Academic literature on the topic 'RG Gynecology and obstetrics ; RM Therapeutics. Pharmacology'

Epithelial ovarian cancer is the deadliest gynaecological cancer: most women die within five years of their diagnoses. Moreover, survival of women with ovarian cancer (OC) has not significantly improved in the past decade. Oncolytic viruses (OVs), a new class of anti-cancer agent, infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs also induce profound immune responses, for example disruption of chemokine and cytokine networks, with potential influence on therapeutic effectiveness. On the other hand, Natural Killer cells (NK cells), a key immune population that surveillance against cancers and viruses, may hinder the spread of OVs or promote anti-tumoural effects of OVs. This work investigated the role of innate immune responses, in particular NK cells and interleukin (IL)-17F, on the efficacy of oncolytic adenovirus in OC. I demonstrated that NK cells were activated by adenovirus-infected OC cells. Activated NK cells then augmented oncolytic adenovirus in eliminating OC via contact-dependent interactions between activating NK receptor DNAM-1 and adenovirus-infected malignant cells. In addition, consistent changes in chemokines and cytokines were observed after wild-type and oncolytic adenovirus infection. In particular, IL-17F, but not IL-17A, was significantly upregulated in different established and primary OC lines after adenovirus infections. Moreover, a range of inflammatory chemokines, including CCL2, CXCL1, CXCL2 and CXCL5, were down-regulated after oncolytic adenovirus infection. This work also revealed the logistical and technical challenges of the use of primary patient materials. I identified that our primary culture method for expanding OC cells was suboptimal. I subsequently evaluated a simple immunohistochemical method to screen for successful primary expansion of malignant cells from OC ascites. I showed that PAX8, but not CK7, was a specific marker of successful ex vivo expansion of HGSOC.

Ovarian cancer is one of the most lethal malignancies and often presents at an advanced stage, resulting in a poor prognostic outlook. Platinum chemotherapy leads to an initial clinical response, however most patients will ultimately relapse and there remains a sub-group who are intrinsically resistant to platinum. I focussed on high grade serous ovarian cancer (HGSOC), the most common subtype of ovarian cancer. The ID8 CRISPR-generated models represented a novel and simple tool to investigate the biology of HGSOC. By using this in vivo model, I aimed to further the understanding of platinum sensitivity in HGSOC by investigating the homologous recombination pathway and the tumour microenvironment. In vitro work showed that sensitivity to PARP inhibitors was clearly correlated with defective homologous recombination but the relationship with platinum sensitivity was more complicated. Using the ID8 derivatives, in vivo cisplatin experiments identified Pten and Nf1 loss to be associated with the worst prognosis with the knockout of Brca1 or Brca2 prolonging survival. A Brca1 mutation in the PALB2 domain compared to the BRCT2 domain was found to be associated with a greater sensitivity to cisplatin. The tumour microenvironment was shown to differ between genotypes and altered with the addition of platinum chemotherapy. Specifically, the loss of Pten was associated with an immunosuppressive microenvironment with increased levels of myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs). The chemokines, Ccl2 and Ccl7 were shown to be significantly increased in the Trp53-/-;Pten-/- genotype. I targeted both the cytokine/chemokine response directly by using a transgenic mouse model (CCR1, 2, 3, 5 receptors knockout) and the PI3K/AKT pathway by using a PI3K inhibitor (p110β) (AZD8186) to attempt to reverse the effect of Pten loss. The transgenic mouse model (GGTACKO) showed encouraging early results with a reduction in MDSCs and TAMs in the knockout mice injected with the Trp53-/-;Pten-/- genotype but a repeat experiment is required before valid conclusions can be made. The AZD8186 in vivo experiment showed a significant reduction in MDSC levels in the ascites following AZD8186 treatment in mice injected with the Trp53-/; Pten-/- genotype and a non-significant decrease in the tumour samples. There was also a reversal in the anaemia previously shown with the loss of Pten and a decrease in Ccl2 and Ccl7 expression. I have used a transplantable in vivo model for HGSOC to investigate potential mechanisms of platinum sensitivity and identified poor prognostic genotypes (Pten, Nf1). I have found Pten loss to be associated with an immunosuppressive microenvironment and highlighted potential therapeutic targets. By targeting the PI3K/AKT pathway I have shown that the effect of Pten loss can be reversed. The next step will be to determine whether this reversal results in a prolonged survival.

The contribution of store-operated calcium entry (SOCE) in the response of human sperm to progesterone, a steroid secreted from the cumulus cells surrounding the oocyte, has not yet been elucidated. The aim of this study was to investigate the presence of SOCE proteins in human sperm and examine the effects of pharmacological modulation of SOCE on the progesterone-induced biphasic intracellular calcium concentration ([Ca\(^{2+}\)]i) response. STIM (stromal interacting molecule) and Orai, proteins of the SOCE system were detected in human sperm in a similar location to intracellular Ca\(^{2+}\) stores. 2-aminoethyldiphenyl borate (2-APB; SOCE modulator) altered SOCE in human sperm in a bimodal manner as seen in other cell types. Furthermore, 5\(\mu\)M 2-APB potentiated the initial progesterone-induced [Ca\(^{2+}\)]i transient within the neck and midpiece, but not in the flagellum. In the sustained phase of the progesterone-induced [Ca\(^{2+}\)]i response both 5\(\mu\)M 2-APB and 10\(\mu\)M loperamide (another modulator of SOCE) potentiated the [Ca\(^{2+}\)]i response. Higher doses of 2-APB (50-200\(\mu\)M) didn’t potentiate the transient [Ca\(^{2+}\)]i and inhibited the sustained response consistent with reported actions on SOCE. Ryanodine receptors were localised to the neck/midpiece region which suggested that they may mobilise intracellular Ca\(^{2+}\) stores in response to progesterone, leading to activation of STIM/Orai and initiating SOCE.

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.

The use of antiretroviral therapy (ART) during pregnancy and lactation has significantly reduced the rate of mother-to-child transmission (MTCT) of HIV. However, pregnancy is known to affect the pharmacokinetics of many drugs, including key antiretroviral (ARV) drugs. In addition, ARV use during lactation raises questions about unintended exposure of breastfed infants to maternal drugs through breast milk. For drugs with significant genetic contribution to observed pharmacokinetic variability, we hypothesised that polymorphisms in drug disposition genes may accentuate or attenuate pregnancy-induced changes and/or breastfed infants’ exposure. HIV positive pregnant women and nursing mothers taking efavirenz (EFV)- or nevirapine (NVP)-based ART were recruited from three hospitals in Benue State, Nigeria. A novel strategy involving a preliminary pharmacogenetic association study was used to investigate the magnitude of pregnancy-induced changes in EFV and NVP pharmacokinetics in women stratified by single nucleotide polymorphisms (SNPs) in disposition genes. EFV apparent clearance (CL/F) was higher and AUC0-24, Cmax and Cmin were significantly lower in pregnant compared with postpartum women. When stratified based on the SNP with the highest predictive power, pregnant women with CYP2B6 516GG genotype were especially at risk. In the NVP cohort, exposure was also significantly lower in pregnant compared with postpartum women. When stratified based on composite CYP2B6 516G > T and 983T > C genotypes, Cmin was below target in most patients with combined CYP2B6 516GG and 983TT during pregnancy and postpartum. Cmin was below target in at least 50% of pregnant women with one or two variant alleles, compared with 0% in postpartum women. The intensive pharmacokinetics of EFV and NVP in breast milk and pharmacogenetic predictors were described for the first time. Breast milk pharmacokinetic parameters of EFV in breast milk differed significantly between patient groups stratified by CYP2B6 516G > T. The median time averaged milk-to-plasma concentration (M/P) ratio was 1.10 (range: 0.57-1.71) and the paediatric dose weight-adjusted exposure index was 4.05% (1.08-13.8). The resulting infant plasma concentration was influenced by CYP2B6 516G > T, highest up to 8 days of age at 1590 ng/mL (190-4631) and decreased by about 90% in the age stratum 9 days to 3 months. NVP AUC0-12, Cmax and Cmin in breast milk were significantly lower in patients with composite CYP2B6 516GG/983TT than those with at least one variant allele. The M/P ratio was 0.88 (0.74-1.2) and paediatric dose weight-adjusted exposure index was 3.64% (1.99-9.88). Infant plasma concentration differed significantly based on CYP2B6 516G > T/983T > C and CYP3A4 20230G > A (*1G), highest in those exposed through both breast milk and post-exposure prophylaxis compared with either alone. A breastfeeding physiologically-based pharmacokinetic (PBPK) model to predict infant exposure to maternal drugs through breast milk was developed and validated, with over 90% of all individual observed data points within the predictive interval. This thesis presents details about five different studies where these findings were observed. Their clinical implications in the context of current knowledge and practice were also explored.