2v. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D. 1979) from the Dept. of Obstetrics and Gynaecology, University of Adelaide

Pregnancy toxaemia (ketosis) is a metabolic disease of ewes which occurs during the late gestation as a result of the inability of the pregnant ewe to maintain an adequate energy balance for the fast growing maternal fetal unit. As a result of energy defi-ciency mobilization of lipid reserves results in a doubling of the plasma free fatty acid (FFA) giving rise to fatty liver and increased ketone bodies β-hydroxybutyrate (BHB) in blood and urine. It is associated with a higher rate of mortality and causes severe economic losses. The objective of this study was directed at investigating the relationship between metabolic parameters and cytokine TNFα, to check the interaction between the TNFα and fat metabolism in late pregnant ewes of different breeds, and whether TNFα play a role in the pathogenesis of pregnancy toxaemia, which may serve as marker to early diagnosis of the disease. In this study, 29 pregnant and clinically healthy ewes (16 Merino, 13 Blackhead) were selected out of a flock of sheep. Blood samples were collected at 5, 3, and 1 week be-fore parturition (b.p.) and also 4 weeks after parturition (a.p.). The average numbers of lambs were 2.18 and 1.58 /ewe for Merino and Blackhead breeds respectively. The blood samples were analyzed for the following:  Concentration of metabolic parameters: glucose, insulin, free fatty acids (FFA), β-hydroxybutyrate (BHB), albumin, total protein (TP), iron (Fe), glutamat-dehydro-genase (GLDH), creatin kinase (CK), gamma-glutamyl-transferase (GGT), choles-terol, haptoglobin.  Haematological parameters: Haematocrite (HK), haemoglobin concentration (HB), erythrocyte count (EC), leukocyte count (LC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).  Cytokine TNFα by using ovine TNFα ELISA assay. The results of glucose concentration of pregnant ewes showed significant increase (3.8 mmol/l) in five weeks b.p. and declined with advancing gestation (2.6 mmol/l) one week b.p. Insulin concentration remained constant with an average of 0.11 nmol/l b.p., and then significantly increased to 0.22 nmol/l four weeks a.p. Maximal FFA concentrations were found at five weeks b.p. (976 µmol/l). The levels of FFA showed high levels b.p. compared with reference range (R.R. < 600 µmol/l), and the FFA levels significantly decreased postpartum (four weeks b.p.). while there was significant increasing (p<0.05) in the level of FFA in Merino sheep than in Black-head sheep b.p. On the other hand there was no significant difference a.p. The mean values of BHB in all periods of sampling, period 1(5 w.b.p.), period 2 (3 w.b.p.), period 3 (1 w.b.p.), and period 4 (4 w.a.p.) were 0.37 mmol/l, 0.23 mmol/l, 0.17 mmol/l and 0.3 mmol/l respectively. The mean of BHB indicated normal levels of BHB before and after parturition compared to subclinical ketosis (BHB > 1 mmol/l) and clinical ketoses (BHB > 1.6 mmol/l), and there was a significant difference (p<0.05) in the values of BHB between Blackhead and Merino breeds before parturi-tion while there was no significant difference after parturition. The concentration of TNFα showed elevated levels in all period of sampling before parturition. The TNFα values were 30.4 (17.2, 785.0) ng/ml (median, first, and third quartiles), 35.6 (13.6, 54.3), and 26.6 (13.0, 39.9) ng/ml in period 1(5 w.b.p.), period 2 (3 w.b.p.), and period 3 (1 w.b.p.) respectively. These values decreased to 19.1 (9.9, 33.8) ng/ml at 4 weeks after parturition. Statistical analysis showed that there was a positive correlation between free fatty ac-ids and TNFα. This correlation means that adipose tissue produces TNFα causing insu-lin resistance, which stimulates the lipolysis and leads to an increase of circulatory free fatty acids levels. It is concluded that fat mobilization occurs in the prepartum clinically healthy ewes with a significant increase in the levels of FFA, and also there is an increase in the proinflammatory cytokine TNFα at late gestation which predisposes ewes to pregnancy toxaemia and can aid in the diagnosis of the disease.

Pregnancy toxaemia (ketosis) is a metabolic disease of ewes which occurs during the late gestation as a result of the inability of the pregnant ewe to maintain an adequate energy balance for the fast growing maternal fetal unit. As a result of energy defi-ciency mobilization of lipid reserves results in a doubling of the plasma free fatty acid (FFA) giving rise to fatty liver and increased ketone bodies β-hydroxybutyrate (BHB) in blood and urine. It is associated with a higher rate of mortality and causes severe economic losses. The objective of this study was directed at investigating the relationship between metabolic parameters and cytokine TNFα, to check the interaction between the TNFα and fat metabolism in late pregnant ewes of different breeds, and whether TNFα play a role in the pathogenesis of pregnancy toxaemia, which may serve as marker to early diagnosis of the disease. In this study, 29 pregnant and clinically healthy ewes (16 Merino, 13 Blackhead) were selected out of a flock of sheep. Blood samples were collected at 5, 3, and 1 week be-fore parturition (b.p.) and also 4 weeks after parturition (a.p.). The average numbers of lambs were 2.18 and 1.58 /ewe for Merino and Blackhead breeds respectively. The blood samples were analyzed for the following:  Concentration of metabolic parameters: glucose, insulin, free fatty acids (FFA), β-hydroxybutyrate (BHB), albumin, total protein (TP), iron (Fe), glutamat-dehydro-genase (GLDH), creatin kinase (CK), gamma-glutamyl-transferase (GGT), choles-terol, haptoglobin.  Haematological parameters: Haematocrite (HK), haemoglobin concentration (HB), erythrocyte count (EC), leukocyte count (LC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).  Cytokine TNFα by using ovine TNFα ELISA assay. The results of glucose concentration of pregnant ewes showed significant increase (3.8 mmol/l) in five weeks b.p. and declined with advancing gestation (2.6 mmol/l) one week b.p. Insulin concentration remained constant with an average of 0.11 nmol/l b.p., and then significantly increased to 0.22 nmol/l four weeks a.p. Maximal FFA concentrations were found at five weeks b.p. (976 µmol/l). The levels of FFA showed high levels b.p. compared with reference range (R.R. < 600 µmol/l), and the FFA levels significantly decreased postpartum (four weeks b.p.). while there was significant increasing (p<0.05) in the level of FFA in Merino sheep than in Black-head sheep b.p. On the other hand there was no significant difference a.p. The mean values of BHB in all periods of sampling, period 1(5 w.b.p.), period 2 (3 w.b.p.), period 3 (1 w.b.p.), and period 4 (4 w.a.p.) were 0.37 mmol/l, 0.23 mmol/l, 0.17 mmol/l and 0.3 mmol/l respectively. The mean of BHB indicated normal levels of BHB before and after parturition compared to subclinical ketosis (BHB > 1 mmol/l) and clinical ketoses (BHB > 1.6 mmol/l), and there was a significant difference (p<0.05) in the values of BHB between Blackhead and Merino breeds before parturi-tion while there was no significant difference after parturition. The concentration of TNFα showed elevated levels in all period of sampling before parturition. The TNFα values were 30.4 (17.2, 785.0) ng/ml (median, first, and third quartiles), 35.6 (13.6, 54.3), and 26.6 (13.0, 39.9) ng/ml in period 1(5 w.b.p.), period 2 (3 w.b.p.), and period 3 (1 w.b.p.) respectively. These values decreased to 19.1 (9.9, 33.8) ng/ml at 4 weeks after parturition. Statistical analysis showed that there was a positive correlation between free fatty ac-ids and TNFα. This correlation means that adipose tissue produces TNFα causing insu-lin resistance, which stimulates the lipolysis and leads to an increase of circulatory free fatty acids levels. It is concluded that fat mobilization occurs in the prepartum clinically healthy ewes with a significant increase in the levels of FFA, and also there is an increase in the proinflammatory cytokine TNFα at late gestation which predisposes ewes to pregnancy toxaemia and can aid in the diagnosis of the disease.:Table of contents 1 Introduction…………….…………………………………………………..1 2 Review of literature……....…………….……………………………….….3 2.1 Metabolic condition of ewes in the late pregnancy………….………………3 2.2 Metabolic disorders subacute and acute pregnancy Toxaemia…….………..4 2.3 The Importance and dynamic of the different biochemical parameters glucose, insulin, free fatty acids, and β-hydroxybutyrate of ewes in the late pregnancy…………………….………..….……………………………10 2.3.1 Glucose……………………………………………………………………..10 2.3.2 Insulin………………………………………………………………………12 2.3.3 β-hydroxybutyrate (BHB)…….……………………………………………16 2.3.4 Free fatty acids……………………………………………………………...20 2.4 TNFα and its role in fat metabolism and pregnancy toxaemia………….….22 3 Animals, material and methods……………………………………..……38 3.1 Animals……………………………………………………………………..38 3.2 Clinical examination………………………………………………………..38 3.3 Collection of blood samples……………………………………………..…38 3.4 Analysis of haematological parameters in blood samples……………….....38 3.5 Determination of biochemical parameters………………………………….39 3.6 Determination of haptoglobin by using haptoglobin assay……………...…39 3.6.1 Haptoglobin assay principle………………………………………………..39 3.6.2 Components………………………………………………………………...40 3.6.3 Additional materials required………………………………………………40 3.6.4 Sample and reagent preparation………………………………….….……..40 3.6.4.1 Samples……………………………………………………………………..40 3.6.4.2 Haemoglobin.................................................................................................40 3.6.4.3 Chromogen/Substrate………………………………………………………40 3.6.5 Manual methods (microplate or spectrophotometric)……………………...41 3.6.5.1 Calibrator…………………………………………………………………...41 3.6.5.2 Test temperature…………………………………………………………....41 3.6.5.3 Procedure………………..………………………………………………….41 3.7 Analysis of TNFα by using ovine TNFα ELISA assay…………………….42 3.8 Statistical analysis…………………………………………………….…….43 4 Results.……………………………………………………………………..44 4.1 Clinical examination (observation)…………………………………….…...44 4.2 Biochemical parameters………………………………………………….…44 4.2.1 Glucose………………………………………………………………….…..44 4.2.1.1 Glucose concentrations in all sheep…………………………..…………….44 4.2.1.2 Glucose concentrations in Blackhead sheep…………………………..…....44 4.2.1.3 Glucose concentrations in Merino sheep………………………...…………45 4.2.2 Insulin……………………………………………………………………….45 4.2.2.1 Insulin concentrations in all sheep………………………………..………...45 4.2.2.2 Insulin concentrations in Blackhead sheep………………………..………..46 4.2.2.3 Insulin concentrations in Merino Sheep…………………………………….46 4.2.3 Free fatty acids……………………………………………………………...47 4.2.3.1 Free fatty acid concentrations in all sheep………………………………….47 4.2.3.2 Free fatty acid concentrations in Blackhead sheep………………………....47 4.2.3.3 Free fatty acid concentrations in Merino sheep………………………….....47 4.2.4 β-hydroxybutyrate (BHB)…….…………………………………………….48 4.2.4.1 β-hydroxybutyrate concentrations in all sheep……..……………………....48 4.2.4.2 β-hydroxybutyrate concentrations in Blackhead sheep……… …………....48 4.2.4.3 β-hydroxybutyrate concentrations in Merino sheep…………………...…...49 4.2.5 Tumor necrosis factor alpha (TNFα)………………….……………….…...50 4.2.5.1 TNFα concentrations in all sheep…………..................................................50 4.2.5.2 TNFα concentrations in Blackhead sheep……………………………….....50 4.2.5.3 TNFα concentrations in Merino sheep………………………………….......51 4.2.6 Haptoglobin………………………………………………………………....51 4.2.6.1 Haptoglobin concentrations in all sheep……………………………..…......51 4.2.6.2 Haptoglobin concentrations in Blackhead and Merino sheep…….………..51 4.2.7 Albumin…………………………………………………………………….52 4.2.7.1 Albumin concentrations in all sheep…………………………..…………...52 4.2.7.2 Albumin concentrations in Blackhead and Merino sheep……………….....53 4.2.8 Creatinkinase (CK)…………………………………………………………53 4.2.8.1 Creatinkinase activity in all sheep………………………………………….53 4.2.8.2 Creatinkinase activity in Blackhead and Merino sheep…………………….53 4.2.9 Gamma-Glutamyl Transferase (GGT)………………………….…………..54 4.2.9.1 GGT activity in all sheep…………………………………………………...54 4.2.9.2 GGT activity in Blackhead and Merino sheep……………………………..55 4.2.10 Glutamat-Dehydrogenase (GLDH)………………………………………...56 4.2.10.1 GLDH activity in all sheep…………………………………………………56 4.2.10.2 GLDH activity in Blackhead and Merino sheep………….…………….......56 4.2.11 Total protein………………………………………………………………..57 4.2.11.1 Total protein concentrations in all sheep………………………………...…57 4.2.11.2 Total protein concentrations in Blackhead and Merino sheep……………..57 4.2.12 Cholesterol……………………………………………………………….…58 4.2.12.1 Cholesterol concentrations in all sheep………………………………...…...58 4.2.12.2 Cholesterol concentrations in Blackhead and Merino sheep…………....….58 4.2.13 Iron………………………………………………………………………….59 4.2.13.1 Iron concentrations in all sheep…………………………...………………..59 4.2.13.2 Iron concentrations in Blackhead and Merino sheep………………….........60 4.3 Haematological parameters………………………………………………...60 4.3.1 Haematological parameters in all sheep……………………………………60 4.3.2 Haematological parameters in Blackhead sheep…………………………...61 4.3.3 Haematological parameters in Merino sheep………………………………61 4.3.4 Statistical analysis of haematological parameters………………………….62 4.3.4.1 Haemoglobin concentration (Hb)…………………………………………..62 4.3.4.2 Haematocrite (HK)…………………………………………………………62 4.3.4.3 Mean corpuscular volume (MCV) …………………………………………62 4.3.4.4 Mean corpuscular haemoglobin (MCH)……………………………………62 4.3.4.5 Mean corpuscular hemoglobin concentration (MCHC)……………………63 4.3.4.6 Thrombocytes volume (THB)……………………..…………………….…63 4.3.4.7 Leukocytes (LC)………..…………………………………………………..63 4.3.4.8 Erythrocytes.……………………………………………………………….63 5 Discussion…………………………………………………………………..65 6 Summary…………………………………………………………………..71 7 Zusammenfassung………………………………………………………...73 8 References…………………………………………………………………75 Acknowledgements.......................................................................................................84

Abstract The aim of this work was to evaluate the outcome of childhood Henoch-Schönlein purpura (HSP), the effectiveness of Cyclosporine A (CyA) for treating severe HSP nephritis (HSN), and more particularly the possibility for influencing the course of HSP disease by early prednisone treatment. A total of 47 adults who had had childhood HSP were evaluated after a mean of 24.1 years (16.4–35.6). The outcome was highly dependent on the renal symptoms at onset, since 7 out of 20 adults (20%) who had severe renal symptoms at onset had renal impairment as adults, compared with 2 out of 27 (7%) with mild or no renal symptoms at onset (relative risk 4.7; 95% CI 1.3–18.7). 70% of pregnancies in women after childhood HSN were complicated by hypertension or proteinuria. The annual incidence of HSN with nephrotic-range proteinuria was 2 per million children under 15 years. After a mean follow-up of 4.6 years, only three patients out of 19 were in complete remission. Kidney biopsy did not predict the outcome in these patients. CyA seemed to be promising for the treatment of severe HSN with nephrotic-range proteinuria, since four out of seven patients treated with CyA achieved stable remission and three had preserved their renal function after a mean follow-up of 6.0 years. Treatment at an early stage in the disease was associated with stable remission. The efficacy of early prednisone treatment was evaluated in a randomized double-blind trial involving 171 patients (84 prednisone, 87 placebo). Prednisone, given at a dose of 1 mg/kg/day for 2 weeks, with weaning over the next two weeks, was effective in reducing the intensity of abdominal pain (pain score 2.5 vs. 4.8; t-test p = 0.029) and shortening its duration (1.5 days vs. 2.7 days; t-test p = 0.028) compared with the placebo. The mean scores for joint pain were lower in the prednisone group (4.6 vs. 7.3; t-test p = 0.030) and the improvement from joint symptoms was faster (log rank p = 0.007). Prednisone did not prevent the development of renal symptoms but it was effective in treating them, since renal symptoms resolved in 61% of the prednisone patients after treatment compared with 34% of the placebo patients (difference 27%, 95% CI 3–47%, p = 0.024). Prednisone was most effective for children aged 6 or more with renal symptoms at onset, since only two patients needed to be treated in order to save one from renal involvement (95% CI's for NNT 2–6). The long-term outcome of HSP is dependent on renal symptoms. Severe renal symptoms indicate early immunosuppressive treatment for HSN, and patients with renal involvement at the acute phase need long-term follow-up, especially women during and after pregnancy. Early treatment with prednisone is effective in reducing the abdominal and joint symptoms involved in HSP and is also effective in altering, but not preventing, the course of renal involvement. / Tiivistelmä Väitöskirjatyön tarkoituksena oli selvittää lapsuusiän Henoch-Schönleinin purppuran (HSP) pitkäaikaisennustetta, Siklosporiini-A:n (CyA) tehoa vaikean HSP-nefriitin hoidossa ja tutkia varhain aloitetun prednisonihoidon hyötyä HSP-taudin oireisiin. HSP:n pitkäaikaisennustetta selvitettiin tarkastamalla 47:n lapsena HSP-taudin sairastaneen aikuisen terveystilanne keskimäärin 24.1 vuoden (16.4-35.6) seuranta-ajan jälkeen. HSP-taudin ennuste oli vahvasti riippuvainen munuaisoireen vaikeusasteesta: 20 % niistä, joilla taudin alussa oli vaikeat munuaisoireet, kärsi vielä aikuisiällä munuaisoireista; vastaava luku munuaisoireettomilla ja niillä, joilla oli ollut vain lievää veri- tai valkuaisvirtsaisuutta, oli 7 %, (RR 4.7; 95 % CI 1.3–18.7). Raskauskomplikaatiot olivat yleisiä lapsuusiällä HSP-taudin sairastaneilla naisilla, sillä 70 % raskauksista komplisoi korkea verenpaine tai valkuaisvirtsaisuus. Vuosittain 2 lasta miljoonasta sairastuu vaikeaan nefroottistasoiseen HSP-nefriittiin Suomessa. Vain kolme nefroottistasoiseen HSP-nefriittiin sairastuneesta 19 lapsesta oli 4.6 vuoden seurannan jälkeen parantunut oireettomaksi. Ensimmäisen munuaisbiopsian vaikeusaste ei ennakoinut selviytymistä. CyA näytti olevan lupaavan tehokas lääke vaikean HSP-nefriitin hoidossa, sillä neljä seitsemästä CyA-hoitoa saaneesta lapsesta, oli oireeton 6.0 vuoden seurannan jälkeen. Mitä aikaisemmin vaikean nefriitin hoito oli aloitettu, sen parempi hoitotulos oli. Varhain aloitetun prednisonihoidon hyötyä HSP-taudin oireisiin selvitettiin satunnaistetulla kaksoissokkotutkimuksella, johon satunnaistettiin 171 lasta (84 prednisoni, 87 lumelääke) saamaan joko prednisonia 1 mg/kg/päivä 2 viikon ajan tai lumelääkettä. Prednisoni vähensi tehokkaasti vatsa- ja nivelkipuja ja se lyhensi merkitsevästi myös niiden kestoa. Prednisoni ei estänyt munuaisoireen kehittymistä lapselle, mutta niiltä, joille se kehittyi, oireet hävisivät merkitsevästi nopeammin lumelääkitykseen verrattuna (61 % versus 34 %, 95 % CI 3–47 %, p = 0.024). Kaikkein tehokkainta prednisoni oli yli 6 vuotiaille lapsille, joilla oli munuaisoire heti taudin alussa (NNT 2, 95 % CI 2–6). Tutkimuksen perusteella voidaan sanoa, että lapsuusiällä HSP-nefriitin sairastaneet lapset tarvitsevat seurantaa aikuisiällä, erityisesti naiset raskauden aikana. HSP-nefriitin varhainen hoitaminen on tärkeää. Varhainen prednisonihoito ei estä munuaisoiretta, mutta hoitaa jo kehittynyttä nefriittiä ja vähentää vatsa- ja nivelkipuja tehokkaasti.

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.

Objectives: To assess the expression of the symptoms of jaw muscle pain and its association with alterations in biochemistry, other symptoms and the carriage of staphylococci. Methods: Three different study populations were assessed. The first was selected and examined by the author and consisted of 43 pain and 41 age and sex matched controls. The second was a study of CFS patients who were blinded to the author and the author subsequently examined the associations between jaw muscle symptom reporting and the standardised biochemistry measures. The third study was also blinded to the author but included an investigation of staphylococci and certain cytokine and biochemistry measures. Results: The three studies clearly establish an association between the carriage of toxicogenic coagulase negative staphylococci and the expression of jaw muscle pain in both males and females. These associations were homogeneous and were found whether the patients were selected on the basis of having jaw muscle pain or selected from within a population of patients selected on the basis of having Chronic Fatigue Syndrome. The studies associated the changes with variations in biochemistry and these were in turn associated with symptom expression within the jaw muscle pain patients. These biochemical alterations included the dysregulation of immune cell counts, cytokines, electrolyte and protein metabolism. These symptoms and biochemical changes were associated with pain severity and illness duration and staphylococcal toxin production. From the data a model was developed which shows the mechanisms involved in the development of chronic pain in the jaw muscles. Conclusions: The carriage of toxicogenic coagulase-negative staphylococci were found to be associated with the expression of jaw muscle pain and the alterations in biochemistry associated with these symptoms.