3. Electronic Theses and Dissertations (ETDs) - All submissions
Permanent URI for this communityhttps://wiredspace.wits.ac.za/handle/10539/45
Browse
4 results
Search Results
Item Association between coagulation factor levels, cytokine profiles, clinical manifestations and genotypic features in factor X deficiency(2011-03-25) Thwala, Cyprian McwayizeniFactor X deficiency is a rare bleeding disorder with an incidence of one in a million in the general population. Patients with the severe form of factor X deficiency suffer from serious bleeds occurring mainly into the joints and the muscle. In the two factor X deficient families currently looked after at the Haemophilia Comprehensive Care Centre, there are definite differences in the bleeding tendencies between and within family members. We hypothesize the differences in genetic mutations and the influence of cytokines to be responsible for these bleeding variabilities. These factors were explored in our study. The study population included a total of fourteen members of the two families with factor X deficiency. Blood for factor X measurement, cytokine studies and genetic studies was collected in the Haemophilia Comprehensive Care Centre of the Charlotte Maxeke Johannesburg Academic Hospital. Each blood was processed according to the test to be performed. Factor X activity levels were measured using the factor X assay, and the information on each patient’s bleeding episodes was obtained from the Haemophiliac Clinic database. Cytokines were analyzed in all patients using the ELISA kits from Biosource. Factor X gene was amplified using PCR and sequenced with Spectrumedix SCE 2410. iv For cytokine studies, high levels of IL-1beta and TNF-alpha were observed in frequent bleeding patients compared to infrequent bleeders. These cytokines are known to be involved in acute inflammatory process leading to cellular infiltrate and joint swelling. This results in synovitis and the creation of massive joint bleeding. The low levels of IL-1beta and TNF-alpha detected in infrequent bleeding patients appear to be related to the high levels of IL-1Ra and IL-10. These anti-inflammatory cytokines are known to inhibit the inflammatory synovitis and lessen the severity of joint bleeding. For genetic studies, differences were observed between the amino acid sequence of the three frequent bleeding patients and the consensus. In addition, a novel mutation Cys350Phe was detected in two of these patients. This mutation is characterized by very low factor X levels which sometimes are not detectable in circulation. The substituted cystine is known to cause defect in the substrate binding, leading to the lost of enzyme activity. From these findings we have concluded that the origin of the heterogeneity of bleeding in factor X deficiency is multifactorial, cytokines and genetic mutations seems to have a role in determining the clinical manifestations of the factor X deficient patients.Item Studies on cytokines as mediators of fever and sickness(2009-05-08T12:26:32Z) Harden, Lois MayThe presence of endotoxin in animals and humans triggers a sequence of acute phase responses, which include the synthesis and release of pro-inflammatory cytokines from immune cells, followed by the development of various symptoms of sickness including fever and an array of behavioural responses, commonly referred to as sickness behaviours. Most experimental investigations examining the mechanisms mediating fever and sickness behaviour responses have used purified lipopolysaccharide (LPS), the glycolipid pyrogenic moiety of the Gram-negative bacterial membrane, to trigger the innate immune system. Results obtained from studies using specific antagonists to block the action of cytokines synthesized following systemic administration of LPS, have uncovered important roles for pro-inflammatory cytokines, such as interleukin (IL)-1b, IL-6, tumour necrosis factor-alpha (TNF-a) and leptin, in mediating fever. Although it has been shown that administration of pro-inflammatory cytokines can induce sickness behaviour in experimental animals, no clear role has been identified for these cytokines as endogenous mediators of sickness behaviours induced following LPS administration. Using rats as experimental animals and endogenous cytokine antagonism, I therefore investigated whether endogenously released IL-1b, IL-6, TNF-a and leptin are physiologically active not only in the generation of fever, but also in the generation of two specific sickness behaviours, lethargy and anorexia, induced by subcutaneous (s.c.) administration of LPS. Lethargy, anorexia and fever were measured as changes in voluntary wheel-running, food intake and body temperature respectively. I antagonized the biological action of these cytokines in the periphery following s.c. administration of LPS by injecting rats intraperitoneally (i.p.) with specific anti-rat sera to one of the following: TNF-a, IL-1b, IL-6 or leptin. Peripherally-released leptin appeared to be an important mediator of both fever and anorexia, as the presence of leptin antibodies in the circulation abolished both the anorexia and fever induced by s.c. administration of LPS. In contrast though, whereas the presence of IL-6 antibodies in the circulation abolished the LPS-induced fever, suppression of voluntary activity was reversed by the presence of IL-6 antibodies only to the extent of 27%, and appetite also was not returned to normal levels in the presence of IL-6 antibodies. Thus, IL-6 may be an essential component of LPS-induced fever, but an additional factor or factors, possibly working in parallel with IL-6, may be required to mediate the lethargy and anorexia induced by s.c. administration of LPS. Injecting rats i.p. with TNF-a antiserum or IL-1b antiserum had no effect on LPS-induced lethargy and LPS-anorexia, indicating that if these cytokines are working with peripherally-released IL-6 to induce sickness behaviour, it is likely due to their synthesis in the brain. Injecting species-homologous rat IL-1β and IL-6 into the brains of conscious rats, I aimed to identify whether either of these two cytokines can act within the brain to induce lethargy and anorexia in the absence of an infection. Intracerebroventricular (i.c.v.) administration of either IL-1β or IL-6 before the night-time active period decreased voluntary activity in the rats in a dose-dependent fashion, whereas only IL-1β decreased food intake and body mass of the rats. It is possible therefore, that increased levels of IL-1β in the brain may be working in parallel with IL-6 released in the periphery to induce lethargy and anorexia following s.c. administration of LPS. Thus I antagonized the biological action of these cytokines endogenously by administering species-specific antiserum to IL-6 (IL-6AS) i.p., and a caspase-1 inhibitor, which prevents the cleavage of pro-IL-1β to biologically active IL-1β, i.c.v. and monitored the symptoms of sickness induced by LPS until they ceased, so as to determine the cytokine involvement not only in the induction of these responses, but also in the resolution of these responses. Pre-treating rats with either IL-6AS i.p. or a caspase-1 inhibitor i.c.v. attenuated the magnitude and the duration of the anorexia and lethargy induced by LPS administration. LPS-induced fever was completely abolished in rats pretreated i.p. with IL-6AS, while it was only partially attenuated in rats pre-treated i.c.v. with a caspase-1 inhibitor. In conclusion, there appears to be some distinct differences in the cytokine-mechanisms regulating fever and sickness behaviours induced by LPS. Identifying the physiological mechanisms mediating fever and sickness behaviours during illness may provide clinicians with more insight into managing not only the thermal, but also the non-thermal responses to infections, responses which may become detrimental to the host if they continue for a prolonged period. My observation that reducing either IL-6 in the circulation or IL-1β in the brain significantly enhances the resolution of anorexia and lethargy, but does not completely prevent them from occurring, appears to indicate that while individual cytokines are possible targets for therapies aimed at alleviating these sickness responses in patients with bacterial infections, to abolish them multiple cytokines may need to be targeted.Item Cytokines associated with insulin resistance in critically ill patients.(2009-02-13T07:38:59Z) Wilgen, UrsAbstract Mortality of patients requiring intensive care treatment for greater than 5 days has been shown to be about 20% worldwide. Hyperglycaemia is common in critically ill patients. Strict glucose control with insulin in critically ill patients was shown to reduce mortality and morbidity significantly. Several interrelated mechanisms are involved in the development of “stress hyperglycaemia” in critically ill patients. These include dextrose containing intravenous infusions and total parenteral nutrition; the counter regulatory hormones (catecholamines, cortisol, glucagon and growth hormone) which oppose the effects of insulin; nervous system signaling; increased insulin clearance; and excess production of cytokines that interfere with intracellular insulin signaling pathways. Aim of study: To determine if the cytokines TNFα, IL-6 and adiponectin are significant determinants of insulin resistance in critically ill patients. Methods: The study was a prospective observational study conducted in the intensive care unit (ICU) at the Chris Hani Baragwanath hospital. Forty sequential adult ICU admissions that met with the inclusion criteria were enrolled. Blood specimens were drawn for adiponectin, TNF, and IL-6 at the time of ICU admission, on day 3, day 7 and on discharge from the ICU. Demographic data and clinical data were recorded, and body mass index (BMI) and APACHE II scores were calculated on admission. Blood glucose was measured every 2 to 4 hours, recorded and a mean value was calculated over the 24 hour period. Insulin infusions were started when the blood glucose values exceeded 6.0mmol/l. Administration of insulin was according to a fixed sliding scale. The total amount of insulin administered intravenously over that 24 hour period was recorded. Other factors known to be related to insulin sensitivity, such as inflammation (as indicated by C-reactive protein), vii triglycerides, insulin, C-peptide and cortisol levels were also drawn in addition to the blood drawn for routine investigations. Results: Duration of stay in ICU correlated with severity of illness as assessed by the APACHE II score (r = 0.44, p = 0.004). There was no significant difference in the mean 24 hour plasma glucose concentration throughout the duration of stay in ICU, there were however significant differences in the amount of insulin administered to maintain normoglycaemia. The amount of administered insulin required was found to peak on day 3 and decline thereafter. The main determinant of insulin administered was mean glucose (r = 0.79, p < 0.00001). The measured insulin concentrations on admission correlated with mean plasma glucose (r = 0.41, p = 0.009) and C-peptide (r = 0.45, p = 0.004) levels. The main determinants of mean plasma glucose levels on admission were BMI (r = 0.38, p = 0.013) and serum cortisol (r = 0.41, p = 0.008) levels. Serum triglycerides levels showed a significant difference from admission to discharge, with values increasing from admission levels. Adiponectin levels showed a significant increase from admission to discharge. IL-6 levels showed a significant decrease. TNFα levels did not show statistically significant changes. No statistically significant correlations were found between the levels of TNFα or IL-6 and administered insulin. Adiponectin concentrations showed a negative correlation with amount of administered insulin on discharge (r = -0.457, p = 0.0049). There were significant gender differences in BMI, administered insulin on admission, serum cortisol and C-peptide concentrations, with females having higher values than males. BMI was shown to account for the gender differences in administered insulin and C-peptide levels. viii There were significant differences in IL-6 and TNFα concentrations between the survivors and nonsurvivors, with higher levels being seen in non-survivors. Adiponectin levels were lower in nonsurvivors, but this did not reach statistical significance. Conclusion: Although there was a demonstrable change in insulin sensitivity during the stay in ICU, there was no statistically significant association between the cytokines TNFα or IL-6 and insulin administration. There was a negative correlation between adiponectin concentrations and administered insulin on discharge. This data also demonstrates that mortality is associated with increased levels of proinflammatory cytokines.Item The pathogenesis of inflammatory muscle pain(2008-02-21T12:17:28Z) Loram, Lisa CaroleABSTRACT The aim of my thesis is to further investigate the mechanisms underlying inflammatory muscle pain. Despite numerous studies investigating the mechanisms of inflammatory hyperalgesia, little is known of the mechanisms underlying inflammatory muscle hyperalgesia. Using rats as experimental animals, I investigated inflammatory hyperalgesia in muscle and compared it to that of inflamed cutaneous tissue. I injected carrageenan, a plant-origin polysaccharide, into leg muscle and into the hind paw of rats, and measured the behavioural response, as well as cytokine changes, in both plasma and inflamed tissue. Carrageenan induced inflammatory hyperalgesia but the cytokine cascade was not the same in muscle and cutaneous tissue. At no time following carrageenan injection was muscle tumour necrosis factor alpha (TNF-&) concentration elevated above that of muscle injected with saline. TNF-& is a key inflammatory mediator in cutaneous tissue, but apparently not in muscle. Interleukin-1) (IL-1)) and interleukin-6 concentrations also were different during muscle inflammation compared to those of cutaneous inflammation. IL-1) and IL- 6 concentrations, following carrageenan injection, were elevated later in muscle compared to in cutaneous tissue. IL-1) is a potent sensitizer of nociceptors in cutaneous tissue, and also may play an important role in sustaining muscle pain, but it is unlikely to be an initiator of the inflammatory muscle hyperalgesia. In the course of comparing muscle hyperalgesia and cutaneous hyperalgesia, I aimed to identify whether these differences in cytokine concentrations were unique to muscle tissue or if similar differences in cytokine concentrations existed between the hind paw and other cutaneous sites. To explore an alternative cutaneous tissue site, I injected carrageenan into the rat tail and measured the behavioural response, changes in cytokine concentrations and histological changes. Elevations of pro-inflammatory cytokines occurred concurrently with the infiltration of leukocytes into the inflamed tail tissue with the thermal and mechanical hyperalgesia similar to that found in the hind paw. Different mechanisms therefore appear to underlie muscle and cutaneous inflammatory hyperalgesia, regardless of the site used to investigate cutaneous inflammation. One of the consequences of the poor understanding of muscle pain is the lack of a reliable regimen for treating human muscle pain, including delayedonset muscle soreness (DOMS). DOMS, which has a partial inflammatory pathogenesis, is not relieved by non-selective cyclo-oxygenase inhibitors. This phenomenon may be that prostaglandins are not produced peripherally or centrally, when muscle tissue is damaged. I investigated the effect of inhibiting cyclo-oxygenase-2, the isoform released during inflammation, on DOMS in human volunteers. I found that rofecoxib, a cyclo-oxygenase-2 inhibitor, did not attenuate DOMS and nor did tramadol, a central-acting analgesic. The neurochemical pathway underlying DOMS therefore appears to be distinct from the pathways which underlie pain and hyperalgesia in other syndromes. Future research should include investigations into the central mechanisms of muscle pain and blocking the action of IL-1) and CINC-1 both peripherally and centrally may prove a beneficial target for the treatment of clinical muscle pain.