/library/oar/handle/123456789/126941 Sat, 27 Dec 2025 09:31:14 GMT 2025-12-27T09:31:14Z /library/oar/handle/123456789/129870 Title: Investigating the role of olfactory receptors in innate immune memory and sepsis Abstract: Sepsis, defined as the abnormal host response to infection causing lethal organ dysfunction, remains one of the leading causes of patient morbidity and mortality worldwide. These mortality rates are thought to be contributed to heavily by the phenomenon of ‘immune paralysis’, a state of sustained immunosuppression. It is postulated that elucidating the molecular mechanisms that govern innate immune memory could in turn translate to effective treatments. The presence of ectopic olfactory receptors (ORs) on innate immune cells has been discovered in numerous studies in the past decade, linking their activity with specific pro- and anti-inflammatory functions. This study aims to test the hypothesis that ectopic ORs are associated with the host response to infection and the induction of innate immune memory. Using publicly available datasets, over 100 ectopic ORs were identified to be differentially upregulated in sepsis. Expression data from cell-sorted blood leukocytes revealed the differential expression of multiple ORsin sepsis, with OR52B2 upregulation being singled out monocytes. Expression data for lipopolysaccharide (LPS)-treated healthy human monocyte-derived macrophages showed differential downregulation of OR8G5 and upregulation of OR52K3P. OR kinetics observed through a human endotoxemia model dataset revealed continuous upregulation of OR2B6, a suspected monocyte-specific OR, over the span most significant to the development of innate immune memory, proving an attractive target for testing. A cohort of 8 healthy volunteers (4 females, 4 males; mean age 21 years) were recruited for this study. Heparinised blood was drawn with consent from each volunteer, from which monocytes were then harvested and enriched. Enriched monocytes were split into two categories, with one being subjected to a single controlled dose of LPS, while another was subjected to two doses with an overnight rest in between. ELISA testing revealed a stronger TNF-α response upon first exposure compared to restimulation, corroborated by quantitative real-time polymerase chain reaction, which showed much stronger TNFA expression in first exposure monocytes. The observed patterns closely mimic those demonstrated in several reports of monocytes in sepsis patients, characterised by sepsis-induced immune paralysis. OR2B6 expression was not reliably detected during both first stimulation with LPS and in endotoxin tolerance (restimulation), suggesting that olfactory receptor gene expression may not be inducible by bacterial pathogen-associated molecular patterns. Although the impact of ectopic ORs on sepsis-induced immune paralysis remains largely unknown, this study provides multiple avenues through which further work may be carried out to elucidate the role of ORs in the host response during sepsis. Description: M.Sc. Biomed. Sc.(Melit.) Mon, 01 Jan 2024 00:00:00 GMT /library/oar/handle/123456789/129870 2024-01-01T00:00:00Z /library/oar/handle/123456789/129869 Title: Development of monoclonal antibodies targeting T cell coinhibitory proteins TIM-3 and TIM-4 Abstract: Immune checkpoint inhibition (ICI) therapy uses antibodies to block checkpoint proteins from binding with their partner proteins to help overcome dysfunctional T cells and promote the recognition and elimination of tumour cells. The exploration of novel immune checkpoint targets, like T cell immunoglobulin mucin 3 and 4 (TIM-3 and TIM-4), stems from trying to improve the efficacy of ICI therapies (e.g. against cytotoxic T-lymphocyte antigen 4 (CTLA-4) and/or programmed cell death protein 1 (PD-1)) which are currently being used to treat certain cancers like breast, colon and lung cancer either as sole agents or in combination with other immune checkpoint inhibitors or alternative treatments. Targeting TIM-3 and TIM-4 may reverse immune tolerance and suppress tumour cell growth and migration but no effective TIM-4 inhibitors have been developed yet. In this study, monoclonal single chain variable fragment (scFv) antibodies against TIM-3-Fc and TIM-4 were identified using phage display biopanning which isolated antibodies from large immunoglobulin gene repertoires displayed on the surface of bacteriophages. Thirty monoclones from each of the polyclonal pools of anti-TIM-3-Fc and anti-TIM-4 scFv antibodies displayed on phages were picked for validation. Enzyme-linked immunosorbent assay was used to determine the specificity of the phage clones to the target proteins TIM-3-Fc and TIM-4 respectively versus non-target proteins severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike S1 and SARS-CoV-2 receptor-binding domain proteins respectively. This revealed 21 anti-TIM-3-Fc scFv antibody phage monoclones with affinity and specificity to TIM-3-Fc and 8 anti-TIM-4 scFv antibody phage monoclones binding with affinity and specificity to TIM-4. Finally, sanger sequencing was used to determine the DNA sequence of the antiTIM-3-Fc and anti-TIM-4 scFv antibodies and to compare the resulting amino acid sequences to confirm that different scFv antibodies were identified for TIM-3-Fc versus TIM-4. Description: M.Sc. Biomed. Sc.(Melit.) Mon, 01 Jan 2024 00:00:00 GMT /library/oar/handle/123456789/129869 2024-01-01T00:00:00Z /library/oar/handle/123456789/127239 Title: Von Willebrand factor analysis in patients with Myeloproliferative neoplasms Abstract: Myeloproliferative neoplasms (MPNs) encompass clonal disorders including essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). These disorders are characterised by clonal proliferation of myeloid lineage components, leading to symptoms such as agranulocytosis, erythrocytosis, and thrombocytosis. Common to these MPNs are mutually exclusive mutations in JAK2, CALR, and MPL genes, and they share a tendency toward myeloproliferation, splenomegaly, potential progression to AML or myelofibrosis, and the occurrence of thrombohemorrhagic events. Moreover, patients, especially those with ET, may develop AVWD, a qualitative defect in von Willebrand factor (VWF). To elucidate the relationship between MPNs and AVWD, this study compared the VWF profiles of MPN patients to healthy controls (HCs) using several assays. Initial analyses included VWF:Antigen (VWF:Ag), VWF:Activity (VWF:Ac), Factor VIII (FVIII), and the VWF Antigen/VWF:Activity (VWF:Ag/VWF:Ac) ratio, providing quantitative and qualitative insights. Further specific tests included VWF multimeric analysis and VWF propeptide (VWFpp) levels, complemented by both the custom reverse Ristocetin Induced Platelet Aggregation (RRIPA) assay and the traditional Ristocetin Induced Platelet Aggregation (RIPA) assay. Our findings revealed a loss of high molecular weight multimers (HMWM) with a corresponding increase in intermediate (IMWM) and low molecular weight multimers (LMWM) within the MPN cohort. VWFpp levels were normal, indicating regular VWF release. However, the VWFpp to VWF antigen ratio (VWFpp:VWFAg) was elevated, suggesting a shortened half-life of plasma VWF and reflecting increased clearance. Measurements of maximum aggregation (MA) from both RRIPA and RIPA assays indicated a defect in aggregation; changes in RRIPA were not statistically significant, indicating that normal platelet function might partially compensate for these VWF abnormalities. Description: B.Sc. (Hons)(Melit.) Mon, 01 Jan 2024 00:00:00 GMT /library/oar/handle/123456789/127239 2024-01-01T00:00:00Z /library/oar/handle/123456789/127238 Title: A comparative in vitro study of the coagulation effects of branded vs generic preparations of the oral anticoagulant apixaban Abstract: The introduction of the direct oral anticoagulants (DOACs) represents a major advancement in the field of anticoagulant therapy. However, scant research has been conducted to study the effects of generic versions of DOACs on in vitro coagulation tests. The aims of this study were: 1) to determine the anticoagulant effects of the branded version of apixaban (Eliquis®) on a panel of coagulation assays, including Prothrombin time (PT), activated Partial Thromboplastin time (aPTT), PT-derived Fibrinogen, Clauss Fibrinogen, Thrombin time (TT), Diluted Russell’s Viper Venom Test (dRVVT), One-stage Factor VII, One-stage and chromogenic Factors VIII and Factor IX, and Thromboelastography (TEG); 2) to compare the effect of the branded version with those of three generic versions of apixaban (MAR- apixaban®, Apigat®, and Apismart®). Plasma from six healthy first-time donors was collected and pooled to produce platelet-poor plasma (PPP) which was spiked with five different concentrations (50 - 750 ng/ml) of each of the four apixaban versions. Each concentration was tested using apixaban-calibrated anti-Xa assay and further confirmed using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry coupled with tandem mass spectrometry (UHPLC-MS/MS). Each assay was then performed in duplicate on each concentration of each version of apixaban. Correlation testing and One-way ANOVA/Kruskal-Wallis tests were used to identify significant correlations between increasing apixaban concentrations and assay results, and any differences in mean results between the apixaban versions, respectively. A positive correlation was identified with increasing concentrations of apixaban and PT, APTT, PT-derived fibrinogen, and dRVVT assays; a negative correlation was observed with all factor assays; and no correlation was observed with Clauss Fibrinogen and TT assays. Inconsistent results were obtained for all Thromboelastography (TEG) parameters due to the high intra-assay variability. This study concluded that branded and generic versions of apixaban exerted identical in vitro effects on the different coagulation assays tested. Description: B.Sc. (Hons)(Melit.) Mon, 01 Jan 2024 00:00:00 GMT /library/oar/handle/123456789/127238 2024-01-01T00:00:00Z