Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2005. / "A dissertation in pharmacology and pharmaceutical sciences." Typescript. Advisor: Deborah A. Scheuer. Vita. Title from "catalog record" of the print edition Description based on contents viewed May 31, 2006. Includes bibliographical references (leaves 238-274). Online version of the print edition.

Topical glucocorticoids (GCs), such as hydrocortisone (HC), are the main drugs used to treat inflammatory skin conditions including eczema and psoriasis, but their longterm use is limited by the onset of side effects such as skin thinning, impairment of wound healing and systemic metabolic dysfunction. For this reason, there is a substantial need for new compounds with the same anti-inflammatory effects but fewer adverse effects. Previous studies have suggested 5α-tetrahydrocorticosterone (5α-THB) as a new, more selective anti-inflammatory compound; this steroid is a metabolite of the major endogenous GC in rodents, corticosterone (B). Preliminary data indicated that 5α-THB may be as effective as HC in reducing mouse irritant dermatitis, but without the local or systemic side effects of HC. The aim of this thesis is to investigate the mechanisms through which 5α-THB delivers more selective anti-inflammatory effects, with the hypothesis that 5α-THB influences distinct signalling pathways from those of B. A mouse model of irritant dermatitis induced by topical application of croton oil on the ear was developed, and the anti-inflammatory properties of 5α-THB were analysed, in comparison with those of B, after 6 and 24 hours of treatment. In inflamed tissue, B reduced tissue oedema and cell infiltration at both time points; in contrast, 5α-THB did so at 24 but not 6 hours, at a dose five-fold higher than B. Real-time analysis at 24 hours showed that B and 5α-THB similarly reduced the croton oil-induced increase of transcripts of genes encoding vascular and cellular adhesion molecules. Interestingly, while B did not affect the abundance of transcripts of the anti-inflammatory gene Dusp1, 5α-THB increased it in croton oil-treated ears, suggesting a different mechanism of action between 5α-THB and B. The experiment was repeated with the injection of the glucocorticoid receptor (GR) antagonist RU486; RU486 relieved the effect of B on swelling but did not attenuate the anti-inflammatory effects of 5α-THB, indicating a further important difference between the two steroids. Angiogenesis is fundamental for the healing process, and it is known that topical GCs impair wound healing in part by inhibiting angiogenesis; for this reason, the effects of 5α-THB on the formation of new vessels, in comparison with B, were tested in a mouse model of inflammatory angiogenesis induced by sub-cutaneous implantation of polyurethane sponges. 5α-THB, at equipotent doses to B for the reduction of macrophage infiltration, inhibited angiogenesis to a lesser extent than its precursor. In addition, B had systemic effects in that it lowered adrenal gland weights, whereas 5α-THB did not. Histological analysis suggested that while B inhibits formation and maturation of new vessels, 5α-THB may affect only the former process. Molecular analysis showed that B reduced the abundance of transcripts of the majority of the tested genes involved in inflammation, angiogenesis and tissue remodelling, but 5α-THB had more selective effects. Ex vivo studies in mouse bone marrow-derived macrophages stimulated with LPS showed that 5α-THB inhibited release of pro-inflammatory cytokines in a weaker manner compared with B. This inhibition was partially prevented by co-incubation of RU486 with B but not with 5α-THB. In in vitro studies, molecular pathways activated by B and associated with adverse side effects were only weakly activated by 5α-THB. In particular, 5α-THB only weakly induced phosphorylation of GR, and activation of expression of GC-responsive reporter plasmids and endogenous metabolic genes. Interestingly, 5α-THB reduced B-induced trans-activation of some of these genes. In summary, 5α-THB effectively reduces skin inflammation, but, unlike B, has only moderate anti-angiogenic properties, and weakly activates molecular mechanisms associated with adverse metabolic side effects. Most importantly, its action may not be due to activation of GR. This work opens the intriguing possibility that GCs work through mechanisms not yet investigated, and this may be of pivotal importance in the search for new safer anti-inflammatory compounds.

In mammals endogenous, self sustained oscillators, known as circadian clocks, have evolved as a result of day night cycles, with a period close to 24 hours, and are involved in many physiological processes; such as sleep wake cycles, metabolic and hormonal activity. The suprachiasmatic nucleus (SCN), is the central oscillator, and is synchronised to the external environment by light, via the eye. It has been demonstrated that peripheral clocks, too, contain the circadian oscillator, with tissues such as the lung, liver, heart and kidney as well as many isolated cell types remaining rhythmic, in culture, for many days. However, these peripheral oscillators require a signal from the central oscillator in order to co-ordinate a synchronised time. Leading candidates in the relay of this information are the circulating glucocorticoid hormones corticosterone (rodents) or cortisol (man), which are known to have potent effects on the peripheral clock, both in-vivo and in-vitro. Further to this, glucocorticoids have been used for many decades to suppress the symptoms of inflammation, a by product of many human diseases.This thesis aims to address the temporal regulation of the peripheral clock by the endogenous glucocorticoid, corticosterone, using a transgenic mouse harbouring a luciferase conjugated clock reporter, and circadian reporter cell lines. It also aims to address the relative contribution of the two closely related nuclear hormone receptors, the glucocorticoid and mineralocorticoid receptors. A further aim of the work with glucocorticoid signalling was to design a flow-though culture system, in order to address the effects of the endogenous pulsatile release of glucocorticoids on the peripheral oscillator. This thesis also aims to characterise the inflammatory response in relation to its circadian characteristics; its relationship with corticosterone and the effect of inflammation on the central clock components. Finally, this thesis aims to investigate a potential input/output of the clock, a member of the family of C/EBP transcription factors, C/EBP alpha, and whether it is under endogenous circadian control and regulated by glucocorticoids.Work in this thesis has shown that glucocorticoids dynamically regulate the peripheral clock at all phases of the circadian cycle and that this regulation occurs mainly through the glucocorticoid receptor; yet the mineralocorticoid receptor does have a function in the immediate response to glucocorticoid administration. Furthermore, as a result of the initial temporal profile after corticosterone addition, on the clock protein PERIOD2, I have shown transient regulation of the clock through Caveolin-1 based signalling. There is also a significant circadian component to the inflammatory response, which appears, at least in part, to be REV-ERB alpha mediated, and the inflammatory response also has profound effects on circadian gene expression in the periphery. A functional flow-through system was designed and a working model produced, albeit with technical difficulties, to address glucocorticoid pulsing and circadian timing but much more work is needed for effects to be fully understood. C/EBP alpha appears not to be under circadian regulation nor under direct glucocorticoid regulation, at least in peripheral models used here.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

No description available.

Glucocorticoids profoundly influence the immune system and pharmacological doses exert potent anti-inflammatory actions. During inflammation, glucocorticoids limit oedema and influence cell trafficking, differentiation programmes and gene transcription in glucocorticoid-sensitive leukocytes. Within cells, glucocorticoid action is modulated by a pre-receptor mechanism; glucocorticoid metabolism by the enzyme 11β- hydroxysteroid dehydrogenase (11β-HSD). Two 11β-HSD isozymes exist: 11β-HSD1, which catalyses amplification of glucocorticoid levels in intact cells by oxo-reduction of intrinsically inert cortisone (11-dehydrocorticosterone in rodents) into active cortisol (corticosterone in rodents) and 11β-HSD2, which performs the opposite reaction. Thus, amplification of intracellular glucocorticoid levels by 11β-HSD1 may represent an endogenous anti-inflammatory mechanism. This hypothesis has been tested in Hsd11b1-/- mice (homozygous for a targeted disruption in the Hsd11b1 gene, encoding 11β-HSD1), using carageenan-induced pleurisy and experimental model of arthritis induced by injection of arthritogenic antibodies. In both models, Hsd11b1-/- mice showed more severe acute inflammation than control mice. During carrageenan-induced pleurisy, Hsd11b1-/- mice recruited more inflammatory cells to the pleural cavity than congenic controls, with a greater proportion of viable cells, at the onset and peak of pleurisy, suggesting a worse inflammatory response. Histological examination suggested impaired resolution of inflammation in Hsd11b1-/- mice with persistence of inflammation in the visceral pleura, activation of lymphoid aggregates, and uniquely in Hsd11b1-/- mice, formation of fibrous adhesions between lung lobes 48h after initiation of pleurisy. During experimental arthritis induced by injection of serum from arthritic K/BxN mice, clinical signs of inflammation occurred earlier in Hsd11b1-/- mice and were slower to resolve than in control mice. Histological assessment of the acute phase (2d) of arthritis showed no difference in joint pathology between genotypes, despite greater oedema and higher clinical scores in the Hsd11b1-/- mice. However, when the inflammation had resolved (21d following injection of serum), compared to control mice, Hsd11b1-/- mice showed more severe exostosis, intense periarticular inflammation, more collagen deposition and uniquely, ganglion cyst formation. At 21d, whereas basal (morning) plasma corticosterone levels were normal in control mice, they remained elevated in Hsd11b1-/- mice, suggesting ongoing inflammation and persistent activation of the hypothalamic-pituitary-adrenal axis. Mast cells are critical in the initiation of an inflammatory response and are essential in this model of arthritis. Mast cells expressed 11β-HSD1 (but not 11β-HSD2) mRNA and activity. Although mast cell number did not differ in joints or peritoneum of Hsd11b1-/- mice, 11-HSD1-deficient mast cells had a lower threshold for degranulation induced by K/BxN arthritogenic serum. As well as implicating a role for mast cell 11β-HSD1 in limiting initial inflammation in arthritis, these findings also have implications for infection, allergy and tolerance. Collectively, these data suggest that 11β-HSD1 deficiency worsens acute inflammation and results in slower resolution. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the acute inflammatory response and programme its subsequent resolution. Increasing leukocyte 11β-HSD1 or local delivery of substrate affords a novel approach for anti-inflammatory therapy.

No description available.

No description available.

Haemostasis is an important aspect in neurosurgical operations for the achievement of good outcome. Bipolar coagulation is an extensively used haemostatic technique in modern neurosurgery but it may also cause iatrogenic brain trauma due to thermal injury. Published studies on coagulation-induced brain injury on a histological level are, however, limited. The present study aimed at investigating the extent of inflammatory and glial responses caused by different settings of bipolar coagulation using an animal model. It also investigated whether and how pre-operative treatment with dexamethasone or progesterone, both known to have neuro-protective effects, would modulate gliosis and macrophage infiltration induced by bipolar coagulation. The study consisted of two parts. The first part investigated the astrocytic and macrophage responses after bipolar coagulation at different power settings. 45 Sprague-Dawley rats received craniotomy, followed by bipolar coagulation at different power output settings (mock operation as control, 20W and 40W) over the rat cortex for a standardized duration of two seconds. On day 3, day 7 and day 28, brain sections were assessed by immunohistochemical staining for GFAP (astrocytes) and ED1 (macrophages). Quantification of outcome by random field cell counting under light microscopy was performed. The second part of the study used another 45 male Sprague-Dawley rats, divided into three treatment groups: Group 1 received the vehicle agents only (Control); Group 2 received progesterone 20mg/kg; Group 3 received dexamethasone 1 mg/kg. All treatments were given intraperitoneally two hours before craniotomy. The animals received bipolar coagulation at 40W for a standardized duration of two seconds. On day 1, 3 and 7, brain sections were assessed by immunohistochemical staining for GFAP and ED1. Quantification of outcome by random field cell counting under light microscopy was performed. T2-weighted magnetic resonance imaging for the animals on day 3 was also performed. The results showed that bipolar coagulation was associated with significant glial and inflammatory responses that correlated with power output. Progesterone and dexamethasone were both effective in reducing the glial hypertrophy and macrophage infiltration associated with bipolar coagulation. Dexamethasone had an additional advantage of reducing brain oedema and cavity formation. The findings suggested that progesterone and dexamethasone could be further explored as potential protective and/or remedial agents for bipolar coagulation-induced brain trauma sustained during neurosurgical procedures. / published_or_final_version / Surgery / Master / Master of Research in Medicine

Long-term glucocorticoid use leads to glucocorticoid-induced osteoporosis (GIOP) and fractures which require proper management. Little is known about the "real-world," long-term costs and effectiveness of anti-osteoporotic treatments. A retrospective analysis of data from the 1996-2004 Medical Expenditure Panel Survey was conducted to evaluate the "real-world" outcomes. Markov modeling with Monte Carlo simulations was used to yield long-term estimates of these outcomes. A total of 5,461 subjects met the study criteria for long-term glucocorticoid users (LTGS, average prednisone-equivalent dose=11.0 mg/day, average length=237 days), which represents 2.3% of the non-institutional U.S. population. The study subjects tended to be middle-aged (49.7 years old), female (61.4%) and white (86.2%). Overall 22.4% of LTGS users reported use of any anti-osteoporotic agent. Hormone replacement therapy (HRT) was the most frequently used in women followed by bisphosphonates, while bisphosphonates and calcitonin were used by men. Analyses of variance indicated some significant differences in characteristics of LTGS users among treatment groups which suggest a selection bias. Female LTGS users had higher prevalence rates (6.8%) of osteoporosis than males (1.0%), but the prevalence rates of osteoporotic fractures were similar (3.0%). The logistic regression analyses indicated that the use of oral glucocorticoid tablets does not significantly change the odds of osteoporotic fractures in study subjects (relative risk (RR)=1.146, 95% confidence interval (CI) 0.901-1.458 for subjects in the WELL state; RR=0.55, 95% CI 0.188-1.621 for subjects in the GIOP state; RR=1.241, 95% CI 0.532-2.893 for subjects in the GIFX state). The estimated 10-year and lifetime incremental cost per osteoporotic fracture avoided are $27,253-$35,692 (10-year) and $84,942-$91,075 (lifetime) in hypothetical female glucocorticoid users. HRT is the most cost-effective option for hypothetical females except that calcitonin is preferred for 65-year-old females receiving lifetime treatments. When HRT is excluded, calcitonin is the next most cost-effective option except that raloxifene is preferred for 30- and 50-year-old females receiving 10-year treatments. Calcitonin is the most cost-effective option for male glucocorticoid users. Bisphosphonates are less cost-effective which may be due to selection bias. Anti-osteoporotic treatments are recommended for all long-term glucocorticoid users, but the preferred option depends on gender, age, length of treatments and budgets.