Prolonging function of the corpus luteum (CL) is a method of suppressing estrus that maintains secretion of endogenous progesterone to keep mares out of heat naturally. The use of oxytocin to prolong CL function is becoming more popular. In these therapies, upregulation of cyclooxygenase-2 is inhibited, which impairs prostaglandin F2α ( PGF2α) production. Intramuscular (IM) administration of 60 IU of oxytocin once daily from 7 to 14 days after ovulation is currently the most common treatment protocol. Although that protocol is efficacious in ≥70% of treated mares, the need for daily administration is a drawback to its use. A proprietary slow-release oxytocin formulation (SR-OT) with a two-injection protocol to prolong CL function was evaluated in the first experiment. Mares were examined to determine the day of ovulation (day 0) and then randomly assigned either to a non-treated control group or an SR-OT treatment group (n = 8 mares/group). Mares in the treated group received 1.0 mL of SR-OT containing 2,400 IU oxytocin IM once on Day 7 and again on Day 10 after ovulation. Jugular blood samples were collected on day 0 and then every M, W, and F continuously. Serum progesterone concentrations were evaluated to assess CL function, which was prolonged in 0/8 (0%) control mares and 6/8 (75%) of the SR-OT treated mares (p< 0.01). In a second study, the ability of the endometrium to synthesize PGF2α was evaluated in mares in a state of prolonged CL function. Mares were designated into groups 50-59, 60-69, or 70-79 days post-ovulation (50s, 60s, 70s) and 14-day post-ovulation controls. PGF2α synthesis was evaluated by measurement of a prostaglandin metabolite in response to a single 10 IU intravenous oxytocin bolus (0 minutes). Blood samples were collected serially from 30 minutes before until 120 minutes after oxytocin administration. The metabolite response was significantly higher in the 70s versus the 50s and 70s versus the 60s groups (p< 0.001; p< 0.02, respectively); and there was no significant difference between the 70s group and the control group (P>0.36). Luteal function was maintained after oxytocin administration in 4/4, 3/4, and 0/3 mares in the 50s, 60s, and 70s groups, respectively. Collectively, these results indicate that the luteolytic mechanism returns approximately 70 days into the period of prolonged CL function.

Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

Background: Psychological and physical stress can either enhance or suppress immune functions depending on a variety of factors such as duration and severity of stressful situation. Chronic stress exerts a significantly suppressive effect on immune functions. However, the mechanisms responsible for this phenomenon remain to be elucidated. Autophagy plays an essential role in modulating cellular homeostasis and immune responses. However, it is not known yet whether autophagy contributes to chronic stress-induced immunosuppression. T cell immunoglobulin and mucin domain 3 (Tim-3) has shown immune-suppressive effects and obviously positive regulation on cell apoptosis. Tim-3 combines with Tim-3 ligand galectin-9 to modulate apoptosis. However, its impact on autophagy and chronic stress-induced immunosuppression is not yet identified. Results: We found remarkably higher autophagy level in the spleens of mice that were subjected to chronic restraint stress compared with the control group. We also found that inhibition of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated chronic stress-induced alterations of pro-inflammatory and anti-inflammatory cytokine levels. We further elucidated that 3-MA dramatically inhibited the reduction of lymphocyte numbers. Moreover, chronic stress dramatically enhanced the expression of Tim-3 and galectin-9. Inhibition of Tim-3 by small interfering RNA against Tim-3 significantly decreased the level of autophagy and immune suppression in isolated primary splenocytes from stressed mice. In addition, α-lactose, a blocker for the interaction of Tim-3 and galectin-9, also decreased the autophagy level and immune suppression. Conclusion: Chronic stress induces autophagy, resulting with suppression of immune system. Tim-3 and galectin-9 play a crucial regulatory role in chronic stress-induced autophagy. These studies suggest that Tim-3 mediated autophagy may offer a novel therapeutic strategy against the deleterious effects of chronic stress on the immune system.

The naked mole-rat possesses several unique physiological and molecular features that underlie their remarkably and exceptional resistance to tissue hypoxia. Elevated pattern of Epo, an erythropoietin (Epo) factor; c-fos; vascular endothelial growth factor (VEGF); and hypoxia-inducible factors (HIF-1α) contribute to the adaptive strategy to cope with hypoxic stress. Moreover, the naked mole-rat has a lower metabolic rate than any other eutherian mammal of comparable size that has been studied. The ability to actively reduce metabolic rate represents a strategy widely used in the face of decreased tissue oxygen availability. Understanding the different molecular and physiological factors that induce metabolic suppression could guide the development of pharmacological agents for the clinical management of stroke patient.

Sepsis-induced immunosuppression increases the risk of chronic infection and reduces survival. Myeloid-derived suppressor cells (MDSCs) expand in the bone marrow and spleen during murine polymicrobial sepsis, contributing to immunosuppression. A better understanding of molecular controls of MDSC production is needed to identify treatment targets. We previously reported that miR-21 and miR-181b couple with transcription factor NFI-A to induce MDSCs during murine sepsis. Here, we expand upon these observations by showing that conditional deletion of the Nfia gene in the myeloid lineage precludes MDSC development. NFI-A-deficient Gr1+CD11b+ myeloid cells are not immunosuppressive and differentiate normally into macrophages and dendritic cells. In contrast, ectopically expressed NFI-A prevents differentiation of these immature Gr1+CD11b+ cells, while converting them into MDSCs. In addition, NFI-A-deficient Gr1+CD11b+ cells decreased, and cells transfected with NFI-A increase expression of miR-21 and miR181b. Our results support a myeloid cell loop in which NFI-A and miR-21 and miR-181b sustain Gr1+CD11b+ MDSC-dependent immunosuppression during sepsis.

Objectives: Mental and physical stress can suppress the immune system in both humans and animals. The mechanism by which stress affects immune responses, however, remains poorly defined. Toll-like receptors (TLRs) play a key role in modulating immune responses and cell survival. The mechanisms by which TLRs modulate chronic stress are largely unexplored. Methods: Six- to 8-week-old male mice were subjected to chronic 12-hour daily physical restraint stress. Apoptotic cells were determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. We examined cytokine levels by enzyme-linked immunosorbent Assay (ELISA). The expression of CYP11A1 was determined by quantitative real-time RT-PCR. Results: TLR9-deficient mice were resistant to chronic stress-induced lymphocyte apoptosis. In addition, in TLR9 knockout (KO) mice, chronic stress-induced upregulation of corticosterone levels was significantly decreased. Notably, lymphocytes from both TLR9 KO and wild-type mice were similarly sensitive to corticosteroid-induced cell apoptosis. Moreover, TLR9 deficiency blocked the chronic stress-induced imbalance in T helper (Th) 1 and Th2 cytokine levels. Conclusion: Taken together, our findings reveal that TLR9 plays an essential role in chronic stress-induced immune suppression.

Objectives: Mental and physical stress can suppress the immune system in both humans and animals. The mechanism by which stress affects immune responses, however, remains poorly defined. Toll-like receptors (TLRs) play a key role in modulating immune responses and cell survival. The mechanisms by which TLRs modulate chronic stress are largely unexplored. Methods: Six- to 8-week-old male mice were subjected to chronic 12-hour daily physical restraint stress. Apoptotic cells were determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. We examined cytokine levels by enzyme-linked immunosorbent Assay (ELISA). The expression of CYP11A1 was determined by quantitative real-time RT-PCR. Results: TLR9-deficient mice were resistant to chronic stress-induced lymphocyte apoptosis. In addition, in TLR9 knockout (KO) mice, chronic stress-induced upregulation of corticosterone levels was significantly decreased. Notably, lymphocytes from both TLR9 KO and wild-type mice were similarly sensitive to corticosteroid-induced cell apoptosis. Moreover, TLR9 deficiency blocked the chronic stress-induced imbalance in T helper (Th) 1 and Th2 cytokine levels. Conclusion: Taken together, our findings reveal that TLR9 plays an essential role in chronic stress-induced immune suppression.

Mounting evidence supports that sepsis-associated immunosuppression increases mortality. As potential contributors to poor sepsis outcomes, myeloid-derived suppressor cells, which are Gr1+ CD11b+ innate-immune cell progenitors unable to differentiate and possess suppressive activities, expand dramatically in septic mice by a process requiring increased microRNA-21 and microRNA-181b expression. The inhibition of these microRNAs in vivo in septic mice restores Gr1+ CD11b+ cell differentiation and maturation and improves survival. Here, we show that during sepsis-induced generation of myeloid-derived suppressor cells, transcription factor nuclear factor 1 A type represses cyclin-dependent kinase inhibitor p21 to arrest differentiation of Gr1+ CD11b+ cells. Our findings include the following: 1) Gr1+ CD11b+ myeloid cells from late septic mice genetically lacking nuclear factor 1 A type cannot suppress CD4+ T cell proliferation and activation; 2) the reconstitution of nuclear factor 1 A type in microRNA-21 and microRNA-181b-depleted Gr1+ CD11b+ myeloidderived suppressor cells inhibits cyclin-dependent kinase inhibitor p21 and restores the immunesuppressor phenotype; 3) ex vivo nuclear factor 1 A type knockdown in Gr1+ CD11b+ myeloid-derived suppressor cells from late septic mice restores cyclindependent kinase inhibitor p21 expression and promotes monocyte and dendritic cell differentiation; and 4) ectopic nuclear factor 1 A type expression in normal Gr1+ CD11b+ cells generates an immunosuppressive phenotype. We suggest that therapeutically targeting nuclear factor 1 A type during late sepsis might improve survival.

Stress can either enhance or suppress immune functions depending on a variety of factors such as duration of stressful condition. Chronic stress has been demonstrated to exert a significant suppressive effect on immune function. However, the mechanisms responsible for this phenomenon remain to be elucidated. Here, male C57BL/6 mice were placed in a 50-ml conical centrifuge tube with multiple punctures to establish a chronic restraint stress model. Serum IL-10 levels, IL-10 production by the splenocytes, and activation of STAT3 in the mouse spleen were assessed. We demonstrate that IL-10/STAT3 axis was remarkably activated following chronic stress. Moreover, TLR4 and p38 MAPK play a pivotal role in the activation of IL-10/STAT3 signaling cascade. Interestingly, blocking antibody against IL-10 receptor and inhibition of STAT3 by STAT3 inhibitor S3I-201 attenuates stress-induced lymphocyte apoptosis. Inhibition of IL-10/STAT3 dramatically inhibits stress-induced reduction in IL-12 production. Furthermore, disequilibrium of Th1/Th2 cytokine balance caused by chronic stress was also rescued by blocking IL-10/STAT3 axis. These results yield insight into a new mechanism by which chronic stress regulates immune functions. IL-10/STAT3 pathway provides a novel relevant target for the manipulation of chronic stress-induced immune suppression.

Myeloid-derived suppressor cells (MDSC)expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBPβ synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBPβ protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBPβ protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.