Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat Type 2 Diabetes Mellitus (T2DM). Clinical trials show that women taking RSG experience more limb fractures than patients taking other T2DM drugs. The purpose of this study is to understand how RSG (3mg/kg/day and 10mg/kg/day) and the bisphosphonate alendronate (0.7mg/kg/week) alter bone quality in the male, female and female ovariectomized (OVX) Zucker fatty rat model over a 12 week period. Bone quality was evaluated by mechanical testing of cortical and trabecular bone. Microarchitecture, bone mineral density (BMD), cortical bone porosity, bone formation/resorption and mineralization were also measured. Female OVX RSG10mg/kg rats had significantly lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented these negative effects in the OVX RSG model. Evidence of reduced bone formation and excess bone resorption was detected in female RSG-treated rats.

Rosiglitazone (RSG) is an insulin-sensitizing drug used to treat Type 2 Diabetes Mellitus (T2DM). Clinical trials show that women taking RSG experience more limb fractures than patients taking other T2DM drugs. The purpose of this study is to understand how RSG (3mg/kg/day and 10mg/kg/day) and the bisphosphonate alendronate (0.7mg/kg/week) alter bone quality in the male, female and female ovariectomized (OVX) Zucker fatty rat model over a 12 week period. Bone quality was evaluated by mechanical testing of cortical and trabecular bone. Microarchitecture, bone mineral density (BMD), cortical bone porosity, bone formation/resorption and mineralization were also measured. Female OVX RSG10mg/kg rats had significantly lower vertebral BMD and compromised trabecular architecture versus OVX controls. Increased cortical porosity and decreased mechanical properties occurred in these rats. ALN treatment prevented these negative effects in the OVX RSG model. Evidence of reduced bone formation and excess bone resorption was detected in female RSG-treated rats.

Fibroblasts continuously strive to organize and compact the surrounding extracellular matrix (ECM). Recent data suggest that this cellular contractility controls interstitial fluid homeostasis in loose connective tissues (CT). The aim of this thesis was to study the effects of prostaglandins on fibroblast contractility and to investigate whether fibroblasts in the interstitial CT surrounding the human endolymphatic duct (ED) can modulate inner ear fluid pressure and endolymph resorption. Paper I shows that prostaglandin E1 (PGE1) and prostacyclin inhibit fibroblast-mediated collagen matrix compaction in vitro and lower the interstitial fluid pressure in vivo in rat dermis. Paper II demonstrates that the inhibition of collagen matrix compaction by PGE1 is protein kinase A-dependent. Furthermore, PGE1 induces a complete but reversible actin depolymerization in human dermal fibroblasts by affecting the phosphorylation state of regulatory actin-binding proteins. Paper III describes that the cells of the interstitial CT encompassing the human ED are organized in a network based on intercellular- and cell-ECM contacts. Paper IV shows that two distinct cell phenotypes populate this interstitial CT: one expressing the lymph endothelial marker podoplanin and the other a fibroblast marker. Furthermore, CT cells isolated from human ED tissues exhibited the same tissue compacting properties in vitro as dermal fibroblasts. In conclusion, PGE1 inhibits fibroblast contractility by interfering with the stability and dynamics of the actin cytoskeleton, which leads to a loss of integrin-mediated adhesion to the ECM. These mechanisms are supposedly involved in edema formation in skin during inflammation and might be involved in the formation of endolymphatic hydrops in the inner ear of patients with Ménière’s disease.

Unrecognized metamorphic complexities can produce erroneous interpretations when using equilibrium thermodynamics and isotope geochronology. Universally employed methods for determining pressure-temperature conditions during regional metamorphism are based on the assumption of chemical equilibrium, and geochronology in metamorphic rocks can suffer from cryptic redistribution of isotopes. In this research, the scales of disequilibrium in regionally metamorphosed rocks and the effects of garnet resorption on Lu-Hf garnet ages were examined through numerical simulations of these processes. Concerning scales of disequilibrium, thirteen porphyroblastic datasets, previously measured using X-ray computed tomography, were examined by numerically simulating diffusion-controlled nucleation and growth of garnet while tracking chemical potential gradients to determine reaction affinity Ar (-[Delta]rG). Maximum nucleation rates are 10⁻¹³̇⁶-10⁻⁹̇⁸ nuclei cm⁻³ s⁻¹, interfacial energies are 0.004-0.14 J m⁻² assuming shape factors of 0.1-1.0, and Al intergranular diffusion (QD = 140 kJ/mol⁻¹) is 10⁻¹⁴̇⁴-10⁻¹¹̇¹ m² s⁻¹ at 600 °C. Limitations in determining crystallization kinetics arise from difficulties in constraining rock-specific properties (e.g., porosity and Al solubility). Ar at the time and location of nucleation is 0.4-5.9 kJ/mol⁻¹ of 12-oxygen garnet ([Delta]T = 4.0-62.0 °C) for the earliest nuclei, and 5.3-29.0 kJ/mol⁻¹ ([Delta]T = 50-125 °C) for nucleation at maximum Ar. The results demonstrate potential for delayed nucleation and metastability that can generate spurious interpretations. The timing of metamorphic events is also critical for understanding geologic history. In the Makhavinekh Lake Pluton aureole, Labrador, garnet resorption caused redistribution of Lu and loss of Hf from consumed rims, creating spuriously young ages. Garnet-ilmenite Lu-Hf geochronology using bulk separates yields apparent ages that young toward the contact from 1876 ± 21 Ma (4025 m) to 1396 ± 8 Ma (450 m). Toward the contact, garnet crystals are progressively more resorbed. Numerical modeling was used to test retention of Lu and loss of Hf during resorption as the dominant control on age. More resorption and Lu retention produce younger apparent ages (false ages). Application of the model to the aureole yields model ages from 1850 Ma to 1374 Ma, younging toward the contact. Thus, Lu-Hf geochronology applied to resorbed garnets requires careful examination of Lu zoning. / text

Patients with inflammatory or infectious conditions such as periodontitis, peri-implantitis, osteomyelitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis display varying severity of destruction in the adjacent bone tissue. Bone loss in inflammatory diseases is considered a consequence of cytokine induced RANKL and subsequent enhanced osteoclast formation. Hence, osteotropic cytokines and their receptors have been suggested to be important for the pathogenesis of inflammation-induced osteolysis. It is, here, suggested that bacterial components, so called “pathogen associated molecular patterns=PAMPs”, may also be involved. Varieties of cells express receptors for PAMPs, including Toll-like receptors (TLRs) which are the first line of defence in the innate immune system. LPS (lipopolysaccharide), fimbria and lipoproteins from pathogenic bacteria such as P. gingivalis, S. aureus are ligands for TLR2 and flagellin from pathogenic flagellated bacteria like S. typhimurium is a ligand for TLR5.   Since the susceptibility to, or the severity of inflammation-associated bone diseases are likely related to differences in the tissue response, and the mechanisms by which PAMPs interact with bone cells are not fully understood, we aimed to elucidate the importance of different TLRs for inflammation induced bone loss by conducting in vitro and in vivo investigations. Activation of TLR2 and TLR5 in organ cultured mouse parietal bones increased bone resorption in a time- and concentration-dependent manner by a process inhibited by OPG and bisphosphonate, showing the crucial role of RANKL-induced osteoclast formation. In addition, the number of osteoclasts, expression of osteoclastic genes and osteoclastogenic transcription factors were increased. In the bones and in osteoblasts isolated from the bones, TLR2 agonists increased the expression of RANKL without affecting OPG, while TLR5 activation resulted in enhanced RANKL and decreased OPG. Activation of both TLR2 and TLR5 stimulated the expression in both bones and osteoblasts of prostaglandins and pro-inflammatory cytokines, known to stimulate RANKL. By blocking the cytokines and prostaglandin, we showed that TLR2 and TLR5 induced bone resorption and RANKL expression are independent of these molecules. Activation of TLR2, but not TLR5, in mouse bone marrow macrophage cultures inhibited RANKL-induced osteoclast formation, an effect not observed in committed pre-osteoclasts. Local administration in vivo of TLR2 and TLR5 agonists on the top of mouse skull bones enhanced local and systemic osteoclast formation and bone resorption. Using knockout mice, we showed that the effects by LPS from P. gingivalis (used as TLR2 agonist) and flagellins (used as TLR5 agonists) are explicit for TLR2 and TLR5 ex vivo and in vivo, respectively. These data show that stimulation of TLR2 and TLR5 results in bone resorption in vitro and in vivo mediated by increased RANKL in osteoblasts and thus may be one mechanism for developing inflammatory bone loss. Interestingly, histological analyses of skull bones of mice treated locally with TLR2 and TLR5 agonists revealed that the bones not only reacted with locally increased osteoclastogenesis (osteoclast formation), but also with locally increased new bone formation. This was observed on both periosteal and endosteal sides of the bones, as well as in the bone marrow compartment. The formation of new bone was seen close to osteoclasts in some parts, but also in other areas, distant from these cells. The response was associated with active, cuboidal osteoblasts, extensive cell proliferation and increased expression of genes coding for bone matrix proteins and osteoblastic transcription factors. In conclusion, activation of TLR2 and TLR5 in osteoblasts results in bone loss associated with enhanced osteoclast formation and bone resorption, as well as with increased osteoblast differentiation and new bone formation, indicating that inflammation causes bone modeling. The data provide an explanation why LPS from P. gingivalis and flagellin from flagella-expressing bacteria can stimulate bone loss. Since TLR2 and TLR5 can be activated not only by bacterial components, but also by endogenous ligands produced in inflammatory processes, the data also contribute to the understanding of inflammation induced bone loss in autoimmune diseases. Hopefully, these findings will contribute to the development of treatment strategies for inflammation induced bone loss.

Age-related bone loss is a serious public health problem affecting 15 to 20 million people in the United States (1). In the last decade, several studies have investigated the effects of repeated bouts of exercise on bone density. Although this increase in bone density is mediated via hormones and/or cellular metabolites few studies have linked the increase in bone density with changes in these hormones or metabolites. Therefore, the purpose of this study was to determine the effect of a single bout of weight bearing exercise on the growth factors that alter bone metabolism. Seven healthy female subjects (age 23 ± 5 yrs.) agreed to participate in this study. A V02 max test was administered to each subject to determine their overall fitness level. A treadmill speed corresponding to 70% of their V02 max was then developed and the subjects ran for 40 minm on the treadmill at that speed. Blood samples were drawn pre and 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 min post-exercise and assayed for serum growth hormone, calcium, parathyroid hormone, and alkaline phosphatase. No significant change was found in parathyroid hormone, calcium, or alkaline phosphatase serum levels. Growth hormone was found to be significantly decreased 30, 45, and 60 min. post-exercise as compared to pre-exercise levels (7.089, 5.399, 4.728, and 11.28 ng/mL, respectively). While this indicates that exercise may cause a depression in growth hormone levels during recovery, the elevated pre-exercise levels of three of the seven subjects masked the exercise stimulated release of growth hormone. Growth hormone stimulates bone absorption via insulin-like growth factors and the GH data suggests that these hormones may have been released well after our last measurement time point. In conchusion, 40 minutes of running exercise at 70% of V02 max does not produce a change in any of the hormones or metabolic factors directly associated with calcium turnover in the bone, suggesting that acute exercise has no immediate effect on bone metabolism. However, the exercise protocol did stimulate GH release which influences bone growth indirectly by its ability to regulate IGF release. Therefore, even though no acute effects were evident, a single bout of exercise may alter the long term control of bone metabolism. / School of Physical Education

Aus der Anwendung von Arzneimitteln bei Lebensmittel liefernden Tieren erwächst das Problem der Rückstandsbelastung tierischer Lebensmittel. Bei der Sicherheitsbewertung von Rückständen im Rahmen der Zulassung von Tierarzneimitteln wird der Aspekt des Einflusses von Verdauungsprozessen auf die Struktur und Bioverfügbarkeit von Rückständen beim Endverbraucher Mensch bisher nicht berücksichtigt. Ziel der vorliegenden Arbeit war es, das präsystemische Schicksal proteingebundener Arzneitmittelrückstände zu untersuchen. Da native Arzneitmittelrückstände nach Art und Menge schwer zu erfassen sind, wurde mit halbsynthetischen Rückständen gearbeitet, die durch Inkubation mit Rattenlebermikrosomen mit der Modellsubstanz Furazolidon gebildet wurden. Nach der Inkubation wurden freie und schwach gebundene Metaboliten durch Lösungsmittelextraktion entfernt, um nur die fest an Proteine gebundenen Rückstände zu gewinnen. Diese proteingebundenen Rückstände wurden in vitro mithilfe von Salzsäure und Pronase E verdaut. Die intestinale Bioverfügbarkeit der verdauten Rückstände wurde im isoliert perfundierten Darm untersucht. Zur Verifizierung der In-vitro-Befunde wurde die Bioverfügbarkeit der gebundenen Furazolidonrückstände außerdem bei Ratten untersucht. Der quantitative Nachweis der proteingebundenen Furazolidonrückstände erfolgte hochdruckflüssigkeitschromatographisch. Es wurden nur Rückstände erfasst, die die intakte Seitenkette des Furazolidons, das 3-Amino-2-oxazolidinon (AOZ), enthielten. Der Nachweis erfordert eine Derivatisierung des AOZ mit Nitrobenzaldehyd unter sauren Bedingungen. Während der Derivatisierung wird AOZ aus den Rückständen säurehydrolytisch freigesetzt. In der vorliegenden Arbeit wurde eine Methode wurde eine Methode entwickelt, die die sichere Unterscheidung zwischen freiem und gebundenem AOZ ermöglicht, da AOZ auch bei der Verdauung der Rückstände abgespalten wird. Nach In-vitro-Verdauung wurden zirka 80% der Rückstände als freies AOZ und nur noch 20% in der ursprünglichen gebundenen Form gemessen. Perfusionsversuche mit reinem AOZ zeigten, dass die Seitenkette des Furazolidons intestinal bioavearfügbar ist. Es scheint, dass AOZ die Darmwand durch passive Diffusion überwindet, da sich im Verlauf der zweistündigen Perfusion in der resorbierten Flüssigkeit eine AOZ-Konzentration aufbaute, die annährend so hoch war wie im Perfusionsmedium. Untersuchungen zur Biotransformation von AOZ in isolierten Enterozyten und Rattendarmmikrosomen gaben keine Hinweise auf einen intestinalen Abbau des AOZ. Hingegen deuten ein geringfügiger Abfall von AOZ in isolierten Hepatozyten und ein limitierter, jedoch signifikanter Abfall in Rattenlebermikrosomen auf hepatische Verstoffwechslung hin. Die Perfusion mit verdauten Furazolidonrückständen zeigte, dass proteingebunde Rückstände die Darmwand mit großer Wahrscheinlichkeit nicht überwinden konnten. Im Unterschied dazu wurde das während der Verdauung freigesetzte AOZ resorbiert. Fütterungsversuche mit Ratten zeigten, dass auch die natürliche Verdauung zur Freisetzung von AOZ aus Rückständen führt. Messungen in Geweben, Organen und Körperflüssigkeiten der Ratten ergaben, dass wahrscheinlich nur freies AOZ resorbiert wird, jedoch keine proteingebundenen Rückstände resorbiert werden und dass AOZ in unveränderter Form renal eliminiert werden kann. Quantitative Aussagen ließen sich aus den Versuchen nicht ableiten. Aus den Befunden wurde geschlossen, dass proteingebundene Arzneimittelrückstände im Gastrointestinaltrakt modifiziert werden können, so dass Verbindungen entstehen, die in tierischen Produkt noch nicht vorhanden sind. Die kovalente Bindung ist offensichtlich nicht der Endpunkt im Prozess der Rückstandsbildung, wie es einige Autoren postulieren. Die neuen Moleküle können bioverfügbar sein. Bei der Sicherheitsbewertung von Arzneimittelrückständen kann nicht a priori angenommen werden, dass sie pharmakologisch und toxikologisch unwirksam sind, da sie eliminiert werden müssen und wahrscheinlich auch biotransformiert werden. Im Fall von Furazolidon ist die wasserlösliche Seitenkette AOZ ein "neuer" Metabolit, der partiell bioverfügbar ist und möglicherweise in geringem Maße verstoffwechselt wird. Die Ausscheidung erfolgt renal, eventuell auch über andere Wege. Der Teil der proteingebundenen Rückstände, der die saure Hydrolyse im Magen übersteht, scheint hingegen im Darm nicht in nenneswerten Maße resorbiert zu werden. Dies könnte auch für den proteingebundenen Restrückstand, der nach Abspaltung von AOZ übrig bleibt, gelten. Zusammenfassend bleibt festzuhalten, dass mit den verwendeten In-vitro-Modellen eine quantitative Aussage über das Schicksal proteingebundener Rückstände im Organismus zu erzielen ist. / The use of veterinary drugs in food producing animals give rise to contamination of animal food with drug residues. At present, the influence of digestion on structure and bioavailability of residues in humans has not been considered within the scope of saftety evaluation of residues during veterinary drug authorization. The aim of this study is the investigation of the presystemic fate of protein-bound drug residiues.

[Truncated abstract] Bone remodeling is intricately regulated by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The elevation in osteoclast number and/or activity is a major hallmark of several common pathological bone disorders including post-menopausal osteoporosis, osteoarthritis, Paget's disease, and tumour-mediated osteolysis. Receptor activator of nuclear factor kappa B ligand (RANKL) is a key cytokine for osteoclast differentiation and activation. The association of RANKL to its cognate receptor, RANK, which is expressed on osteoclast precursors and mature osteoclasts, is essential for osteoclast formation and activation. The intimate interaction between RANKL and RANK triggers the activation of a cascade of signal transduction pathways including NF-κB, NFAT, MAPK and PI3 kinase. Although osteoclast signaling pathways have been intensively studied, the precise molecules and signaling events which underlie osteoclast differentiation and function remain unclear. In order to dissect the molecular mechanism(s) regulating osteoclast differentiation and activity, this thesis herein explores the key RANKL/RANK-mediated signaling pathways. Four truncation mutants within the TNF-like domain of RANKL [(aa160-302), (aa160-268), (aa239-318) and (aa246-318)] were generated to investigate their potential binding to RANK and the activation to RANK-signal transduction pathways. All were found to differentially impair osteoclastogenesis and bone resorption as compared to the wild-type RANKL. The impaired function of the truncation mutants of RANKL on osteoclast formation and function correlates with their reduced ability to activate crucial RANK signaling including NF-κB, IκBα, ERK and JNK. Further analysis revealed that the truncation mutants of RANKL exhibited differentially affinity to RANK as observed by in vitro pull-down assays. ... It is possible that Bryostatin 1 acts via upregulation of a fusion mechanism as the RANKL-induced OCLs are morphologically enlarged, exhibiting increased nuclei number expressing high level of DC-Stamp. Furthermore, Rottlerin was shown to inhibit NF-κB activity, whereas Bryostatin 1 showed the opposing effects. Both inhibitor and activator were also found to modulate other key osteoclastic signaling pathways including NFAT and total c-SRC. These findings implicate, for the first time, Protein Kinase C delta signaling pathways in the modulation of RANKL-induced osteoclast differentiation and activity. Taken together, the studies presented in this thesis provide compelling molecular, biochemical and morphological evidence to suggest that: (1) RANKL mutants might potentially serve as peptide mimic to inhibit RANKL-induced signaling, osteoclastogenesis and bone resorption. (2) A cross talk mechanism between extracellular Ca2+ and RANKL exist to regulate on osteoclast survival. (3) TPA suppressed RANKL-induced osteoclastogenesis predominantly during the early stage of osteoclast differentiation via modulation of NF-κB. (4) Selective inhibition of Protein Kinase C signaling pathways involved in osteoclastogenesis might be a potential treatment method for osteoclast-related bone diseases. (5) Protein Kinase C delta signaling pathways play a key role in regulating osteoclast formation and function.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.