Les alphavirus arthritogènes de la rivière Ross (RRV) et du chikungunya (CHIKV) sont des arbovirus à l’origine de maladies inflammatoires musculosquelettiques chez l'homme. Ils sont largement distribués dans le monde et provoquent périodiquement des épidémies explosives. Les principaux signes cliniques lors d’une infection par un alphavirus arthritogène sont les myalgies, polyarthrites et arthralgies intenses pouvant persister plusieurs mois après l'infection. Les mécanismes de développement de l’infection et des manifestations persistantes sont peu connus. Pour étudier la pathogenèse de l'infection par RRV, nous avons généré un virus recombinant exprimant une nouvelle luciférase brillante et brillante. Nous avons montré que les monocytes humains, malgré une faible susceptibilité à l'infection in vitro par RRV, étaient capables de maintenir une réplication virale jusqu'à 45 jours post infection indiquant leur rôle potentiel dans les formes chroniques. Grâce un modèle expérimental de l’infection par RRV, nous avons suivi les phases aiguë et chronique de la maladie in vivo. Nous avons montré que les cinétiques de réplication du virus recombinant étaient proches de celles du virus parental. Nous avons également observé un tropisme musculaire et articulaire et une corrélation entre le signal bioluminescent et la charge virale confirmant ainsi la relevance de ce modèle. En étudiant la dissémination virale, nous avons montré que le Bindarit, une molécule anti-inflammatoire diminuant le développement de la maladie dans le modèle murin, induit une plus grande réplication dans le tissu cardiaque. Enfin, nous avons pu observer une réplication virale dans les tissus musculaires durant la phase chronique de la maladie et avons montré le rôle de la dose inoculée dans le développement de la persistance virale. Suite à un traitement immunosuppresseur, nous avons observé une légère augmentation du signal bioluminescent indiquant un contrôle de la réplication virale persistante par la réponse immunitaire adaptative. Ce nouveau modèle d’imagerie in vivo permet un suivi en temps réel de la dissémination virale permettant des études de pathogenèse et l'évaluation de stratégies thérapeutiques. / Ross River virus (RRV) and chikungunya virus (CHIKV) are mosquito-transmitted viruses that cause musculoskeletal inflammatory diseases in humans. They are widely distributed and periodically cause explosive epidemics. After infection with RRV, patients experience fever, maculopapular rash, myalgia and intense pain in the peripheral joints. Approximately 30% of patients develop a chronic form of the disease with myalgia and poly-arthralgia persisting for months to years after infection. The mechanisms underlying these persistent symptoms remain unclear. To study the dynamics and pathogenesis of RRV infection in vitro and in living animals, we generated a recombinant virus expressing a novel small and bright luciferase. First we showed that human monocytes, despite a low susceptibility to RRV infection, were able to maintain viral replication in vitro up to 45 days post infection. Then, using a murine model of RRV infection, we monitored the acute and chronic phases of the disease. We observed near native replication kinetics and a muscular/articular tropism after infection with our recombinant virus. Moreover, the bioluminescent signal correlated with the viral load further confirming the relevance of this new imaging model. After monitoring of the viral dissemination in live mice, we showed that Bindarit, an anti-inflammatory molecule known to prevent the development of the alphaviral disease in a mouse model, induces a higher replication in the cardiac tissue; thereby indicating that caution must be used before treatment of patients. We were also able to observe viral replication in the muscles during the chronic stage of the disease when using a low inoculation dose. Finally, following an immunosuppressive treatment, we observed a slight increase in the bioluminescent signal indicating a control of remnant viral replication by the adaptive immune response. This new model provides a non-invasive real-time assessment of viral replication and dissemination allowing pathogenesis studies and therapeutic strategies evaluation.

In patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth.

Exercise tolerance, measured as peak oxygen consumption (VO₂ peak), is very low in patients with end-stage renal failure undergoing maintenance haemodialysis. Due to their associated anaemia and low peak heart rates during maximal exercise it has been argued that the reduced blood oxygen carrying capacity and central cardiovascular limitations are primarily responsible for the poor exercise tolerance of these patients. However, others suggest that peripheral (skeletal muscle) limitations including impaired substrate utilization, muscle weakness caused by peripheral neuropathy and myopathy, malnutrition and general physical deconditioning are responsible for the poor exercise tolerance. The present thesis was therefore designed to study whether central cardiovascular function or anaemia or muscle weakness causes patients with end-stage renal failure to terminate exercise at workrates well below those achieved by healthy controls.

Coronary artery disease has become a world wide medical problem. There is an overwhelming association between coronary atherosclerosis, angina pectoris, acute myocardial infarction and sudden death. The narrowing of coronary arteries is thought to damage the heart by limiting appropriate changes in coronary blood flow and by causing myocardial ischemia. This thesis attempts to examine the coronary circulation in patients who present with chest pain with and without coronary artery disease. One of the aims of this thesis is to validate the use of a short-lived radionuclide to study changes in regional myocardial perfusion. This technique has been applied in clinical medicine in an attempt to describe the disturbances of regional myocardial perfusion that occur in patients with coronary artery disease. These disturbances of perfusion have been related to the patients' symptoms, the electrocardiogram and the stenosed arteries seen in the arteriogram. Krypton-81m in solution is an inert freely diffusible gas (half-life 13 seconds) which emits a single 190 kev gamma ray. This tracer, a special catheter and a gamma camera have been developed in experiments to measure changes in regional myocardial perfusion. The systematic and rand-Om errors of the method have been defined in experiments. The results show that the mixing and delivered arterial concentration of krypton-81m are stable within a useful physiological range of changes in heart rate, blood pressure and coronary blood flow. Correlations with a reference technique have shown that the method can measure changes in regional myocardial perfusion between O and 3 ml/ml/min. The invasive method, the planar imaging and the need for calibration with washout at high levels of perfusion are investigated and described as limitations that must be considered. Eighty patients presenting with chest pain have been investigated by routine clinical methods, precordial mapping of the electrocardiogram during exercise and coronary arteriography. Changes in regional myocardial perfusion at rest and during atrial pacing has been measured using krypton-81m. The results have shown that stable mixing and delivered arterial concentration of krypton-81m can be achieved in the patients. Fifteen patients with negative exercise tests all demonstrated uniform increases in regional myocardial perfusion with pacing. The remaining 65 patients with positive exercise tests and significant coronary artery disease all showed both regional increases and decreases in myocardial perfusion during atrial pacing. In 16 of the 65 patients the jeopardized segment of ventricular myocardium showed significant increases in perfusion during the first 4 to 7 minutes of pacing. Th e increase stopped and regional perfusion in the affected segment then decreased progressively until the pacing was stopped. In 23 of the 65 patients the affected segment showed no changes in perfusion for 5 to 7 minutes of atrial pacing and then showed progressive decreases in regional myocardial perfusion until the pacing was stopped. Finally, in 26 of the 65 patients the affected segment showed immediate and progressive decreases of regional myocardial perfusion starting with the commencement of atrial pacing. In all the patients with disturbed perfusion ST segment depression in the electrocardiogram appeared after (140 ± 14 sec) the regional decrease of myocardial perfusion in the affected segment. Chest pain always appeared later at 220 ± 19 sec after the appearance of disturbed myocardial perfusion. Regional myocardial perfusion returned to normal in all the patients after the atrial pacing was stopped. There was a spatial relationship between the region of the ventricles affected by disturbed perfusion and the region of the precordium showing abnormal electrocardiographic signs during the exercise test. In conclusion, this clinical study has shown that patients with chest pain who have coronary artery disease suffer decreases of regional myocardial perfusion in affected segments of the ventricles during episodes of angina pectoris induced by atrial pacing. Regional perfusion may increase, remain stable or decrease in the affected segment following the onset of a stress test such as atrial pacing. This probably represents the amount of reserve function and adaptation left in the diseased coronary circulation and may be a useful physiological indicator of the severity of coronary disease and of patients at high risk. ST segment depression and pain have a close temporal relationship to the decreases of regional myocardial perfusion that occur in these patients. These studies suggest that there is a close relationship between myocardial perfusion and metabolism in health and disease. Both myocardial perfusion and metabolism will have to be affected by any rational therapy for angina pectoris and ischemic heart disease.

Fatigue and exercise intolerance are common symptoms experienced by patients with chronic heart failure (CHF). Historically it has been argued that central cardiopulmonary factors including pulmonary congestion and reduced lung compliance cause dyspnoea that limits the exercise tolerance of such patients. But recent studies have indicated that exercise capacity in patients with CHF may not be limited solely by central cardiorespiratory factors. Rather the focus has shifted to aspects of the peripheral circulation and skeletal muscle function as possible factors limiting the exercise tolerance of patients with CHF. However there are few studies describing both the structural and functional abnormalities in the skeletal muscle of patients with CHF. In the first study of this dissertation, 11 patients with end-stage heart failure (NYHA class Ill-IV) and 10 healthy control subjects (C) underwent i) graded exercise to exhaustion for determination of peak oxygen consumption (VO₂ peak) and peak work load (Wlpeak); ii) isometric and isokinetic tests of skeletal muscle function and iii) radionuclide angiography for determination of ejection fraction (EF%). VO₂ peak (12.5 ± 1.0 vs 34.3 ± 3.5 mlO₂fkg/min; p<0.001), Wlpeak (73 ± 10 vs 224 ± 14 W; p<0.001), total work performed by the quadriceps muscles (TWQ) in a 30 sec isokinetic test (TWQ; 1565 ± 166 vs 2892 ± 345 J; p<0.05), and hamstring muscles (TWH) (TWH; 604 ± 163 vs 2003 ± 326 J; p<0.05), maximum voluntary isometric contraction (MVC) of the quadriceps muscles (MVC; 134 ± 12 vs 194 ± 11 Nm; p<0.001) and isokinetic peak torque of the ~uadriceps (PKTQ) (PKTQ; 133 ± 15 vs 203 ± 23 Nm; p<0.05) and hamstring muscles (PKTH) (PKTH; 60 ± 8 vs 108 ± 16 Nm; p<0.05) and time to fatigue during a test of isometric endurance (68 ± 12 vs 100 ± 10 sec; p<0.05) were all significantly lower in patients with CHF. However when corrected for the reduced lean thigh volume (muscle mass) in patients with CHF, PKTQ, PKTH and MVC were no longer different from control values. But the total work performed by the quadriceps and hamstring muscles in a 30 second isokinetic test was reduced even when corrected for the reduced lean thigh volume in patients with CHF. Furthermore, patients with CHF terminated progressive cycle exercise to exhaustion at heart rates, rates of ventilation, respiratory exchange ratios and blood lactate concentrations that were significantly lower than values achieved by control subjects during maximal dynamic exercise. These data suggest that skeletal muscle functional abnormalities including a decreased resistance to the development of fatigue exist in patients with severe CHF. In the second study of this dissertation, 10 patients with CHF who participated in the first study and eight control subjects underwent a skeletal muscle biopsy of the vastus lateralis muscle for light and electron microscopic analysis. Significant histological and ultrastructural changes were found in all SM biopsies from patients with CHF. These included atrophy and hypertrophy of fibres, fibre splitting, internalized nuclei, nuclear knots, moth-eaten fibres, increased lipid droplets. Electron microscopy showed a large variety of nonspecific abnormalities, including mitochondrial changes, Z-band degeneration and accumulation of intracellular glycogen. Ultrastructural morphometry revealed capillary basement membrane width significantly increased in the SM of patients with CHF, (409 ± 13 vs 121 ± 3 nm; p<0.01). A novel, blinded, impartially scored method for grading SM pathology showed that SM biopsies of patients with CHF had higher scores for myopathic changes compared to C (12.0 ± 1.5 vs 1.6 ± 1.0 arbitrary units; p<0.05). SM pathology score correlated significantly with VO₂ peak, Wlpeak, and TWQ (p<0.05 to p<0.02) but not with EF%. EF% did not correlate with either VO₂ peak, Wlpeak or TWQ. These data support the hypothesis that: i) severe SM structural and functional abnormalities may limit exercise capacity in patients with CHF; ii) the severity of SM pathology but not resting systolic cardiac function, predicts exercise performance in patients with CHF.

No description available.

No description available.

Um estudo prévio demonstrou em pacientes com choque séptico a presença de exossomos plasmáticos. Nossa hipótese é que exossomos circulantes poderiam contribuir para a disfunção contrátil da sepse. Foi colhido sangue de 55 pacientes com choque séptico. A incubação de corações isolados com exossomos de pacientes sépticos reduziu significantemente as derivadas da pressão ventricular e aumentou as concentrações de nitrato no miocárdio. A exposição de músculos papilares a exossomos reduziu a tensão desenvolvida. Concluímos que exossomos de pacientes sépticos induzem disfunção inotrópica em corações isolados e músculos papilares, possivelmente contribuindo para a disfunção miocárdica da sepse. / A previous report identified in plasma of septic shock patients the presence of exosomes. We hypothesized that circulating exosomes in sepsis could contribute to inotropic dysfunction. We collected blood samples from 55 patients with septic shock. Incubation of isolated heart preparations with exosomes induced a significant decrease in the derivatives of ventricular pressure and increased myocardial nitrate content. Exposure of isolated rat papillary muscles to exosomes reduced developed tension. We conclude that exosomes from septic patients induce myocardial dysfunction in isolated heart and papillary muscle preparations. Thus, they may contribute to myocardial dysfunction of sepsis.

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

(Abstract shortened by UMI.) / The conclusion of the studies reported herein can be summarized as follows: (1) PI in TCD correlates well with WMC volume and helps to differentiate those with and without WMC in stroke patients. (2) Post-stroke cognitive complaints are not related to severity of WMC among lacunar stroke patients. (3) The ARWMC scale correlates with objective cognitive performances and the operational definitions of ARWMC scale improves inter-rater reliability on CT. (4) Cognitive impairment in patients with confluent WMC is mediated by global and frontal cortical atrophy. Predictors for cognitive progression are cortical atrophy, absence of hyperlipidemia, low BP, and low cognitive scores. / With an aging population, prevalence of dementia is expected to escalate in the coming decades. The burden is especially great in developing countries like China. Similar to Alzheimer's pathology (e.g. amyloid plaque), age-related white matter changes (WMC) are important substrates of dementia. Since WMC are considered to be of ischemic origin, dementia related to WMC is believed to be more preventable than Alzheimer's disease. Yet, studies focusing on WMC have been relatively few. The thesis will cover 4 aspects of WMC and cognitive impairment. / Xiong, Yunyun. / Adviser: Vincent Mok. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 198-244). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese; some appendixes in Chinese.