Characterisation of Embryonic Dermal Precursor Cells

Skin is an attractive organ for the acquisition of stem cells due to its accessibility, size and potential for autologous transplants. Research into skin development has implications for the isolation of stem cell populations, for example skin-derived precursors (SKPs), as well as the treatment of skin conditions, such as fibrosis. This study centred on the early development, differentiation and stem cell potential of the dermis in embryonic mouse skin. Based on microarray data, the expression of specific Wnt family members was examined using RT-PCR and immunohistochemistry. No evidence of Wnt protein expression was observed in the dermis, but more embryonic stages and Wnt family members need to be explored to better understand Wnt signalling and its role in dermal development. Our main focus was to investigate the plasticity of the common dermal fibroblast precursor population present at E13.5. We hypothesised that the dermis contains a common precursor capable of producing all cell types of the dermis and could harbour a high proportion of mesenchymal stem cell (MSC)-like precursors. This E13.5 dermal cell (DC) population was investigated by exploring its differentiation potential when cultured in adipogenic and osteogenic media. These experiments indicated the E13.5 DCs contained a small subpopulation of MSC-like progenitors. However, when E13.5 DCs were cultured to produce SKPs and, subsequently, pushed to adipogenic and osteogenic lineages, they differentiated less than expected. Most research regarding SKPs has used adult and older embryonic skin, therefore the findings here are novel in that SKPs were not expected at a younger age. However, RT-PCR revealed differences between the gene expression profiles of early and late embryonic dermal SKPs. Moreover, neither displayed the expected differentiation potential. The possible reasons for these unexpected findings include the potential role of hair follicle induction and/or a later migration of neural crest progenitors into the dermis

Novel luminescent compounds for immunoassay

The thesis describes the synthesis of ruthenium bipyridyl and phenanthro1ine compounds that are luminescent. Several of the compounds were suitably derivatised for protein conjugation i.e. [Please see inside of thesis for formulas] The excitation and emission spectra were recorded for the above compounds and their luminescent lifetimes determined. Some of the above compounds were conjugated to antibody samples and their luminescent characteristics recorded. It was found that the complex-antibody conjugates had similar properties to their parent complexes possessing a large Stoke's Shift (about 130-140 nm) and a long luminescent lifetime (125-1200 ns). Hence these compounds may be of use as protein labels in time resolved immunoassays. The thesis also describes the synthesis of some luminescent chromium and aluminium compounds i.e. [Please see inside of thesis for formulas] Their luminescent characteristics were investigated. The preparation of several oxine derivatives was also attempted with varying success. The thesis ends with an epilogue discussing science as a God-given activity

Sepsis biomarkers in unselected patients on admission to intensive or high-dependency care

Although many sepsis biomarkers have shown promise in selected patient groups, only C-reactive protein and procalcitonin (PCT) have entered clinical practice. The aim of this study was to evaluate three promising novel sepsis biomarkers in unselected patients at admission to intensive care. We assessed the performance of pancreatic stone protein (PSP), soluble CD25 (sCD25) and heparin binding protein (HBP) in distinguishing patients with sepsis from those with a non-infective systemic inflammatory response and the ability of these markers to indicate severity of illness. METHODS: Plasma levels of the biomarkers, PCT and selected inflammatory cytokines were measured in samples taken from 219 patients during the first six hours of admission to intensive or high dependency care. Patients with a systemic inflammatory response were categorized as having sepsis or a non-infective aetiology, with or without markers of severity, using standard diagnostic criteria. RESULTS: Both PSP and sCD25 performed well as biomarkers of sepsis irrespective of severity of illness. For both markers the area under the receiver operating curve (AUC) was greater than 0.9; PSP 0.927 (0.887 to 0.968) and sCD25 0.902 (0.854 to 0.949). Procalcitonin and IL6 also performed well as markers of sepsis whilst in this intensive care unit (ICU) population, HBP did not: PCT 0.840 (0.778 to 0.901), IL6 0.805 (0.739 to 0.870) and HBP 0.607 (0.519 to 0.694). Levels of both PSP and PCT reflected severity of illness and both markers performed well in differentiating patients with severe sepsis from severely ill patients with a non-infective systemic inflammatory response: AUCs 0.955 (0.909 to 1) and 0.837 (0.732 to 0.941) respectively. Although levels of sCD25 did not correlate with severity, the addition of sCD25 to either PCT or PSP in a multivariate model improved the diagnostic accuracy of either marker alone. CONCLUSIONS: PSP and sCD25 perform well as sepsis biomarkers in patients with suspected sepsis at the time of admission to intensive or high dependency care. These markers warrant further assessment of their prognostic value. Whereas previously published data indicate HBP has clinical utility in the emergency department, it did not perform well in an intensive-care population

Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay

Background: Testosterone concentrations in normally cycling women are assumed to be elevated around the time of ovulation. The clinical relevance of changing testosterone concentrations during the menstrual cycle, however, is unclear. Poor performance of current direct immunoassays for testosterone at low concentrations confounds this issue. Therefore, our objective was to assess daily testosterone fluctuation during the menstrual cycle by a thoroughly validated isotope dilution-liquid chromatography-Tandem mass spectrometry (ID-LC-MS/MS) method and to evaluate whether an ARCHITECT® 2nd Generation Testosterone fully automated immunoassay is equally suited for this purpose. Methods: Testosterone was measured in serum obtained daily during the menstrual cycle of 25 healthy women, characterized by biochemical and physical examination. Results: Performance of the ID-LC-MS/MS method was concordant with a published reference method (y = 1.007x - 0.056 nmol/L; r = 0.9998). Comparison of the immunoassay to ID-LC-MS/MS yielded y = 1.095x + 0.104 nmol/L (r = 0.9031). Overall, testosterone concentrations were higher mid-cycle, but a peak was not discernible in each individual. Apart from a persistent positive bias, the immunoassay measured the same testosterone profiles as the ID-LC-MS/MS method. The reference interval in women was 0.30-1.69 nmol/L (8.7-48.7 ng/dL) for ID-LC-MS/MS and 0.50-2.00 nmol/L (14.4-57.7 ng/dL) for the immunoassay. Conclusion: The elevation of mid-cycle testosterone concentrations is statistically significant, although not clinically relevant since day-To-day variation is higher and independent of the menstrual cycle. In this light, a single testosterone measurement might not be reflective of the overall testosterone status in an individual. Measurements obtained using the 2nd generation immunoassay gave comparable results across the menstrual cycle. © 2012 Elsevier Inc. All rights reserved

Testosterone, free testosterone, and free androgen index in women: Reference intervals, biological variation, and diagnostic value in polycystic ovary syndrome

Objective: The objective of our study was to determine reference intervals and biologic variation for testosterone (T), free testosterone (fT), and free androgen index (FAI) in women with accurate methods and to test the discriminative value of these parameters in a polycystic ovary syndrome (PCOS)-population. Methods: Serumwas obtained daily during a normal menstrual cycle from 25 healthy women (677 data-points). A single serum sample was obtained from 44 PCOS-patients. T was measured by LC-MS/MS and by Architect® 2nd generation T Immunoassay. Sex hormone-binding globulin was measured to calculate fT and FAI. Results: Reference intervals which were established in healthy women with an ovulatory menstrual cycle were T = 0.3-1.6 nmol/L and 0.5-2.0 nmol/L, fT = 5.2-26 pmol/L and 7.2-33 pmol/L, and FAI = 0.4-2.9 and 0.6-4.4, by LC-MS/MSand immunoassay, respectively. T, fT and FAIwere higher in PCOS patients than in controls (p b 0.0001). The areas under the curve of receiver operator characteristic (ROC) plots were not different for T, fT, or FAIwhen Twasmeasured by LC-MS/MSversus immunoassay based on prediction of PCOS. FAI and fTwere the strongest predictors of PCOS. Conclusions: When based upon the appropriate reference intervals and ROC analysis, LC-MS/MS and second generation immunoassay have equivalent clinical utility for the diagnosis of PCOS