Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Mar 3

Techn. Hochsch., Diss., 2003--Aachen.

Universiẗat, Diss., 2004--Kiel.

Background: COPD is associated with an increased lung macrophage burden. Whilst lung macrophages may self-renew, recruitment of peripheral blood monocytes from the systemic circulation is considered to represent their principal means of replenishment. Through modulating expression of monocytic chemokines CCL2/CCL3 and their respective receptors (CCR2/CCR1+CCR5), IL-6 could play a key role in facilitating the recruitment of monocytes to the lungs of COPD patients. COPD is associated with enhanced pulmonary and systemic IL-6 levels; concentrations of the soluble IL-6 receptor sIL-6R may be an important determinant of IL-6 signalling in COPD. Trans-signalling through sIL-6R, IL-6 may facilitate recruitment of monocytes in COPD by influencing chemokine and chemokine receptor expression. Aims: 1) To compare levels of IL-6, sIL-6R, CCL2 and CCL3 in the plasma and sputum of COPD and controls. 2) To examine of the effects of IL-6 stimulation on monocyte chemokine receptor gene expression (CCR1, CCR2 and CCR5). 3) To compare subtypes (CD14++CD16-, CD14+CD16+, CD14-CD16++) and chemokine receptor expression (CCR1, CCR2, CCR5) of monocytes in COPD (paired stable & exacerbating) and controls. 4) To compare the migratory ability of monocytes from COPD and controls. 5) To compare numbers of marginated CX3CR1+ monocytes in the pulmonary microvasculature and proliferation status (Ki67 positivity) of alveolar macrophages in COPD and controls. Methods: 1) MSD soluble marker analysis was performed on plasma and sputum supernatant. 2) Monocytes underwent stimulation with IL-6 and sIL-6R; chemokine receptor expression was determined by quantitative PCR. 3) Flow cytometry was performed on whole blood to determine monocyte subtype and chemokine receptor expression. 4) Monocyte migration towards sputum supernatant was assessed using a transwell system incorporating fluorescence based detection of DNA from migrated cells. 5) Immunofluorescence and immunohistochemistry was performed on lung tissue (obtained from patients undergoing surgical resection of lung carcinoma) to identify marginated (CX3CR1+CD14+, CX3CR1+CD16+) monocytes and proliferating alveolar macrophages (Ki67) respectively. Results and Conclusion: Levels of sIL-6R were increased in the lungs and systemic circulation of COPD patients implying potential for enhanced IL-6 trans-signalling: monocytes cultured in the presence of IL-6+sIL-6R upregulated expression of the CCR5 gene. A greater proportion of circulating COPD CD14++CD16- and CD14+CD16+ monocytes were demonstrated to express CCR5 compared to controls indicating that CCR5 ligands may have an important influence over monocyte migration in COPD. Levels of CCR5 ligand CCL3 were significantly elevated in COPD sputum supernatant; IL-6 levels were positively associated with CCL3 indicating that IL-6 trans-signalling may mediate lung chemokine expression. Nevertheless, COPD monocytes demonstrated impaired migration towards sputum supernatant and reduced margination to pulmonary microvessels. Despite this, the number of alveolar macrophages in COPD was increased; however this was not likely to be related to self-replication owing to low alveolar macrophage Ki67 expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 1 and Type 2 diabetes (T1D/T2D) share a common etiology that involves an increase in oxidative stress that leads to dysfunction and subsequent β cell death. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids to form lipid metabolites involved in a variety of biological functions including cellular oxidative stress response. On the other hand, Interleukin 6 (IL-6) signaling has been demonstrated to be protective in islets. In this study, we explored the effect of lipoxygenase enzymes 12-Lipoxygenase, 12/15 Lipoxygenase and IL-6 on β cell function and survival in mice using both STZ and high-fat diet (HFD) models of diabetes. Alox12-/- mice showed greater impairment in glucose tolerance following STZ and HFD compared to wild-type mice (WT), whereas Alox15-/- were protected against dysglycemia. These findings were accompanied by evidence of islet oxidative stress in Alox12-/- mice and reduced oxidative stress in Alox15-/- mice, consistent with alterations in the expression of antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12-/- mice showed a compensatory increase in Alox15 gene expression and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. IL-6 was able to significantly attenuate the generation of reactive oxygen species by proinflammatory cytokines in human pancreatic islets. Furthermore, we find that IL-6 regulates the master antioxidant response protein NRF2. Collectively these results show that loss of Alox12 activates a compensatory increase in Alox15 that sensitizes β cells to oxidative stress and signaling by IL-6 is required for maximal antioxidant response under conditions of increased ROS formation, such as obesity.

Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS. / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

No description available.

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

Background Nutritional treatment in the intensive care unit (ICU) has evolved from meeting nutritional requirements to manipulating patient outcome. Pharmaconutrition, referring to nutrients that are applied for their pharmacological properties, forms part of the standard nutritional care plan. The most abundant amino acid in the body, glutamine, is also the most-researched pharmaconutrient. It is an independent predictor of mortality in ICU patients, at both deficient and very high levels. Glutamine supplementation is recommended in the ICU setting for its proven outcome benefits. However, recent data showed that glutamine supplementation increases mortality risk in certain patient groups. Moreover, it suggested that not all ICU patients are glutamine deficient. Therefore, the main aim of this study was to investigate the plasma glutamine levels of adult ICU patients, on admission to the ICU. In addition, to elucidate the profile of ICU patients that can be expected to present with a glutamine deficiency or excess, with regards to gender, diagnosis and inflammatory markers. Methods In this observational, cross-sectional study, 60 mixed ICU adult patients admitted to two hospitals in the North West province were included in the study group. Blood sampling was conducted within 24 hours following ICU admission, to determine plasma glutamine, interleukin (IL)-6 and C-reactive protein (CRP) levels. Plasma glutamine levels were compared with those of a control group of healthy individuals, matched by age, race, and gender. Gender-related differences in plasma glutamine levels were investigated, as well as differences between patients with various medical conditions. The relationship between plasma glutamine levels and IL-6 or CRP was examined. Additionally, a CRP concentration cut-off point at which glutamine becomes deficient was determined by means of a receiver operating characteristic (ROC) curve. Results and discussion Intensive care unit patients had significantly lower plasma glutamine levels than healthy individuals on day one of ICU admission (p < 0.0001). However, only 38.3% (n = 23) had deficient plasma glutamine levels (< 420 μmol/L), while 6.7% (n = 4) presented with supra-normal levels (> 930 μmol/L). No significant difference could be detected between the plasma glutamine levels of male and female ICU patients (p = 0.116). Likewise, levels between diagnosis categories were also not significantly different (p = 0.325). There was a significant inverse association between plasma glutamine levels and CRP concentrations (r = -0.44, p < 0.05), and a trend towards an inverse association with IL-6 (r = - 0.23, p = 0.08). A CRP cut-off value of 95.5 mg/L was determined, above which plasma glutamine values became deficient; however, more research is needed to confirm this result. Conclusion and recommendations This research therefore showed that ICU patients, when compared with healthy individuals, had lower plasma glutamine levels on day one of admission to the ICU. However, not all were glutamine deficient, as the majority had normal and some presented with supra-normal plasma glutamine levels. An individualised approach should therefore be followed in identifying candidates for glutamine supplementation. The patients‟ condition alone may not be sufficient to predict glutamine status, but an association between plasma glutamine levels and CRP was firmly established, as well as a cut- off CRP-value above which glutamine can be expected to become deficient, which could be of use in this regard. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2015