Academic literature on the topic 'QR180 Immunology ; RG Gynecology and obstetrics'

Reproduction is a crucial process, required for bringing the next generation into the world. In preparation for pregnancy, and throughout pregnancy itself, reproductive tissues recruit specific populations of immune cells that are thought to contribute in a variety of ways to successful reproduction. Pregnancy culminates in parturition, an inflammatory process characterised by an influx of inflammatory cells into reproductive tissues. Effective healing of reproductive tissues in the post-partum period is vital for continued reproductive success, and it too is thought to involve specific populations of immune cells. For leukocytes to effectively perform their functions in the reproductive system and elsewhere, migration to the right place at the right time is crucial. Key regulators of leukocyte homing are the chemokine family of chemoattractants and their G-protein coupled receptors. The chemokine network is complex and controls migration of leukocytes from the Bone Marrow (BM) into the blood and from the blood into tissues. Chemokines influence leukocyte position within tissues, and orchestrate their departure. Very little is known about the types of leukocytes present within reproductive tissues in the post-partum period, or the chemokines and receptors that could be involved in their migration. Exploring these processes is critical for an understanding of how tissues are repaired in readiness for subsequent pregnancies. In this thesis I have examined the leukocyte populations in reproductive and peripheral tissues of mice during the post-partum period and compared them to those found in Non-Pregnant (NP) mice. This analysis has encompassed a range of myeloid cell types, and also the complex populations of CD3+ cells that exist in reproductive tissues. I was also interested in how these cells are instructed to enter reproductive tissues, and in particular on the role of CC chemokine Receptor 2 (CCR2), a receptor associated with the recruitment of monocytes and T cells into tissues. My work has clearly identified cells expressing CCR2 both in reproductive tissues and elsewhere, and defined the impact of the genetic deletion of this receptor on leukocyte populations during the post-partum period. These experiments exploited a variety of standard techniques including histology, quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR), Enzyme-Linked ImmunoSorbent Assay (ELISA) and Luminex, but they also required the development of challenging multiparameter flow cytometry protocols that allowed the simultaneous analysis, and definitive identification, of several leukocyte populations in various tissues and at specific reproductive time-points. Chapter 3 describes detailed experiments that focussed on characterising the myeloid cell populations across a variety of tissues in NP, 1 Day Post Partum (DPP) and 7DPP mice. Most strikingly, this revealed a profound accumulation of several myeloid cell populations in reproductive tissues at 1DPP, including inflammatory Ly6Chigh (hi) monocytes and neutrophils. Moreover, many of these myeloid cells expressed active CCR2 and remarkably CCR2 deletion was associated with a dramatic reduction in myeloid cell abundance in the uterine horn one day after birth. Thus, CCR2 appeared to be required for myeloid cell recruitment to the post-partum uterine horn. Chapters 4 and 5 describe changes in CD3+ cell populations over the post-partum period. Interestingly, the main finding from reproductive tissues was that the large majority of CD3+ cells lacked expression of CD4 and CD8, and were thus termed CD3+ Double-Negative (DN) cells. Three main CD3+ DN cell populations were described. CD3+CD25+NK1.1+TCRβ+ DN cells, likely to be Natural Killer T (NKT) cells, which were mainly found in reproductive tissues and blood. All tissues studied were found to contain CD3+NK1.1-TCRβ+ DN cells, likely to be ‘true’ DN T cells and CD3+NK1.1-TCRβ-TCRγδ+ DN cells, which were consistent with a γδ T cell phenotype. CD3+ DN cells were also found to increase in number at 1DPP, compared to NP tissues, driven by an increase in DN T cells. In contrast to myeloid cells CCR2 was not required for this change. However, at 1DPP there was a CCR2-dependent increase in the proportion of CD3+ DN cells in the blood. Finally, in Chapter 6, hormonal influences on leukocyte populations in reproductive and peripheral tissues were considered. This work had two major components: analysing sex differences in myeloid and T cell populations and exploring the effect that lactation has on these cell subsets over the post-partum period. Females were found to have an increased proportion of eosinophils in their blood, whereas males had a higher proportion of monocytes. I also found that female and male reproductive tissues, as well as peripheral tissues, have very similar CD3+ DN cell populations, suggesting that these cells serve roles in reproductive tissues that are not unique to one sex. Finally, CD3+ cell populations in the post-partum period were found to be controlled to some extent by lactation. Collectively, this work has significantly extended our understanding of leukocytes in various tissues in the post-partum period, and revealed the importance of chemokines in the regulation of these cells. It has laid the groundwork for future investigations aimed at dissecting the functions of these cells in reproductive tissues in the post-partum period.

Epidemiology has linked preeclampsia (PET) to vitamin D deficiency. To date, studies have focused upon serum 25-hydroxyvitamin D3 (25(0H. )D3) alone as the marker of vitamin D status. We provide strong evidence comprehensive analysis of vitamin D metabolites in pregnancy is highly informative, particularly within the context of PET. Uniquely, analysis of maternal urinary metabolites provides a novel insight into vitamin D and the kidney, with lower 25(0H)D3 and 24,25(0H)2D3 excretion early indicators of a predisposition towards PET. Since vitamin D is a potent regulator of immune function, and the decidua appears a key extra-renal site for vitamin D metabolism, we investigated effects of 1 ,25(0H)2D3 upon decidual uterine natural killer cells and macrophages. We show both express a functional vitamin-D system and demonstrate differential sensitivity to 1 ,25(0H)2D3 compared to their peripheral counterparts. To understand the functional impact of vitamin D, whole transcriptomic analysis of 1,25(0H)2D3-mediated effects upon uNK and macrophages was performed. We show the actions of vitamin D extend far beyond simple immuno-regulation, targeting major cellular functions including migration, adhesion and apoptosis. In particular, our data support effects highly relevant to decidualisation. We anticipate these findings to be highly relevant within the context of vitamin D deficiency, mal placentation and PET.

The bacteria Ureaplasma has long been associated with a wide range of adverse health implications, including preterm birth, preterm premature rupture of the membrane and lung disorders, such as bronchopulmonary dysplasia in neonatal infants, but still, little is known about the pathogenic properties of Ureaplasma and possible direct association with adverse health complications. Estimated prevalence of Ureaplasma colonisation in sexually active adults is between 40 – 80%, therefore further understanding of its pathogenic properties and its ability to initiate an immune response is crucial. Specifically selected human cell-lines were examined in vitro to determine whether an innate immune response could be activated by Ureaplasma infection. If inflammatory immune responses were detected in human cell-lines, pathogenic properties of Ureaplasma would be confirmed, and its role in pregnancy and neonatal complications could be supported. Using a range of techniques, activation of immune response pathways were examined, as too were the production of detrimental pro-inflammatory cytokines that would strengthen the suggested associations of Ureaplasma infection with the above-mentioned complications. Myeloid-derived leukocytic monocytes, human bronchial epithelial cells and human amniotic epithelial cells were examined, as these would be the most relevant cell lines to determine if Ureaplasma could induce preterm birth, preterm premature rupture of the membrane and bronchopulmonary dysplasia. All cell lines studied showed immune response and inflammatory cytokine production after stimulation with Ureaplasma. This supports that Ureaplasma is capable of causing tissue damage in neonatal respiratory tracts that may lead to bronchopulmonary dysplasia and damage to the amniotic and chorion membranes that may lead to preterm premature rupture of the membrane. Ureaplasma was detected at the cell surface of human amniotic epithelial cells (HAECs) by TLR2 and TLR2/6 heterodimers. Results suggest that Ureaplamsma multiple banded antigen (MBA) is the strong ligand for TLR2 and TLR6 and stimulation of HAECs with MBA alone caused an immune response. TLR9 was responsible for the detection of internalised Ureaplasma, which is also able to initiate an immune response and inflammatory cytokine production. v Ureaplasma stimulation results in the production of the inflammatory cytokines TNF-α, IL-8 IL-6 via the NF-κB signaling pathway. Production of the potent inflammatory cytokine IL-1β was also observed, which would suggest the formation of inflammasome complexes. NLRs were investigated to find which NLR inflammasome were activated. It was shown that genetically knocking down NLRP7 significantly reduced the amount of IL-1β that was produced after Ureaplasma stimulation, suggesting that NLRP7 inflammsones are activated by Ureaplasma. Reduction in IL-1β was also observed, but to a lesser extent, when NLRP3 was knocked down. We decided to investigate the role of NLRP7 further and found a novel immune pathway, where NH3 causes activation and formation of the NRLP7 inflammasone. NH3 is produced as a bi-product of urease activity, which an essential process for Ureaplasma. The addition of a potent urease inhibitor to HAECs being stimulated with Ureaplasma significantly reduced the production of IL-1β, strongly supporting that NH3 plays a significant role in the detection of Ureaplasma infection and is responsible for causing the tissue damage that contributes to preterm premature rupture of the membrane leading to preterm birth. This investigation strongly supports that Ureaplasma is responsible for causing preterm birth and health complications in neonates, and that more robust treatment and monitoring of Ureaplasma is required, especially in pregnant women. These undertakings will hopefully reduce the rates of preterm birth and the associated health implications, in addition to reducing rates of bronchopulmonary dysplasia in neonates.

Parallel increases in many inflammatory diseases including atopy over the last 40 years suggest that common environmental changes may be promoting inflammatory immune responses. Modern diets have become increasingly rich in n-6 polyunsaturated fatty acids (PUFAs) and relatively deficient in n-3 PUFAs. These dietary changes are believed to promote a pro-sensitisation, pro-allergic and pro-inflammatory environment. Exposure to such an environment during pregnancy and in the very early life period is considered to influence subsequent patterns of the immature and developing neonatal immune system, and this may contribute to the increase in allergic disease in early life. As allergic diseases often first manifest in infancy, prevention strategies need to be targeted early, even in utero. Epidemiologic and experimental data provide a plausible link between dietary changes and increased incidence of childhood atopic disease. Although there have been studies examining the potential benefits of giving n-3 PUFA-rich fish oil supplements during pregnancy, there are no studies examining the effects of increased consumption of oily fish in pregnancy on neonatal immune responses and subsequent clinical outcomes. The Salmon in Pregnancy Study (SIPS) is the first randomised controlled trial of oily fish intervention during pregnancy. The hypotheses being investigated in SIPS is that increased intake of salmon, a source of long chain (LC) n-3 PUFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), in pregnancy will a) increase maternal LC n-3 PUFA intake, b) increase maternal and infant blood LC n-3 PUFA status, c) modulate fetal/neonatal immune responses and d) lower the risk of infant atopy determined at 6 months of age. The primary outcome measures of SIPS were the clinical signs of atopy in the offspring. Pregnant women (n=123) at high risk of having atopic offspring, and with low habitual intake of oily fish (≤ 2/month) were randomised at 20 weeks of pregnancy to either consuming 2 portions/week of farmed salmon (n=62) or continuing their habitual diet (n=61) until the end of pregnancy. The woman attended a clinic at 20 (n=123), 34 (n=110) and 38 (n=91) weeks of gestation at which fasting blood was collected and a food frequency questionnaire (FFQ) was administered (at 20 and 34 weeks). At delivery umbilical cord blood was collected (n=101) for fatty acid and immunological analysis. Infants attended a clinic at 6 months of age (n=86) for assessment of allergic sensitisation by skin prick testing (SPT) using various allergen extracts and of atopic dermatitis (SCORAD index). Maternal and cord plasma and cord blood mononuclear cell (CBMC) fatty acid compositions were determined by gas chromatography. Neonatal (cord) immune cell subsets were identified by flow cytometry. Ex-vivo cytokine production by CBMC in response to stimulants (allergen, mitogen, and toll-like receptor (TLR) ligands) was determined by cytometric bead array and flow cytometry. Ex-vivo prostaglandin E2 production by CBMC was determined by enzyme-linked immunosorbent assay. Immunoglobin E concentration was measured in cord blood plasma and in 6 month infant blood plasma. Eating oily fish twice a week during pregnancy resulted in a higher maternal intake of LC n-3 PUFAs (both EPA and DHA) and in higher maternal and cord blood plasma status of LC n-3 PUFAs (both EPA and DHA). LC n-3 PUFA content of CBMC was not significantly affected. CBMC production of interleukins-2, -4, -5, and -10 and tumour necrosis factor-α was lower in the salmon group. There was no effect of salmon on the atopic outcomes assessed at 6 months.

Dr James A. Thomson reached a milestone discovery in 1998, as he managed to isolate and in vitro expand embryonic stem cells originating from human blastocysts. Since then, human embryonic stem (hES) cells have served as excellent tools for the understanding of a plethora of events that take place during embryogenesis. A full and comprehensive analysis of the molecular mechanisms that regulate both pluripotency and differentiation procedures will ultimately allow these cells to be utilised for therapeutic purposes. The first part of the present thesis is dedicated to investigating the implication of ADP-Ribosylation Factor 6 (ARF6) in TGFβ signalling. ARF6 is a low molecular weight GTPase involved in various cellular functions. Our preliminary data indicate that ARF6 interacts with SMAD4. Building on that, we uncover novel interactions of ARF6 with proteins SMAD2/3 and the interconnection between nucleotide status and downstream signalling events. The connection between ARF6 and TGFβ signalling led us to hypothesize a role for the GTPase in hES cells. In that system, we characterise the effects of ARF6 activation or knockout on both Activin A and BMP4 signalling. In addition, we uncover a novel role for the GTPase during mesendoderm specification. In the last part of the thesis, we utilise a broad transcriptomic approach to reveal novel candidates that are implicated in early differentiation of hES cells to mesendoderm. The assay has been carried out using a novel culture system, based on the ability of Activin A to preserve pluripotency and BMP4 to initiate differentiation.

Tolerance of the semi-allogeneic fetus presents a significant challenge to the maternal immune system. The effect of pregnancy on maternal cellular immunity was established by assessing maternal effector and regulatory T-cell subsets during human pregnancy. This demonstrated that an increase in maternal peripheral regulatory T-cells or a shift from a Th1 to Th2 phenotype was not a requirement for normal pregnancy. We also determined the profound impact of maternal Cytomegalovirus seropositivity on maternal T- cell dynamics. T-cells with specificity for fetal epitopes have been detected in women with a history of pregnancy but it has been thought that such fetal specific cells were deleted during pregnancy. We identified, using MHC-peptide multimers, fetal-specific CD8 T-cells in half of all pregnancies. The fetal-specific response increased during pregnancy and persisted in the post natal period. Fetal-specific cells demonstrated an effector memory phenotype and retained functional potential. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy. Women with recurrent miscarriage were found to have abnormal T-cell function, with increased IFN\(\gamma\) and Il-17 production. Fetal specific T-cells were also detected in this cohort and progesterone attenuated their function, which may have therapeutic implications.