TNF-α, IL-1RA, IL-10, and MCP-1/CCL2 levels were associated with poor prognosis in patients with mTBI. In addition, a systematic review was conducted to identify the inflammatory cytokines associated with adverse psychological outcome in population with mTBI. The results show that IL-6, TNF-α, IL-10, and CRP are associated with PTSD and/or depression in the population with mTBI, particularly in the chronic stages. Collectively, these studies show that all symptomatic patients with head trauma, whether or not they meet the subjective criteria of mTBI, should be managed and offered early rehabilitation to avoid long tern adverse consequences. In addition, this thesis supports the neuro-inflammatory hypothesis of mTBI and identifies inflammatory cytokines that could be potentially utilized as prognostic biomarkers and for the development of mTBI treatment. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Glucocorticoids are widely used in horses for a variety of autoimmune and inflammatory conditions. Its potent antiinflammatory properties have been associated with the suppression of a number of different inflammatory cytokines. The purpose of the study was to evaluate the effect of dexamethasone treatment in horses on mRNA cytokine expression, including interleukin-1Î , interferon-gamma, interleukin-4 and interleukin-6, during a five day treatment period and a five day post treatment period. A randomized complete block design was performed on 16 healthy horses. Group I (8 horses) received 0.1 mg/kg of dexamethasone sodium phosphate by intravenous injection once daily for 5 days. Group II (8 horses) received an equivalent volume of sterile saline by intravenous injection daily for 5 days. A sample of 5x10 mililiters of blood in acid citrate dextrose was obtained prior to initial treatment. Thirty minutes after each treatment injection (placebo or dexamethasone) a sample of blood was obtained during the 5 day treatment period and 24, 48, 72, 96 and 120 hours after the last treatment injection was administered. Peripheral-blood mononuclear cells were isolated from the blood samples and stimulated with concavalin A. RNA was isolated using the QIAGEN RNeasy kit. cDNA first strand synthesis was achieved using QIAGEN's OMMISCRIPT RT KIT. cDNA was also constructed for the house keeping gene Î actin. Primer pairs specific for each cytokine were designed using equine cytokine sequences available on Genbank. cDNA for each cytokine and Î -actin was amplified using Real Time PCR technique. Interleukin-4, interleukin-6 and interferon-gamma mRNA expression was statistically significant suppressed in horses treated with dexamethasone when compared to control horses. Interleukin-1Î was only significantly suppressed on day 5. Interleukin-4, interleukin-6 and interferon-gamma mRNA expression suppression was initially observed on day 2 and lasted 24 hours after the last dose of dexamethasone was administered. Interleukin-6 mRNA expression was significantly higher when compared to control group on day 10. Our results suggest that dexamethasone treatment of healthy horses suppresses mRNA expression of several cytokines, including interleukin-4, interleukin-6 and interferon-gamma. This effect could explain part of corticosteroid's mechanism of action for controlling inflammation in a variety of disease conditions. The time-course effect of dexamethasone showed that the effect on mRNA cytokine expression suppression is only observed on day 2 of treatment and mRNA suppression is maintained for 24 hours after discontinuation of treatment. / Master of Science

Titre de l'écran-titre (visionné le 16 janvier 2024) / Les mégacaryocytes (MK), outre leur rôle de producteurs des plaquettes, sont des cellules de plus en plus étudiées pour leur contribution dans la réponse immunitaire. Les MK humains expriment plusieurs récepteurs immunitaires, dont celui de la région constante des immunoglobulines G (IgG), le FcγRIIA, et divers toll-like receptors (TLR) incluant TLR4, tous impliqués dans la réponse innée. Il est possible que les MK humains participent à l'inflammation via des interactions directes avec les récepteurs FcγRIIA et TLR, dont TLR4. Pour étudier l'implication des MK dans la réponse inflammatoire, des souris humanisées exprimant le récepteur FcγRIIA (FcγRIIA$^\textup{TGN}$) et des souris sauvages ont été utilisées. Une technique de culture et d'isolation des MK murins a été développée, permettant d'obtenir à partir de la moelle osseuse des MK matures et viables. Ces MK ont été soumis à des challenges immunitaires in vitro à l'aide de complexes immuns IgG, ou du ligand de TLR4, le lipopolysaccharide (LPS). La libération de cytokines inflammatoires a été mesurée par analyse ELISA et essai multiplex afin de dresser un profil cytokinique. Ces analyses ont révélé chez les MK FcγRIIA$^\textup{TGN}$ et sauvage en contact avec le LPS une augmentation de libération de plusieurs chimiokines inflammatoires impliquées dans le recrutement des neutrophiles. La mise en présence des MK de souris FcγRIIA$^\textup{TGN}$ avec les complexes immuns a conduit à une augmentation importante de libération de MIP (protéine inflammatoire macrophagique)-2, une chimiokine inflammatoire aussi impliquée dans l'activation des neutrophiles. De plus, dans ces mêmes conditions, une tendance à plus de libération de PF4 (cytokine platelet factor 4) a aussi été observée. En plus d'établir des conditions de culture et d'isolation des MK, les travaux ont permis d'étudier l'implication des récepteurs FcγRIIA et TLR4 dans la réponse inflammatoire médiée par les MK et leur libération de chimiokines qui sont également liées à l'activité des neutrophiles.

Le neutrophile est une cellule caractérisée par sa «plasticité» qui, dans des conditions pathologiques, lui permet d'acquérir des nouvelles fonctions qui sont impliquées dans la pathogenèse des maladies inflammatoires chroniques comme dans l’arthrite auto-immune (AAI). Les études suggèrent que le neutrophile peut être impliqué dans la résorption osseuse qui accompagne la maladie. Pour mieux analyser cette possibilité, nous avons mis des neutrophiles en présence d’un cocktail de cytokines présentes dans l’AAI afin d’analyser la modulation des gènes liés à la différenciation en cellule de type ostéoclaste. Les résultats ont démontré que le neutrophile est capable d’acquérir de nouvelles fonctions en présence de cytokines, notamment des fonctions importantes dans l’orchestration du système immunitaire et la réponse inflammatoire. Le neutrophile est également une source de molécules permettant la résolution de l’inflammation, notamment l’élafine, ce qui pourrait être un agent thérapeutique prometteur dans les maladies inflammatoires chroniques comme l’AAI. / The plasticity of neutrophils is a very useful property that in pathological conditions allow these cells to acquire new functions involved in the pathogenesis of chronic inflammatory diseases such as autoimmune arthritis (AIA). Studies suggest that neutrophils may be involved in bone resorption accompanying chronic inflammatory diseases. To better analyze this possibility, we have exposed neutrophils to a cocktail of cytokines present in AIA and studied their transcriptome to decipher the modulation of genes related to the differentiation into an osteoclast cell type. The results demonstrated the ability of neutrophils to acquire new functions when exposed to cytokines, including important functions involved in the orchestration of the immune system and inflammatory response. We also demonstrate that the neutrophil is a source of molecules involved in the resolution of inflammation, especially elafin, which seems to be a promising therapeutic agent in chronic inflammatory diseases and perhaps in AIA.

Amplification of hematopoietic stem cells (HSCs) from human cord blood has applications for a variety of cell therapy protocols. The purpose of this thesis (performed in collaboration with ViaCell, Inc.) was to analyze differential gene expression (especially related to cytokines) during the process of human HSC amplification in vitro. When applied to markers previously shown to be specific for HSC's and/or progenitor cells, the analysis validates ViaCell's cellular product. Total cellular RNA was isolated from cord blood samples at various stages of amplification and used to synthesize cDNAs as probes for hybridization arrays. mRNA candidates increased in cell populations enriched for stem cells were first identified using hybridization arrays, then confirmed by RT-PCR. Restriction mapping confirmed RT-PCR amplicons. The results identified several developmentally interesting cytokines (CD117, Jagged-2, Manic Fringe, and Notch) upregulated in stem cell enriched fractions. Analysis of one candidate previously shown to be a marker for HSCs and progenitors, CD117, was extended using Western blots to show a CD117-related protein upregulation. The observed upregulations did not contain many inflammatory cytokines, which could hinder survival of HSC grafts. The future hope for the non-CD117 candidates is as potential growth modifiers for stem cell samples isolated by clonogenic amplification.