NK cells control viral replication through cytotoxicity and IFNγ production. These functions were assessed in chronic HCV infected patients undergoing treatment. Aboriginals have genetic polymorphisms that may enhance NK cell function suggesting more effective clearance of chronic HCV than Caucasians. NK cell function was similar at baseline between ethnicities. At 3 months of treatment, Caucasian had higher NK killing potential compared to Aboriginal patients. This had no effect on treatment outcomes. NK cell cytotoxicity negatively correlated with viral loads while NK IFNγ production, particularly within the CD56bright subset, positively correlated with viral load suggesting that viral loads control NK cells function through an unknown mechanism. NK cell killing reflect fibrosis, but not liver damage measured by liver enzymes. IFNγ production,by NK cells does not reflect fibrosis nor liver enzymes levels. Lastly, NK cell function does not associate with therapeutic outcomes of chronic HCV infection suggesting that they do not directly play a role in therapeutic clearance of HCV.

NK cells control viral replication through cytotoxicity and IFNγ production. These functions were assessed in chronic HCV infected patients undergoing treatment. Aboriginals have genetic polymorphisms that may enhance NK cell function suggesting more effective clearance of chronic HCV than Caucasians. NK cell function was similar at baseline between ethnicities. At 3 months of treatment, Caucasian had higher NK killing potential compared to Aboriginal patients. This had no effect on treatment outcomes. NK cell cytotoxicity negatively correlated with viral loads while NK IFNγ production, particularly within the CD56bright subset, positively correlated with viral load suggesting that viral loads control NK cells function through an unknown mechanism. NK cell killing reflect fibrosis, but not liver damage measured by liver enzymes. IFNγ production,by NK cells does not reflect fibrosis nor liver enzymes levels. Lastly, NK cell function does not associate with therapeutic outcomes of chronic HCV infection suggesting that they do not directly play a role in therapeutic clearance of HCV.

The goal of this project was to determine the key regulators of Mesenchymal Stromal Cell (MSC) potency as part of a cell-based therapy to treat inflammatory and autoimmune diseases. The immunomodulatory capacity of MSCs is dictated by multiple, interacting conditions that take place during the biomanufacturing of these cells, as well as after they are transplanted. Variables such as the source of MSCs and the inflammatory cues in their microenvironment are critical regulators of potency that can be manipulated and optimized prior to their use for an enhanced cell-based therapy. Herein, I took a systematic approach to isolating a single variable in the microenvironment of MSCs to determine its effect on the key immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). I then manipulated these variables and applied them across multiple MSC donors to determine how their effect varied between cells isolated from different individuals. Finally, I conducted an in vitro potency assay with MSCs and Peripheral Blood Mononuclear Cells (PBMCs) to determine how enhanced IDO due to these variables translated to immune suppression for an enhanced cell product. Upon transplantion, different disease settings have altered microenvironments that can hinder the efficacy of an MSC therapy. The microenvironment in obesity and type 2 diabetes (T2D) has elevated levels of the fatty acid palmitate which shifts the phenotype of MSCs from immune suppressive to pro-inflammatory. I demonstrated that manipulating the microenvironment of MSCs to enhance IDO protein concentration prior to transplant reverses the pro-inflammatory effects of palmitate and restores immune suppression by MSCs. My finding was that the appropriate environmental cues, along with a potent donor, yields a cell-based therapy that can overcome challenges in many disease settings such as obesity and T2D.

La cryptosporidiose est une zoonose intestinale qui affecte les ruminants nouveau-nés et les individus immunodéficients (enfants, immunodéprimés) et pour laquelle les traitements sont limités et ne sont pas totalement efficaces. Le développement du parasite peut être contrôlé par une réponse immunitaire protectrice dans laquelle les productions d’IL-12 et d’IFNγ sont prépondérantes. Les laboratoires AIM, IPV et la société VitamFero ont décidé de mettre leurs compétences en commun pour évaluer si l’administration d’une souche vaccinale de Toxoplasma gondii atténuée (Toxo Mic1-3KO) pouvait permettre de stimuler efficacement le système immunitaire des nouveau-nés et favoriser le contrôle de la cryptosporidiose. La première partie des travaux de ma thèse Cifre a permis d’obtenir une preuve de concept. En effet, des expérimentations préliminaires sur souriceaux et agneaux dans lesquelles la souche Toxo Mic1-3KO s’était développée suffisamment montraient une diminution de la charge parasitaire suite à une infection d’épreuve par C. parvum. Fort de ces résultats, nous avons souhaité développer deux axes complémentaires pour nous aider à améliorer la souche vaccinale et son utilisation chez les nouveau-nés. / Cryptosporidiosis is a zoonotic disease that affects newborn ruminants and young or immunocompromised individuals and for which treatments are limited and are not fully effective. Parasite development can be controlled by a protective immune response in which IL-12 and IFN-gamma productions are essentials. Laboratories AIM, IPV and VitamFero Start-up Company decided to pool their skills to evaluate whether the administration of an attenuated vaccine strain of Toxoplasma gondii (MIC1-3KO) could stimulate the immune system of neonates and favor the control of cryptosporidiosis. The first part of the work of my Cifre thesis was performed to obtain a proof of concept. Indeed, preliminary experiments on mice and lambs in which MIC1-3KO strain had developed sufficiently showed a decrease in parasite burden following an infectious challenge with C. parvum. Based on these encouraging results we decided to develop two complementary approaches to further improve the vaccine strain and our knowledge on newborn immune response to T.

Weaning is defined as the physical separation of the cow-calf pair and the end of milk feeding. Natural weaning occurs between 7 and 14 months and is a gradual process. However, domestic weaning occurs between 6 and 8 months and occurs rapidly. Calves that are abruptly separated from their dam respond with increased vocalization and walking, and decreased eating and resting. The psychological stress the calf undergoes during weaning causes elevated glucocorticoid and catecholamine hormone concentrations that may predispose to increased morbidity and/or mortality from infectious diseases such as Bovine Respiratory Disease Complex. As an attempt to counter these changes, alternative weaning methods have been implemented and normally occur in two stages. Two-stage weaning begins with the cessation of milk feeding for approximately one week with the calf maintaining some contact with their dam and then permanent separation occurs. One of these methods uses a single fence to separate the cow-calf pair; this process allows the calf to see, hear and smell their dam, but does not allow the calf to suckle from its dam. Increases in cortisol, a glucocorticoid, have been linked to immunological alterations. Most notably, elevated cortisol concentrations decrease neutrophil function by down regulating the gene expression of CD62L and Fas. Cortisol also alters lymphocyte phenotype by decreasing ?δ T cells and increasing°? T cells in the circulation. Lastly, increases in cortisol can modify T cell cytokine production. The cytokines IL-12 and IFN? are secreted from T helper 1 cells while T helper 2 cells secrete IL-4 and IL-10; these T cells subsets also inhibit one another. During higher cortisol concentrations, these T cells are biased toward T helper 2 cytokine production. All these changes in immune function can lead to increased susceptibility to disease around the time of weaning. Therefore, two trials were conducted to test the hypotheses that abrupt weaning results in elevated concentrations of cortisol and subsequently alters immunological functions, and that fenceline weaning alleviates the increase in cortisol and alterations to immune function associated with weaning. In the fall of 2008, 12 Angus and Angus-X heifers (186°21 kgs; 174°16 days of age) were blocked by age and weight and randomly allotted into two groups, fenceline and abrupt. Blood samples were taken on day -7, 0, 7, 14, 21, and 42; fecal samples were taken on day -7, 0, and 3. All calves were weighed on day -7, 0, 7, 14, and 42. On day -1 all calves were separated from their dam and transported for 2 hours to another facility. On day 0 all calves were vaccinated with Brucella abortus (strain RB51). Serum was analyzed for IFN? and IL-4 as well as IgG1 and IgG2 specific antibodies to RB51. Fecal samples were analyzed for cortisol metabolites. Both IgG1 and IgG2 antibodies to RB51 increased from day 0 to day 14 (P<0.05), however no differences were detected between treatment groups. Fecal cortisol metabolites were higher on day 0 in abruptly weaned calves (P< 0.001) but did not differ between groups on day -7 or day 3. Fenceline calves had higher concentrations of IFN? in the serum on day -7 and day 0 as compared to the abruptly weaned calves (P<0.04). In the fall of 2009, forty-four Angus and Angus-X calves (19 heifers and 25 steers; 181°27 kgs; 148°17 days old) were blocked by age and gender and randomly allotted within block into two treatment groups, fenceline (FL) and abrupt (AB). Approximately half the fenceline calves were separated from their dams by a single fence at day -7 and the rest of the fenceline group at day -6; all calves were removed from their dam at day 0. Calves were vaccinated with Histophilus somni on day 1. Blood samples were taken at day -6, 1, 3, 8, 15, and 22. Fecal samples were taken on day -7, -6, 1 and 3. All calves were weighed on day -7, 0, 8, and 22. Serum samples were analyzed for IgG1 and IgG2 specific-H. somni antibodies, white blood cells were analyzed for lymphocyte phenotypes, and gene expression using 18S as the housekeeper gene. Fecal samples were analyzed for cortisol metabolites. Abruptly weaned calves had higher concentrations of cortisol metabolites in the feces than fenceline calves at day 1 (P<0.0001). No difference in average daily gain or H. somni specific antibodies between treatment groups was detected. There was a treatment*date interaction in lymphocyte and neutrophil populations (P<0.05); neutrophils from fenceline calves dropped from day -6 to day 1, but increased from day 1 to day 3, while abrupt calves decreased from day -6 to day 3. Lymphocytes from fenceline calves increased from day -6 to day 1, but decreased from day 1 to day 3, while lymphocytes from abrupt calves increased from day -6 to day 3. No difference in treatment groups was detected for lymphocyte phenotypes or gene expression; however, a date effect was detected. The CD4 and CD8 cell populations increased over time (P<0.0001) and WC1 and TcR1 decreased over time (P=0.0243 and P=0.0027 respectively) for both treatment groups. A decrease was detected over time for expression of GAPDH and CD62L (P<0.0001). The gene expression for the cytokines IFN?, IL-4 and IL-10 had no change over time. Results from the two studies suggest that fenceline weaning decreases the cortisol response associated with cow-calf separation, but does not have a significant effect on immunological parameters measured in this study. / Master of Science

No description available.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

Background: Adherent-invasive Escherichia coli (AIEC) is a pathogen isolated from the ileum of patients with CD. IFNγ is a key mediator of immunity, which regulates inflammatory responses to microbial infections. Previously, we showed enterohemorrhagic E. coli prevents STAT1 activation. Aims: To determine; 1) whether activation of STAT1 by IFNγ was prevented following AIEC infection, and 2) define the mechanisms used. Methods: Human epithelial cells were infected with AIEC strains or other pathogenic and commensal E. coli strains. Following infection, cells were stimulated with IFNγ. Activation of STAT1, was monitored by immunoblotting. Results: AIEC strains prevented STAT1 phosphorylation in response to IFNγ. Effect required live bacteria with active protein synthesis. A bacterial product was responsible for blocking STAT1 signalling and interfered with downstream signalling cascades. Conclusion: Suppression of epithelial cell STAT1 signal transduction by AIEC strains represents a novel mechanism by which the pathogen evades host immune responses to the infection.

No description available.

Survival of patients with poor prognosis or relapsed haematopoietic malignancies can be markedly improved by allogeneic haematopoietic stem cell transplantation (HSCT). HSCT reconstitutes the immune and haematopoietic systems after myeloablative conditioning and inhibits the recurrence of the malignancy by a graft-versus-leukaemia (GVL) response mediated by donor T cells. However, significant post-transplant complications such as graft-versus-host disease (GVHD) continue to plague the event-free survival of this curative procedure. GVHD is facilitated by donor T cells that recognise histocompatibility antigens on host antigen presenting cells (APC), such as dendritic cells (DC). Current treatment options for GVHD are focused on these T cells. However, these treatments result in an increased incidence of infection, graft rejection and relapse. A novel means of immunosuppression in GVHD is the use of multi-potent, mesenchymal stromal cells (MSC). MSC are non-immunogenic cells that actively suppress T cell function in vitro, and can resolve steroid-refractory GVHD in the clinic. Despite their use in the clinic, there is a paucity of pre-clinical data. Our aim was to investigate the in vivo efficacy of MSC to control GVHD while maintaining the beneficial GVL effect, and to begin to understand the mechanism by which MSC exert their immunosuppressive effects. We isolated and characterised MSC from murine bone/bone marrow and demonstrated that they suppressed T cell proliferation in vitro, even at low ratios of 1 MSC per 100 T cells. This was true of both donor-derived MSC, and MSC derived from unrelated donors (third party). Importantly, we observed that MSC significantly reduced T cell production of the pro-inflammatory cytokines TNFα and IFNγ in culture supernatants and that IFNγ plays a key role in the ability of MSC to suppress T cell proliferation. In vivo, we examined the effects of donor-derived MSC on GVHD severity and onset in two myeloablative murine models of HSCT. A major histocompatibility complex (MHC)-mismatched donor-recipient pair combination was used as a proof–of-principle model [UBI-GFP/BL6 (H-2b)àBALB/c (H-2d)], and an MHC-matched, minor histocompatibility antigen (miHA) mismatched donor-recipient pair combination was used to mimic MHC-matched sibling transplantation [UBI-GFP/BL6 (H-2b)àBALB.B (H-2b)]. We examined a number of variables related to MSC infusion including timing, dose and route of injection. We found that early post transplant infusion of MSC by the intraperitoneal injection was most effective at delaying death from GVHD, compared to pre-transplant infusion or intravenous injection. Furthermore, we found that the dose of MSC was critical, as infusion of too few MSC was ineffective and infusion of too many MSC exacerbated the development of GVHD. Taken together, these results suggest that timing, dose and route of injection are all important factors to be considered to ensure successful therapeutic outcome. To investigate the in vivo mechanism of action, we conducted timed sacrifice experiments in the MHC-mismatched model to determine if MSC altered cytokine secretion and cellular effectors, such as DC, known to play a key role in GVHD. Despite the fact that MSC given post-HSCT enter an environment full of activated DC and IFNγ levels, by day 3 and 6 post infusion, these activated DC and IFNγ levels are decreased compared to controls or mice infused with MSC pre-transplant (p<0.05). This confirmed our in vitro data that IFNγ played an important role in MSC-mediated immunosuppression. In addition, when we removed a major source of IFNγ production in vivo by administering the T cell depleting antibody KT3 to mice with or without MSC, we found that although T cell depletion prolonged survival, MSC were unable to further enhance this effect. This was also true when MSC were used in combination with the conventional immunosuppressant cyclosporine. Finally, we examined whether the infusion of MSC would compromise the GVL effect. We found that whilst MSC could delay the onset of GVHD, in our model they did not alter the anti-tumour effects of the donor T cells. Overall, we have shown that MSC can delay but not prevent death from GVHD when administered at an appropriate time and dose and that IFNγ is required for MSC-mediated immunosuppression in our model. These data suggest that patients undergoing HSCT should be monitored for IFNγ, and administered MSC when high levels are reached. Whilst MSC may be a promising therapy for patients with severe GVHD, we highlight that further investigation is warranted before MSC are accepted for widespread use in the clinic. The risks and benefits for transplant recipients should be carefully considered before utilising MSC to treat or prevent GVHD.