In Canada alone, there were an estimated 800,000 visually impaired people in 2007, costing the federal government an annual amount of $15.8 billion in services, treatments and lost revenue. These costs are estimated to double by the year 2032, as the population ages. The leading causes of visual impairment and blindness is retinal degeneration, characterized by the progressive death of retinal cells. The research presented in this PhD thesis aimed to prevent retinal degeneration by over-expressing the X-linked Inhibitor of Apoptosis (XIAP) in retinal cells using plasmid and adeno-associated viral vectors. The work is divided into four sequential chapters targeted at developing an anti-apoptotic gene therapy strategy to prevent retinal cell death. The first chapter examines XIAP gene therapy in the treatment of Leber’s Hereditary Optic Neuropathy (LHON). In vitro studies using the 661W cone-photoreceptor cell line showed that XIAP over-expression significantly lowers cell death when 661W cells are exposed to a number of apoptotic stimuli. In a mouse model of Leber’s Hereditary Optic Neuropathy (LHON), XIAP expression in retinal ganglion cells (RGCs) protected the ultrastructure of the RGC axons within the optic nerve, in addition to providing evidence of functional protection. The second and third chapters further examine the potential for XIAP gene therapy in the treatment of retinal disease by developing an in vivo model of retinal detachment in cats, followed by evaluating the efficacy of XIAP gene therapy intervention. When XIAP was over-expressed in the photoreceptor cells, there was significant structural protection and trends in preservation of function in this model of degeneration. Finally, the fourth chapter explores an alternate method to viral gene therapy by evaluating the efficacy and toxicity of chitosan microparticles as a protein delivery system to the retina. Results show that chitosan microparticles are mucosal-adhesive and are non-toxic at low concentrations in vitro in 661W cells and in vivo in rats. This thesis work provides strong evidence that XIAP gene therapy is an effective method for preventing retinal degeneration, and works as a broad spectrum gene therapy strategy that can be applied to different forms of retinal degeneration.

INTRODUÇÃO: A cardiopatia chagásica crônica (CCC) apresenta características específicas, tais como: disfunção autonômica e atividade inflamatória exacerbada. Esta fisiopatologia sugere que alguns parâmetros metabólicos podem estar alterados em pacientes chagásicos. O objetivo deste estudo foi avaliar os parâmetros metabólicos e inflamatórios nas diversas formas evolutivas de doença de Chagas e sua correlação com medidas de avaliação do Sistema Nervoso Autônomo (SNA). MÉTODOS: Foram avaliados 60 indivíduos divididos em 4 grupos (n=15): Grupo controle (GC), Grupo FI - forma indeterminada, Grupo ECG- cardiopatia chagásica com alteração eletrocardiográfica sem disfunção ventricular e Grupo IC - cardiopatia chagásica com disfunção ventricular e insuficiência cardíaca. Todos os grupos foram pareados de acordo com sexo, idade e índice de massa corporal. Os pacientes realizaram dosagens sanguíneas de insulina, leptina, adiponectina, interleucina-6 (IL- 6) e fator de necrose tumoral-alfa (TNF-alfa) pelo método de ELISA. O SNA foi avaliado através da variabilidade da frequência cardíaca no holter 24 horas e no teste de inclinação postural. Os valores de RMSSD, pNN50 e do componente alta frequência (AF) foram utilizados como estimativa da atividade parassimpática. Os valores do componente de baixa frequência (BF) estimaram a atividade simpática. A análise estatística foi feita utilizando-se a ANOVA ou teste de Kruskal-Wallis para a comparação entre os grupos, o coeficiente de Spearman para a análise das correlações e a regressão linear múltipla para a análise multivariada. RESULTADOS: A leptina e insulina não apresentaram diferenças significativas entre os grupos [Leptina: GC=3,42 (7,43); FI=3,03 (6,53); ECG=5,56 (6,2); IC=2,86 (2,67) ng/ml; p=0,626. Insulina: GC=3,41 (1,98); FI=4,31 (2,85); ECG=4,30 (3,06); IC=4,58 (2,88) ng/ml; p=0,901] A adiponectina apresentou níveis maiores nos grupos ECG e IC [GC=4766,5 (5529,5); FI= 4003,5 (2482,5); ECG= 8376,5 (8388,5); IC= 8798 (4188) ng/ml; p < 0,001]. IL-6 e TNF-alfa foram maiores no Grupo IC [IL-6: GC=1,85 (6,41); FI=1,58 (1,91); ECG=1,0 (1,57); IC= 31,44 (72,19) pg/ml; p=0,001. TNF-?: GC=22,57 (88,2); FI=19,31 (33,16); ECG=12,45 (3,07); IC=75,15 (278,57) pg/ml; p=0,04]. A insulina, leptina e TNF-alfa não apresentaram correlações significativas com medidas de avaliação do SNA. A adiponectina apresentou correlação positiva com o componente AF (r= 0,336; p= 0,009) e correlação negativa com o componente BF (r= -0,336; p= 0,009). A interleucina-6 apresentou correlação positiva com o componente AF (r= 0,419; p=0,004) e correlação negativa com o componente BF (r= -0,393; p= 0,007). Porém, na análise multivariada apenas a adiponectina apresentou correlação significativa com medidas de função do SNA. CONCLUSÃO: A adiponectina foi maior nos grupos ECG e IC. A IL-6 e o TNF-alfa foram maiores no grupo IC. O aumento dos níveis de adiponectina esteve associado a diminuição da atividade simpática e predomínio da atividade parassimpática. / BACKGROUND: Chagas disease (CD) has specific characteristics such as autonomic dysfunction and increased inflammatory activity. This pathophysiology suggests that metabolic parameters can be altered in patients with CD. The aim of this study was to evaluate the metabolic and inflammatory parameters in different forms of CD and their correlation with Autonomic Nervous System (ANS) measures. METHODS: We evaluated 60 subjects divided into 4 groups (n=15): control group (CG), group IF (indeterminate form); group ECG (ECG abnormalities and normal left ventricular function in echocardiogram) and HF group (heart failure with left ventricular dysfunction). All groups were matched for age, sex and body mass index. The patients underwent insulin, adiponectin, leptin, interleukin-6 (IL-6) and tumor necrosis factor-alfa (TNF-alfa) measurements by ELISA. The Autonomic Nervous System was assessed by heart rate variability in 24-hour Holter and tilt test. RMSSD, pNN50 and High Frequency (HF) component values were used to estimate parasympathetic activity and low frequency (LF) components were used to estimate sympathetic activity. Statistical analyses were performed using ANOVA or Kruskal- Wallis tests to compare groups. Spearman coefficient was used for correlation analysis and linear regression for multivariate analysis. RESULTS: No significant differences were observed in leptin and insulin levels between groups. [Leptin: CG=3.42 (7.43); IF=3.03 (6.53); ECG=5.56 (6.2); HF=2.86 (2.67) ng/ml; p=0.626. Insulin: CG=3.41 (1.98); IF=4.31 (2.85); ECG=4.30 (3.06); HF=4.58 (2.88) ng/ml; p=0.901]. Adiponectin was higher in ECG and HF groups. [CG=4766.5 .(5529.5); IF= 4003.5 (2482.5); ECG= 8376.5 (8388.5); HF= 8798 (4188) ng/ml; p < 0.001)]. IL-6 and TNF-alfa were higher in HF group. [IL-6: CG=1.85 (6.41); IF=1.58 (1.91); ECG=1.0 (1.57); HF= 31.44 (72.19) pg/ml; p=0.001. TNF-alfa: CG=22.57 (88.2); IF=19.31 (33.16); ECG=12.45 (3.07); HF=75.15 (278.57) pg/ml; p=0.04]. Insulin, leptin and TNF-alfa did not correlate with autonomic dysfunction. Adiponectin correlated positively with HF component (r=0.336; p= 0.009) and inversely with LF component (r= -0.336; p=0.009). IL-6 correlated positively with HF component (r= 0.419; p=0.004) and inversely with LF component (r= -0.393; p= 0.007). However, in multivariate analysis only adiponectin correlated significantly with ANS measures. CONCLUSION: Adiponectin levels were higher in ECG and HF groups. IL-6 and TNF-alfa were higher in HF group. Higher levels of adiponectin were associated with reduced sympathetic activity and predominance of parasympathetic activity

<p>Reumatoidni artritis (RA) je hronično inflamatorno oboljenje zglobova koji nastaje usled poremećaja u regulaciji imunskih mehanizama. TNF-alfa jedan je od ključnih medijatora inflamacije u RA, a koji preko složenih mehanizama podstiče aktivnost osteoklasta koji dovodi do poremećaja u procesu ko&scaron;tanog remodelovanja u pravcu povećane ko&scaron;tane resorpcije koji se klinički može pratiti određivanjem nivoa markera ko&scaron;tane resorpcije i ko&scaron;tanog formiranja u urinu i serumu. Primenom TNF inhibitora započeo je novi koncept lečenja RA. Cilj rada: Utvrditi razliku mineralne ko&scaron;tane gustine (BMDg/cm2) i vrednosti ko&scaron;tanih biohemijskih markera-prokolagen tip 1N-terminalni propeptid (P1NP) i beta-crosslapsa pre uvođenja terapije, i nakon godinu dana sprovedene terapije TNF inhibitorima. Metode: Studija je sprovedena u Specijalnoj bolnici za reumatske bolesti Novi Sad jednim delom kao retrospektivno, a drugim delom prospektivno istraživanje, koje je obuhvatilo 50 bolesnica sa dijagnozom reumatoidnog artritisa kod kojih je postojala indikacija za uvođenje lekova iz grupe TNF inhibitora. Da bi u&scaron;le u studiju bolesnice su morale da ispune određene uključne/isključne kriterijume koji su bili vezani za dužinu trajanja RA i menopauze, način lečenja RA, stepen o&scaron;tećenja zglobova i prisutnost drugih oboljenja sa reperkusijom na ko&scaron;tano tkivo. Pored reumatolo&scaron;kog i fizikalnog pregleda određivani su faktori rizika za osteoporozu i prelome. Na početku i na kraju godinu dana po uvođenju terapije TNF inhibitora rađena je osteodenzitometrija na aparatu tipa &bdquo;Lunar&ldquo; merena na lumbalnoj kičmi i kuku kao i određivanje biohemijskih markera u serumu prokolagen tip 1 N-terminalni propeptid (P1NP) i betacrosslapsa ECLIA metodom. Rezultati: Prosečna starost bolesnica bila je 51,5 godina koje su u 84%, bolovale od RA do 5 godina kod kojih je u najvećem procentu dužina trajanja menopauze bila do dve godine, a u svojoj terapiji pored metotreksata su imale uključen TNF inhibitor, Etanercept 34%, Adalimubam 46%, Golimubam 9% i 2% Infliksimab.Pre uvođenja biolo&scaron;ke terapije najveći broj bolesnica 80% imalo je osteopeniju, 14% normalan nalaz, dok je osteoporoza zabeležena kod 6% bolesnica. Na kraju jednogodi&scaron;nje primene TNF inhibitora 18% bolesnica je imalo normalan osteodenzitometrijski nalaz, 78 % osteopeniji, a 4% osteoporozu. Ova promena je statistički značajna ( p=0,000). Nakon jednogodi&scaron;nje primene TNF inhibitora nije do&scaron;lo do smanjenja vrednosti BMD (g/cm&sup2;) merenog na lumbalnom delu kičme i kuka. Beleži se statističko značajno povećanje vrednosti T- skora (SD) merenog na lumbalnom delu kičme i vratu butne kosti. Vrednost ko&scaron;tanih biohemijskih markera P1NP i beta crosslapsa značajno su povećani nakon jednogodi&scaron;nje primene TNF inhibitora, pri čemu se beleži veće povećanje biohemijskog markera ko&scaron;tane sinteze, P1NP. Zaključak: Savremeni pristup lečenja reumatoidnog artritisa podrazumeva primenu biolo&scaron;kih lekova kao &scaron;to su TNF inhibitori koji značajno suzbijaju inflamaciju i dovode do smanjenja odnosa RANKL/OPG sistema, čime se inhibira dejstvo osteoklasta i sprečava gubitak mineralne ko&scaron;tane gustine. Primena TNF inhibitora nakon godinu dana sprečila je pad vrednosti BMD (g/cm&sup2;), povećana je vrednost T- skora (SD) i vrednosti ko&scaron;tanih biohemijskih markera, posebno markera ko&scaron;tane sinteze. Uprkos velikom broju studija vezanih za dejstvo TNF inhibitora na kost, za sada nema dovoljan broj istraživanja o njegovom uticaju na sprečavanju osteoporoze i preloma kostiju i nivou vrednosti ko&scaron;tanih biohemijskih markera posebno u dužem periodu praćenja, &scaron;to će biti verovatno predmet daljih istraživanja.</p> / <p>Rheumatoid arthritis (RA) is a chronic inflammatory joint disease resulting from compromised regulation of immune mechanisms. TNF-alpha is one of the key inflammation mediators in RA that, through complex mechanisms stimulates osteoclast activity, thereby modifying the bone remodeling process in the direction of increased bone resorption that can be clinically monitored by determining the level of bone resorption and bone formation markers in urine and serum. Use of TNF has initiated a new concept in RA treatment. Aims: To determine the differences in bone mineral density (BMD, g/cm2) and values of biochemical markers in bone procollagentype 1 N-terminal propeptide(P1NP) and betacrosslaps before and after yearlong TNF inhibitor therapy. Methods: The study was conducted at the Special Hospital for Rheumatic Diseases Novi Sad partly as retrospective and partly as prospective research, which involved 50 female patients diagnosed with rheumatoid arthritis in whom introduction of medications from the TNF inhibitor group was indicated. To be included in the study, patients had to meet certain inclusion/exclusion criteria related to RA and menopause duration, RA treatment, degree of joint impairment, and presence of comorbidities with repercussions for bone tissues. In addition to rheumatological and physical examinations, risk factors for osteoporosis and fractures were determined. At the beginning and one year after commencing TNF inhibitor therapy, osteodensitometry was performed using &ldquo;Lunar&rdquo; apparatus, taking measurements on lumbar spine and hip, and serum levels of biochemical markers procollagentype 1 Nterminal propeptide(P1NP) and beta-crosslaps were determined via ECLIA method. Results: Mean patient age was 51.5 years, 84% of whom suffered from RA for up to 5 years, and in the greatest percentage experienced menopause for two years, receiving therapy that in addition to methotrexate included a TNF inhibitor, Etanercept 34%, Adalimumab 46%, Golimumab 9%, and 2% Infliximab. Prior to commencing biological therapy, majority of patients 80% suffered from osteopenia, 14% had normal findings, and osteoporosis was recorded in 6% of patients. At the end of yearlong TNF inhibitor therapy, 18% of patients had normal osteodensitometry findings, 78% had osteopenia and 4% osteoporosis. This change was statistically significant (p = 0.000). As a result of yearlong TNF inhibitor therapy no reduction occurred in BMD (g/cm&sup2;) values in lumbar spine and hip. Statistically significantly higher T scores (SD) pertaining to lumbar spine and femur were measured. Values of biochemical markers P1NP and beta-crosslaps significantly improved after yearlong TNF inhibitor therapy, whereby a greater increase was recorded in the biochemical bone synthesis marker, P1NP. Conclusion: Advanced rheumatoid arthritis treatment involves the use of biological compounds such as TNF inhibitors that significantly suppress inflammation and reduce the RANKL/OPG ratio, thereby inhibiting osteoclast activity and preventing bone mineral loss. TNF inhibitor therapy after one year prevented reduction in the BMD (g/cm&sup2;) levels, while increasing the T score (SD) and bone biochemical marker values, bone synthesis marker in particular. Despite a large number of studies related to the TNF inhibitor effect on bone, there is presently not enough research on its influence on osteoporosis and bone fracture prevention and bone biochemical marker levels, especially over longer periods, which will likely be the topic of further research.</p>

There is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction, including the induction of aberrant angiogenesis. While leukocytes have been described as mediators of inflammation-associated angiogenesis, the effects of direct chronic endothelial activation have not been addressed in this context. Using an uncleavable mutant of the transmembrane form of tumor necrosis factor-α (TNF-α), we have established models of stable TNF-α expression in endothelial cells in vitro and in transgenic mice in vivo. In the in vitro model, continuous endothelial activation leads to increased leukocyte cellular adhesion molecule expression and intracellular reactive oxygen species, hallmarks of a proinflammatory and dysfunctional endothelium. In addition, stable expression of TNF-α in endothelial cells increased angiogenic sprout formation in the presence but also in the absence of angiogenic growth factors. The partial neutralization of this effect by TNF-α antibodies and the inability of conditioned media from stable TNF-α-expressing endothelial cells to induce angiogenic activities in control endothelial cells suggest that this effect does not require expression of additional autocrine factors, but is an autonomous effect of the transmembrane TNF on the endothelial cells. Furthermore, using the Matrigel plug assay in vivo, increased angiogenesis was observed in endothelial TNF-α-expressing transgenic versus control mice. In conclusion, chronic inflammatory changes mediated by TNF-α can induce angiogenesis in vitro and in vivo, suggesting endothelial cell activation as a direct link between inflammation and angiogenesis. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

SMOFLipid® is a commonly used fat emulsion for parenteral nutrition. We investigated how enrichment of SMOFLipid® with n-3 polyunsaturated fatty acids (PUFA) in a form of second fat emulsion, Omegaven® , changes fatty acid composition of total plasma phospholipids and erythrocyte phospholipids, cytokine concentrations in serum and in supernatant from in vitro whole blood culture stimulated with lipopolasaccharide (LPS) and we evaluated also changes in oxido- reductive balance. Eight patients on long-term home parenteral nutrition recieved both emulsions, SMOFLipid® (6 weeks) and SMOFLipid® +Omegaven® (4 weeks), one by one. We observed no significant differences in common laboratory and clinical parameters between these two types of diet. Enrichment of SMOFLipid® with Omegaven® led to an increase in eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) in total plasma phospholipids and there was also an increse in proportion of EPA in erythrocyte phospholipids, while proportion of DHA remained unchanged. These changes were in both phospholipids of plasma and erythrocyte compensated for a decrease in proportion of linoleic and arachidonic acid (n-6 PUFA). There were elevated IL-6 and TNF-α serum concentrations in patients after both diets. There was a decrease in IL-6 production by 36% with SMOFLipid®...

The cytokine tumor necrosis factor α (TNFα) stimulates the NF-кB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting Rip1 and Traf2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that Rip1 links the TNFR1 to the IкB kinase (IKK) complex, whereas Traf2 couples the TNFR1 to the SAPK/JNK cascade. We found TNFα-induced p38 MAP kinase activation and interleukin-6 (IL-6) production is impaired in rip1-/- murine embryonic fibroblasts (MEF) but unaffected in traj2-/- MEF, demonstrating that Rip1 is also a specific mediator of the p38 MAP kinase response to TNFα. Moreover, we demonstrate that endogenous Rip1 associates with the MAP3K, Mekk3 in response to TNFα and that TNFα-induced p38 MAP kinase activation is impaired in mekk3-/- cells, indicating that Rip1 may mediate the p38 MAP kinase response to TNFα by recruiting Mekk3. We also demonstrate that Rip1 is phosphorylated and ubiquitinated in response to Tnfα and that Rip1 phosphorylation is not required for ubiquitination of Rip1. Furthermore, TNFα-induced ubiquitination of Rip1 is impaired in Traf2-/- cells, suggesting that Traf2 is the E3 ubiquitin ligase responsible for the TNFα-dependent ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in TNFR1 signaling.

The generation of memory T cells following primary exposure to a pathogen is a critical feature of the vertebrate immune system which has evolved as a protective mechanism in order to defend the host against repeated assaults by the patnogen. Memory T cells are long-lived, undergo rapid proliferation upon re-activation, mediate a robust secondary response and clear the pathogen much more efficiently. These aspects have made the generation of memory T cells an attractive goal for the production of both prophylactic and therapeutic vaccines. However, the degeneracy of the T cell receptor, whereby a given T cell recognizes more than one epitope, allows the T cell to be modulated by epitope variants which could be self-ligands, ligands related to the original epitope but altered in sequence, or completely unrelated epitopes. Experiments in both mice and humans show that such cross-reactive stimulation of memory T cells results in complete, partial, or no activation of T cells, and in some cases, even alters the functional identity of the T cell (for example, T helper 1 cells start secreting IL-4, IL-5 and become part of a T helper 2 response). In the context of secondary infection of immune organisms with pathogens containing mutated or related T cell epitopes, such alterations at the cellular level translate into drastic changes in the overall clinical outcome of the infection. Thus, the presence of cross-reactive T cells in the memory population implies that the protective or pathologic nature of the secondary immune response is a consequence of the host's infection history. Although several murine models of heterologous infection resulting in altered pathological outcome have been studied, the exact immune correlates of protection versus immunopathology are still unclear. This thesis addresses this issue in dengue virus infections in humans. Dengue fever (DF) and Dengue Hemorrhagic Fever (DHF) are two disease manifestations caused by infections of humans by the dengue viruses. These are a group of 4 serologically distinct flaviviruses (D1-4) which often co-circulate among endemic populations. While primary infection with any of the four serotypes can result in the more severe clinical disease characterized by DHF, epidemiological data from several outbreaks show that 80% - 90% of DHF cases occur among individuals with secondary infection. This implies that prior immunity to dengue is actually a risk factor for developing severe disease. In these DHF cases, there are increased numbers of CD69+ CD8+ T cells in circulation, with increases observed in the frequency of epitope-specific T cells, and the serum levels of several T cell produced cytokines, chemokines, and immune receptors are highly elevated. Since the four serotypes share 65% - 75% amino acid sequence homology, the possibility that unconserved T cell epitope sequences stimulated cross-reactive responses was borne out in in vitroexaminations. In these studies, peripheral blood mononuclear cells (PBMC) and cloned T cells from both vaccinated and infected donors contained large populations of memory T cells that were cross-reactive for heterologous viral serotypes in proliferation and CTL assays. These data suggest that the severity of disease seen in DHF patients can be attributed to an immunopathologic secondary response during heterologous infection, and highlight a role for serotype cross-reactive T cells in this process. This thesis addresses the hypothesis that the recognition of the natural variants of dengue virus T cell epitopes by serotype cross-reactive CD8+ T cells of a dengue-immune donor results in an altered secondary response profile, with the changes reflected in both the quantitative and qualitative nature of the response. In order to compare the functional profile of the secondary response of dengue-immune PBMC re-activated with heterologous serotypes, we focused on a panel of 4 donors who were vaccinated with live attenuated monovalent vaccines corresponding to D1, D2, or D4 serotypes. We screened a panel of peptides predicted to bind to HLA-A*0201 for cytokine responses and identified 4 novel epitopes that were highly immunogenic in all four donors. Direct ex vivo stimulation of donor PBMC with the heterologous sequences of these epitopes also showed sizeable serotype cross-reactive T cell populations. CFSE- and intracellular staining for cytokines and chemokines showed that these cross-reactive T cells not only expanded but also produced IFNγ, TNFα, and MIP-1β. Multi-parameter staining revealed functionally diverse populations comprised of single cytokine (IFNγ+, TNFα+, MIP-1β+, double cytokine (IFNγ+TNFα+, IFNγ+MIP-1β+, TNFα+MIP-1β+, and triple cytokine (IFNγ+TNFα+MIP-1β+ secreting sub-sets. Stimulation with the epitope variants altered the magnitude of the overall response as well as the relative sizes of these sub-sets. The patterns of responses revealed the effects of epitope immunogenicity, infection history and donor-specific variability. All 4 donors showed the highest cytokine response to a -single epitope (NS4b 2353). The same two peptide variants (D2 NS4a 2148 and D3 NS4b 2343) induced the highest response in all 4 donors regardless of the serotype of primary dengue infection. Interestingly, the epitope variants which showed the highest immunogenecity in our donors corresponded to the D2 and D3 serotypes which have been documented as being more virulent as well as a viral risk factor for DHF. In one donor, the response to all peptide variants was dominated by the same cytokine sub-sets. These data suggested that the dengue-immune memory T cell repertoire was functionally diverse and underwent alterations in size after secondary stimulation. Therefore, we also investigated the effect of epitope variants on dengue-specific CD8+T cell clones isolated from vaccinated and infected donors in order to determine if epitope variants induced altered functional outcomes at the clonal level. The epitope variants functioned either as strong agonists (particularly the D2 and D3 sequences), partial agonists, or null ligands. Some variants were able to induce cytolysis but not other effector functions at low concentrations. The variant ligands also influenced the hierarchy of cytokine responses within each clone. The third part of this thesis focused on the characterization of the frequency and phenotypic profile of epitope-specific CD8+ T cells in patients with DHF and DF at different times in the disease course in order to better understand the kinetics of the response and delineate any differences between the immune profile of severe vs. moderate disease. Tetramer staining for a previously identified HLA-B*07 restricted epitope was combined with staining for activation markers (CD69, CD38, HLA-DR), homing receptors (CCR7, CD62L), and programmed death receptor 1 (PD-1). The DHF subjects had early T cell activation with higher frequencies of tetramer+CD69+ cells as compared to DF subjects, in whom T cell frequencies peaked around the time of defervescence. While each subject had a unique phenotypic profile of tetramer+ cells, there was a difference between DF and DHF subjects in terms of CCR 7 expression; all subjects expressed low levels of CCR7 during acute illness but only the DHF subjects did not show upregulation of CCR7 on tetramer+ cells during convalescence. These data suggest that there is a sustained alteration in memory phenotype in those who recovered from severe dengue disease. A majority of the tetramer+cells also expressed PD-1 during acute illness but not during convalescence. Double-staining with variant tetramers allowed us to directly visualize serotype cross-reactivity of the epitope-specific population, and showed that secondary stimulation did induce the expansion of cells with low avidity for that secondary serotype and higher avidity to the variant. Furthermore, the ratios of these sub-sets changed during the course of the response. Taken together, these studies suggest that the immune response to heterologous secondary dengue infection is mediated by a heterogeneous population of serotype-cross reactive T cells that have different functional avidities to epitope variants and is influenced by the serotype of the secondary infection as well as the prior infection history of the individual. The preferential expansion of clones which secrete IFNγ but not inflammatory MIP-1β or TNFα or a repertoire characterized by a higher ratio of cytolytic to cytokine producing clones could limit immune mediated damage while efficiently clearing the virus. This information will be useful in the design of vaccine strategies aimed at inducing protective cross-reactive responses against all 4 dengue serotypes while preventing immunopathological outcomes following secondary infection.

No description available.

No description available.

Transplantation of multipotent mesenchymal progenitor cells is a valuable option for treating tendon disease. Tenogenic differentiation leading to cell replacement and subsequent matrix modulation may contribute to the regenerative effects of these cells, but it is unclear whether this occurs in the inflammatory environment of acute tendon disease. Equine adipose-derived stromal cells (ASC) were cultured as monolayers or on decellularized tendon scaffolds in static or dynamic conditions, the latter represented by cyclic stretching. The impact of different inflammatory conditions, as represented by supplementation with interleukin-1β and/or tumor necrosis factor-α or by co-culture with allogeneic peripheral blood leukocytes, on ASC functional properties was investigated. High cytokine concentrations increased ASC proliferation and osteogenic differentiation, but decreased chondrogenic differentiation and ASC viability in scaffold culture, as well as tendon scaffold repopulation, and strongly influenced musculoskeletal gene expression. Effects regarding the latter differed between the monolayer and scaffold cultures. Leukocytes rather decreased ASC proliferation, but had similar effects on viability and musculoskeletal gene expression. This included decreased expression of the tenogenic transcription factor scleraxis by an inflammatory environment throughout culture conditions. The data demonstrate that ASC tenogenic properties are compromised in an inflammatory environment, with relevance to their possible mechanisms of action in acute tendon disease.