Background: Knee osteoarthritis (OA) is the most common form of arthritis in older adults. Knee OA is associated with limitations in physical function. Functional limitations are also associated with another geriatric condition, frailty. Frailty is characterized by reduced strength, endurance and physiological function. Purpose: The primary purpose of this study is to determine if there is a difference in radiographic or symptomatic knee OA severity between non-frail and pre-frail women with knee OA. Secondary objectives include: a) the relationship between radiographic and symptomatic OA severity with serum inflammatory cytokines, and b) if there is a difference in inflammatory cytokines between non-frail and pre-frail women with knee OA. Methods: We included 21 community-dwelling women with knee OA. Frailty was assessed using the Fried Frailty Phenotype. Knee OA severity was characterized by the Kellgren and Lawrence (KL) score and the Knee Injury and Osteoarthritis Outcome Questionnaire (KOOS). Inflammatory cytokines included serum interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis alpha (TNF α) and C reactive protein (CRP). Results: Data from 20 participants (66.1 [9.6] years, BMI 29.7 [4.9] kg/m2, non-frail=55%; pre-frail=45%) were analyzed. Radiographic severity was not different between frailty groups (p= 0.11). There was no difference in symptomatic knee OA severity, measured using the KOOS subscales, between frailty groups (p>0.17). Radiographic OA severity and inflammatory markers were not associated (p>0.30). There was a negative relationship between TNF α and self-reported pain (r=0.26), no relationships between inflammatory cytokines with any other KOOS sub-scales. Lastly, there was no difference in any inflammatory cytokines between non-frail and pre-frail groups. Conclusion: Despite the relatively young age, nearly 50% of our participants were pre-frail. Pre-frailty was unrelated to the severity of the knee OA, or inflammatory cytokines. TNF α may be involved in the experience of pain in these women. While it appears women with knee OA frequently demonstrate pre-frail status, more work is necessary to examine the link between these diseases. / Thesis / Master of Science (MSc) / Arthritis is a chronic disease that has a debilitating effect on the lives of more than 4.6 million Canadians. In 2015, the cumulative economic burden of osteoarthritis was 195.2 billion dollars and is expected to increase significantly in the next two years. Knee osteoarthritis is the most common form of arthritis in older adults. Knee osteoarthritis is associated with increased pain, decreased physical function and decreased quality of life (QOL). In vulnerable older adults increased exhaustion, decreased physical function and muscle loss can increase the risk of developing frailty. Frail older adults are at higher risk of adverse health outcomes such as falls, hospitalization and death. Previous research has shown that older adults with knee OA are at higher risk of developing frailty however, it is not understood what underlying mechanisms increase this risk. This thesis provides fundamental information aimed at understanding potential mechanisms associated with knee osteoarthritis and frailty in women. Our study found that despite their relatively young age, nearly half of the women with knee OA are pre-frail. This data shows that inflammatory cytokines in particular, tumor necrosis factor alpha is related to symptomatic knee osteoarthritis severity in particular, self-reported pain. Overall, early detection of frailty is important when managing this condition. These data suggest that chronic knee pain associated with OA may be a useful trigger for early assessments of frailty in women.

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The mesolimbic dopamine (DA) system originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens (NAc) and other areas including the basolateral amygdala (BLA), prefrontal cortex, and the hippocampus. Drug use induces reward and leads to dysregulation in these brain areas and eventually to substance use disorders (SUDs). Chapter 1 introduces the mesolimbic DA system and its relationship to drug use and their relevance to each chapter. Chapter 2 explores opioid effects on BLA circuitry which is known to play a role in the emotional response including anxiety and stress in SUDs. We showed that morphine induced an inhibitory effect on GABAergic lateral paracapsular cells (LPCs). These cells inhibit BLA principal neuron output and are influenced by opioids. Opioid activation in LPCs leads to upregulated BLA output, and activation in the NAc and central amygdala which may have important implications for stress/anxiety response for patients with SUDs. Chapter 3 explores the effect of interleukin-10 on the mesolimbic DA system. Specifically, cell-attached recordings of VTA DA neurons increase their firing rate in the presence of IL-10, and in vivo studies showed increased DA release in the NAc. Interleukin-10 receptors were expressed in VTA DA neurons and signals through the phosphoinositide 3-kinase. Surprisingly, IL-10 induced conditioned place aversion in mice which may be related to depression- and anxiety-like behaviors reported by others. Thus, IL-10 appears to be regulating the mesolimbic DA system and its association with reward which may be important in understanding the relationship between inflammation and SUDs. Chapter 4 explores the DA transporter (DAT) kinetics in the presence of psychostimulants using DA iontophoresis. We showed that iontophoretic DA delivery increased DA concentration and clearance rates compared to evoked release making it an important tool in measuring DAT kinetics. Cocaine was bath applied and slowed DAT reuptake at high concentrations and D2 stimulant quinpirole slowed the reuptake process but did not show any effect on DAT trafficking, and D2 antagonist eticlopride showed no change in reuptake or DAT trafficking. Cocaine-injected mice increased locomotion and reduced anxiety-like behavior, and iontophoresis experiments slowed reuptake with bath-applied cocaine. Thus, DA iontophoresis is useful in studying DAT blocker kinetics but has limitations in studying the effects of DAT trafficking. Chapter 5 discusses the impact these studies have on society, the limitations of each chapter, and future directions for this dissertation. Together these studies explore the reward system and its relationship with SUDs. The overarching aim has been to understand the involvement of DA in motivation and reward in the context of SUDs and the influence of opioids, cytokines, and psychostimulants.

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Interleukin-10 (IL-10) is an important regulator of immune responses secreted by different cell types. Previous results from our group suggested that the biological effects of this cytokine critically depend on its cellular source. Recent studies reported IL-10 dependent immunosuppressive functions of a specialized subset of regulatory B cells and mast cells. These results relied on adoptive cell transfers, a technique which can potentially introduce artifacts. Therefore, we aimed to readdress these questions in independent models using IL-10 transcriptional reporter mice and various conditional IL-10 mutant mice. Findings in IL-10 reporter system suggested prominent IL-10 transcription in regulatory B cells upon LPS administration. Exposure of mice to contact allergen revealed robust reporter expression in CD8 T cells, moderate to mild reporter expression in CD4 T cells and dendritic cells (DC) respectively, and lack of reporter expression in B cells, mast cells and NK cells in allergen challenged ears. We generated cell-type specific IL-10 mutants by Cre/LoxP-mediated conditional gene inactivation. Efficiency and specificity of Cre-mediated recombination was demonstrated by Southern blot and PCR methods. Various immunogenic challenges in conditional IL-10 mutants did not reveal a role for B cell-derived IL-10 in restraining innate TLR or T cell-dependent inflammatory responses. Likewise, mice with selective inactivation of the il10 gene in mast cells exhibited normal CHS responses and unaltered immune response to CpG oligodeoxynucleotides. On the other hand, DC-specific IL-10 mutants developed excessive inflammatory responses to contact allergens, while innate responses to TLR ligands were not altered. This indicates a non-redundant role for DC-derived IL-10 in contact allergy. Thus, the conditional IL-10 ‘‘knockout’’ mice combined with the novel transcriptional IL-10 reporter system can serve as ideal tools to understand the cell-type specific contributions to IL-10-mediated immune regulation.

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in inflammatory bowel disease. For the in situ delivery of IL-10 by Escherichia coli as carrier chassis, a modified transporter was designed with the ability to secrete biologically active IL-10. De novo DNA synthesis comprised a 561-bp fragment encoding the signal sequence of the E. coli outer membrane protein F fused in frame to an E. coli codon-optimized mature human IL-10 gene under control of a T7 promoter. The construct was overexpressed in E. coli laboratory strains, E. coli BL21 (DE3) and E. coli MDS42:T7. The mean concentrations of human IL-10 in the periplasm and culture supernatant of E. coli BL21 (DE3) were 355.8 ± 86.3 and 5.7 ± 1.7 ng/ml, respectively. The molecular mass of the recombinant E. coli-derived human IL-10 was 19 kDa, while under non-reducing conditions the native IL-10 dimer could be demonstrated. Reduction of tumor necrosis factor-α secretion in lipopolysaccharide-stimulated mouse macrophages and detection of the activated form of the transcription factor signal transducer and activator of transcription protein 3 proved the biological activity of the bacteria-produced human IL-10. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

Neste trabalho nós mostramos que células dendríticas (DCs) de camundongos BALB/c expressam receptor para o PAF (Fator ativador de Plaquetas) e que sua ativação promove um fenótipo tolerogênico, associado à produção de IL10 e PGE2. O bloqueio do PAF-receptor por antagonistas aumentou a capacidade das DCs induzirem proliferação de linfócitos T. O antagonista WEB2170 potencializou a resposta imune in vivo a concentração de anticorpo IgG2a OVA-específico aumentou 30 vezes no grupo tratado; a concentração de IgG1 foi semelhante nos dois grupos. O bloqueio do PAFR em camundongos imunizados com OVA em adjuvante completo de Freund, aumentou a produção de IgG1 e IgG2a OVA-específicos. Em camundongos imunizados com OVA/alum o antagonista não alterou a produção de IgG1. Estes resultados indicam que a ativação do PAFR em DCs modula a sua função apresentadora de antígenos pela produção de IL10 e PGE2. O bloqueio do PAFR pode ser útil na ativação das DCs em protocolos de vacinação com DCs e/ou como co-adjuvante em protocolos de imunização. / In the present work we show that BALB/c mice dendritic cells (DCs) express the PAF (platelet-activating factor) receptor and that its activation promotes a tolerogenic phenotype via IL10 and PGE2 production. Blocking PAFR by selective antagonists markedly enhanced DCs ability to induce T cell proliferation. The antagonist WEB2170 potentiated the in vivo immune response the IgG2a OVA-specific levels were 30 fold increased in the treated group; IgG1 concentration was similar for both groups. The PAFR blockade in mice immunized with OVA in complete Freunds adjuvant enhanced both IgG1 and IgG2a OVA-specific antibody production. In OVA/alum immunized mice, the antagonist did not change IgG1 production. These results suggest that PAFR activation in DCs modulates their antigen-presenting function through IL10 and PGE2 production. Blocking PAFR may be useful to induce DCs activation in DCs-based vaccination protocols and/or as a co-adjuvant in immunization protocols.