Rozprawy doktorskie na temat „Blood – Diseases”

Actually, there is a strong incidence of psychiatric diseases, representing a 13% of total burden diseases and 450 million of people affected. The etiology of psychiatric diseases remains unknown. However, scientific evidences suggest a maldevelopment of nervous system (NS). The diagnosis is inaccurate, and international manuals (ICD-10 and DSM-IV) identify pathologies according to a list of symptoms but no underlying biological cause of disease. The aim of the thesis is to identify potential biomarkers -related to the development of NS- in peripheral blood of psychiatric patients diagnosed as different mental diseases, such as autism spectrum disorders (ASD), schizophrenia and bipolar disorders. It is intended to contribute with the improvement of diagnostic, prognostic and treatment of subjects. The thesis is divided into 4 chapters: 1) study of neurotrophins in ASD, where the results show the relationship of this family of molecules with the disease, 2) study of Latrophilin-3 (LPHN3) in the TEA, which was obtained in association with lower cognitive level of ASD, 3) study of the Eph-receptor A4 in the pathology of schizophrenia and bipolar disorder, results of which show no association, and finally 4) study of Ankyrin-3 (ANK3) in schizophrenia and bipolar disorder, which shown a relationship with bipolar disorder but not with schizophrenia.
Actualment, hi ha una forta incidència de les patologies psiquiàtriques, representant un 13% del total de les malalties i 450 milions de persones afectades. L’etiologia de les patologies psiquiàtriques és desconeguda. Tot i així, evidències científiques suggereixen un mal desenvolupament del sistema nerviós (SN). El diagnòstic és poc precís, i els manuals internacionals (ICD-10 i DSM-IV) identifiquen les patologies d’acord a un llistat de símptomes, però sense cap causa biològica subjacent de la patologia. L’objectiu de la tesi és la identificació de biomarcadors potencials –relacionats en el desenvolupament del SN- en sang perifèrica de pacients diagnosticats amb diferents patologies mentals, com ara els trastorns de l’espectre autista (TEA), esquizofrènia i desordres bipolars. És pretén contribuir amb la millora del diagnòstic, el pronòstic i el tractament de les persones que les pateixen. La tesi s’estructura en 4 capítols: 1) estudi de les neurotrofines en els TEA, on els resultats evidencien la relació d’aquesta família de molècules amb la patologia, 2) estudi de la Latrofilina-3 (LPHN3) en els TEA, on s’ha obtingut associació amb el nivell cognitiu més baix dels TEA, 3) estudi del receptor EPH A4 en les patologies d’esquizofrènia i desordres bipolars, resultats del qual no mostren associació i, per últim 4) estudi de la Ankirina-3 (ANK3) en l’esquizofrènia i els desordres bipolars, en el qual si que es troba una relació amb els desordres bipolars, però no amb l’esquizofrènia.
Actualmente, hay una fuerte incidencia de las patologías psiquiátricas, representando un 13% del total de las enfermedades y 450 millones de personas afectadas. La etiología de las patologías psiquiátricas es desconocida. Aún así, evidencias científicas sugieren un mal desarrollo del sistema nervioso (NS). El diagnóstico es poco preciso, y los manuales internacionales (ICD-10 y DSM-IV) identifican las patologías de acuerdo a un listado de síntomas, pero sin ninguna causa biológica subyacente de la patología. El objetivo de la tesis es la identificación de biomarcadores potenciales –relacionados con el desarrollo del SN- en sangre periférica de pacientes diagnosticados con diferentes patologías mentales, como son los trastornos del espectro autista (TEA), esquizofrenia y desordenes bipolares. Se pretende contribuir en la mejora del diagnóstico, el pronóstico i el tratamiento de las personas que las padecen. La tesis se estructura en 4 capítulos: 1) estudio de las neurotrofinas en los trastornos del espectro autista (TEA), en el cual los resultados evidencian la relación de esta familia de moléculas con la patología, 2) estudio de la Latrofilina-3 (LPHN3) en los TEA, donde se ha obtenido una asociación con el nivel cognitivo más bajo de los TEA, 3) estudio del receptor EPH A4 en las patologías de la esquizofrenia y los desordenes bipolares, resultados del cual no muestran asociación y, por último 4) estudio de la Ankirina-3 (ANK3) en la esquizofrenia y los desordenes bipolares, en el cual si que se ha encontrado una relación con los desordenes bipolares, pero no con la esquizofrenia.

Identification of blood-based biomarkers for psychiatric disease risk and development has emerged as an important area of translational research in medicine, offering a means to supplement or replace current interview-based me thods for psychiatric diagnosis. The aim of this thesis is to assess the utility of genome-wide blood transcriptome profiling for the prediction, diagnosis and treatment of patients with psychiatric diseases. Some parts of this work are of a more methodological nature and geared towards the discovery of blood-based biomarkers and gene networks, while others consider mechanistic and translational implications. Overall, this work contributes to understanding the pathophysiology of major psychiatric diseases and to the development of new biomarkers and treatments. Part I discusses the current transition from interview-based psychiatric diagnostics towards genomic-based interventions (Chapter 1) prior to introducing experimental methodologies (Chapter 2) and statistical approaches (Chapter 3) that may provide favorable translational avenues for blood biomarker discovery in psychiatry. Part II contains four investigations (summarized in Chapter 4) that apply genome-wide transcriptome profiling of patient blood samples in pursuit of blood-based biomarkers and gene networks implicated in posttraumatic stress disorder (Chapter 5), acute psychological stress (Chapter 6), methamphetamine-associated psychosis (Chapter 7) and treatment response in bipolar disorder (Chapter 8). Part III proposes a set of rules or postulates for accelerating the identification of reliable and accurate blood-based biomarkers in patients with psychiatric diseases (Chapter 9).

Pulmonary hypertension is an abnormal physiological state associated with a variety of medical conditions. However, the ability to accurately phenotype disease subtypes within this heterogeneous syndrome is limited. In this thesis, I utilised advanced phenotyping techniques, guided by pathophysiological processes known to be dysregulated in pulmonary vascular diseases; immunity and metabolism. I used flow cytometry based immunophenotyping to study circulating leukocyte subpopulations and metabolomic analysis to study metabolite profiles in circulating blood. I hypothesised that there would be differences between disease and health, and differences between disease subgroups. In the immunophenotyping studies, I identified an immune cell signature in Idiopathic Pulmonary Arterial Hypertension (IPAH) and Heritable Pulmonary Arterial Hypertension (HPAH) characterised by increased frequencies of T follicular helper (Tfh) cells, plasmablasts and PD1-expressing CD8+ T cells. This signature was not found in Chronic Thromboembolic Pulmonary Hypertension (CTEPH). These findings support the hypothesis that dysfunctional immune activation may be implicated in IPAH pathobiology, and that IPAH and HPAH may have shared immunopathological mechanisms. In the metabolomic studies, I identified wide ranging metabolic changes in pulmonary vascular disease, including evidence of disrupted energy metabolism, increased cellular proliferation and reduction in antioxidant metabolites. Additionally, by comparing paired samples from different anatomical sites, it was possible to differentiate metabolic perturbations which are localised to specific anatomical sub-compartments. Key to the clinical applications of this research, I have demonstrated immunological and metabolic alterations which are a shared feature amongst different pulmonary vascular disease subgroups, but also some changes which are specific to disease subsets. Future advances in disease phenotyping may facilitate effective new targeted therapy for pulmonary vascular diseases.

Natural exposure to Plasmodium falciparum’s asexual blood-stage results in protection against severe disease, but no vaccine using the widely-studied blood-stage antigens apical membrane antigen 1 (AMA1) or merozoite surface protein 1 (MSP1) has proven convincingly protective in clinical trials. Challenges include antigenic polymorphism, the apparent requirement for exceptionally high antibody concentrations for protection, and clinical-grade production of conformationally-accurate recombinant protein antigens followed by formulation with a human-compatible adjuvant. This thesis describes the generation of viral-vectored vaccines targeting ten less-studied blood-stage antigens, focusing upon antigens implicated in erythrocyte invasion. These vaccines were immunogenic in mice and rabbits. The rabbit antibodies raised were functionally active in the in vitro assay of parasite growth inhibitory activity (GIA). GIA with antibodies against one antigen, RH5, exceeded that achieved with antibodies against the ‘gold standard’ AMA1 or MSP1 antigens. This antigen’s amino acid sequence is relatively conserved between parasite strains. Importantly, and unlike anti-AMA1 and MSP1 antibodies, the GIA effects transcend genetically diverse strains. It was hypothesised that blockade of the interaction of RH5 with its receptor basigin was likely to be a mechanism of action of anti-RH5 antibodies. Vaccine-induced polyclonal anti-RH5 serum was found to be capable of blocking this interaction, as well as merozoite attachment to erythrocytes. A panel of RH5-specific monoclonal antibodies were raised: those which block the RH5-receptor interaction were capable of neutralising parasites. Minimal linear epitopes recognised by these antibodies were mapped, and are likely to be within or close to RH5’s receptor binding site. These data support prompt clinical testing of RH5-based vaccines, and shed light upon the mechanism of action of anti-RH5 antibodies. However substantial challenges remain in establishing whether this antigen, selected on the basis of the in vitro assay of GIA, will be capable of achieving in vivo protection against P. falciparum. Further work presented in this thesis addresses the use of quantitative PCR data to assess blood-stage vaccine efficacy in experimental human challenge with P. falciparum, and the use of surface plasmon resonance to establish more detailed characterisation of vaccine-induced antibody responses. Finally, the results of P. falciparum challenge of RH5-vaccinated Aotus nancymaae non-human primates are presented.

In this study, we investigated the role of Ang II in pathological vascular remodeling. We sought to determine whether the humoral or the mechanical effects of Ang II are the dominant factor driving the remodeling process. The following experimental groups were used: control group (untreated mice), mice treated with an angiotensin receptor blocker (Candesartan, 0.5 mg/kg/day,SQ), an ACE inhibitor (Captopril, 6 mg/kg/day), and a calcium-channel blocker (Amlodipine, 7.5 mg/kg/day). All mice (n=6 per experimental group) were from the C57Bl/6 background. The carotid ligation model was implemented to study the differences in vascular remodeling. Additionally, multiple time points (7-, 14-, and 21-days post-surgery) were used to track the progression of remodeling. In Day-7 analysis, all three treatment groups yielded similar remodeling patterns as evidenced by a significant reduction in neointimal area, medial thickening and hypertrophy compared with the control group. Histomorphometric analysis of carotid sections collected 1mm below the ligation demonstrated that the Amlodipine group had 26% reduction in total vessel area, Candesartan a 36% reduction, and Captopril a 28% reduction (p less than 0.05 in all groups compared with Control), as well as a parallel 38-40% drop in medial thickness. In Day-14 analysis, no significant differences between the Controls and treatment groups were observed, although differences were emerging between the treatment groups. Candesartan was found to reduce the extent of negative remodeling observed between the 7- and 14-day Control data, whereas the Captopril group did not exhibit this trend. All treatment groups exhibited less neointimal formation than Controls, similar to Day-7. By the 21-day time point, the Captopril group underwent positive remodeling, resembling the Candesartan and Amlodipine groups. Although total vessel area was analogous among all groups, neointimal areas were significantly decreased in the treatment groups. Blood pressure plays a pivotal role in the modulation of vascular remodeling in response to mechanical injury. Although intermediate timepoint analysis suggests that humoral aspects of ACE inhibition or angiotensin-receptor blockade yielded unique effects on the overall vessel caliber, upon reaching the late, 21-day time point, the mechanical factors became predominant. These data support the importance of blood pressure control in the attenuation of pathological vascular remodeling.

This thesis provides the first description of the formation and purification of Fab-peptide conjugates applied to the detection of HIV -1 and HIV -2 antibodies, in a novel, autologous red cell agglutination immunoassay. A new method was also developed for the large-scale production and purification of bispecific antibodies, which were used for the detection of heartworm antigen in dogs and cats. The conjugation method for both forms of bispecific conjugate involved blocking the hinge sulphydryls of an Fab' antibody fragment with 5,5'dithiobis(2-nitrobenzoic acid), DTNB, followed by the addition of TNB-blocked fragment to an antibody or peptide containing free thiols, resulting in conjugate formation via disulphide exchange. The reaction steps and purification conditions allowed conjugates to be produced at low cost since reaction times were short (0.5 to 2 hours) and the purification steps could process large-scale, gram quantities of antibodies. Conjugate was also produced in high yields (50-65%) and the process was reproducible, making the methods compatible with large-scale manufacture in a commercial environment.

Reliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases.

Solid organ transplantation is a rare health intervention that saves lives, improves quality of life and, where there is an alternative such as dialysis, is cost-effective. However, organ transplantation is a complex intervention that is limited by the number of organ donors, and requires life-long immunosuppression of the recipient. Infections are a common complication of organ transplantation, and a major cause of death for transplant recipients. Donor-derived infections, while rare, are of particular concern for transplant clinicians, as a potentially avoidable consequence of transplantation. Donation practice must balance the risks of infection transmission with the risks of overly restrictive donation policies that could limit this life-saving intervention. Post-transplant, clinicians must remain vigilant for new recipient infections, whether donor-derived, reactivating or arising anew. In this thesis, I generate novel data regarding aspects of infections affecting solid organ donation and transplantation where current practice can be improved, focusing in particular on blood-borne viruses and preventable post-transplant infections. Theme 1: Assessment of potential organ donors for blood-borne virus transmission risk All potential solid organ donors are rigorously screened for their suitability to donate. One reason potential donors may be declined is where there is perceived to be an increased risk of transmission of infectious diseases, including blood-borne viruses (BBV) such as hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency virus (HIV). Unexpected BBV transmissions have indeed led to devastating recipient outcomes. However, testing and treatment for these conditions have evolved substantially in recent years and decades. Nucleic acid testing (NAT) is now both rapidly available and highly sensitive for infection. Implementation of NAT reduces serological window periods to smaller eclipse periods. Meanwhile, HCV has become curable in the vast majority of cases, HBV can be prevented with vaccination, or effectively suppressed, and even HIV now has effective suppressive treatments. Given these developments, a reassessment of suitability of potential organ donors with BBV, or risk factors for their acquisition, is warranted. The first aim of this thesis was to understand whether transplant clinicians can correctly interpret hepatitis serology and then consistently judge potential donor transmission risk and donation suitability. This is explored in Chapter 2 through an anonymous, self-completed, cross-sectional survey distributed electronically to Australian and New Zealand clinicians involved in kidney transplantation (2014-2015). We compared respondents’ interpretation of clinical scenarios with paired donor and recipient hepatitis B and C serology to recommendations in clinical practice guidelines. We then used logistic regression modelling to investigate characteristics associated with decisions on transplant suitability in scenarios with poor (<50%) guideline concordance. The 110 survey respondents in our sample had demographic characteristics representative of the target workforce. While donor and recipient hepatitis statuses were largely well understood, transplant suitability responses varied among respondents. For an HBV surface antigen (HBsAg) positive donor and vaccinated recipient, 44% of respondents suggested the donor was unsuitable for transplant (guideline concordant) but 35% suggested the donor was suitable with prophylaxis (guideline divergent). In four scenarios with transplant suitability guideline concordance of <50%, acute transplant care involvement predicted guideline concordant responses (OR [odds ratio] 1.69, p=0.04). Guideline concordant responses were chosen less by hepatologists, intensive care doctors (OR 0.23, 0.35 respectively, p=0.01), and New Zealanders (guideline concordant responses OR 0.17, p<0.01; alternative responses OR 4.31, p<0.01). Hence, we conclude that despite broadly consistent interpretations of hepatitis serology by respondents, resulting transplant suitability decisions vary, and often diverge from guidelines. Having established variability between clinicians, this thesis next aimed to describe current practice regarding potential donors with increased risk for BBV transmission in Chapter 3. Specifically, we aimed to describe the size and characteristics of this group, whether they proceeded to donate, and reasons not donating. To do this, we conducted a cohort study of all potential organ donors referred in New South Wales, Australia between 2010 and 2018. Baseline risk potential donors were compared to potential donors with increased BBV transmission risk, due to a personal history of HIV, HCV or HBV and/or behavioural risk factors. We found 624/5749 potential donors (10.9%) perceived to have increased BBV transmission risk. This included 298/5749 (5.2%) with HCV (including HBV co-infections) and 239/5749 (4.2%) with increased risk behaviours (no known BBV). Potential donors with HCV and those with increased risk behaviours were younger and had fewer comorbidities than baseline risk potential donors (p<0.001). Many potential donors (82 with HCV, 38 with risk behaviours) were declined for donation purely due to perceived BBV transmission risk. Most were excluded prior to BBV testing. When potential donors with HCV did donate, they donated fewer organs than baseline risk donors (median 1 versus 3, p<0.01), especially kidneys (OR 0.08, p<0.001) and lungs (OR 0.11, p=0.006). In summary, many potential donors were not accepted due to perceived increased BBV transmission risk, without viral testing, and despite otherwise favourable characteristics. This suggests the potential for transplantation rates to be substantially increased if more potential donors with HCV and/or increased risk behaviours were accepted. Taken together, the findings of variable donor suitability decision-making, and the rejection of otherwise high-quality potential donors without viral testing, suggest there may have been missed opportunities to donate from some potential donors with positive serology or increased risk behaviours. Supporting clinicians to identify such previously rejected potential donors with an acceptable risk profile, and using these organs for transplantation, may have the potential to significantly increase donation rates. Theme 2: Estimating the actual risks of BBV transmission To support change in clinical practice, estimates of the risks posed by increased risk donors are helpful. Blood-borne virus rates vary internationally, and no data from the Australian context was available. We synthesised existing literature to produce Australian estimates of the actual risk posed by potential donors with increased risk behaviours for BBV only (Chapter 4). We conducted a systematic review and meta-analysis of cohorts reporting incidence and prevalence of HIV, HCV and HBV among increased risk groups in Australia. The residual risks of window period infections were estimated in the setting of negative serology and NAT. Residual risk of HIV was found to be highest among men who have sex with men at 4.8 per 10,000 persons testing negative with serology (95% CI: 2.7-6.9), and 1.5 per 10,000 persons with additional negative NAT (95% CI: 0.9-2.2). Residual risk of HCV was highest among injecting drug users (IDU) with a residual risk of 289 per 10,000 persons (95% CI: 191-385) with negative serology, and 20.9 per 10,000 persons (95% CI: 13.8-28.0) with additional negative NAT. Residual risk for HBV was highest in IDU with 98.6 window period infections per 10,000 with negative serology (95% CI: 36.4-212.7) and 49.4/10,000 with additional negative NAT (95% CI: 18.2-106.9). We concluded that the absolute risks of window period infection are low among Australian groups with increased risk but negative viral testing. These findings inform shared decision-making by clinicians and recipients and have the potential to increase organ donation rates from increased risk donors where risks are considered tolerable. To understand the magnitude of transmission risks from donors with positive hepatitis serology, we next examined a cohort of linked donors and recipients (Chapter 5). The aims of this work included describing the rates of transmission and non-transmission of BBV from donors with BBV to recipients, and identifying any previously unrecognized transmissions from donors perceived to pose a baseline transmission risk. To do this, we linked transplant registries with administrative health data for all solid organ donor-recipient pairs in New South Wales, Australia, 2000-2015. All new recipient notifications of HBV, HCV or HIV after transplant were identified. Proven/probable donor-transmissions within 12 months of transplant were classified according to a published US algorithm. Of 2,120 organ donors, there were 72 with a viral infection (9/72 active, 63/72 past). These 72 donors donated to 173 recipients, of whom 24/173 already had the same infection as their donor, and 149/173 did not and so were at risk of donor-transmission. Among those at risk: 3/149 recipients had proven/probable viral transmissions (1 HCV, 2 HBV); none were unrecognised by donation services. There were no deaths from transmissions. There were no donor-transmissions from donors without known BBV. Our findings confirm the safety of organ donation in an Australian cohort, with no unrecognised viral transmissions and most donors with viral infections not transmitting the virus. The results support targeted increases in donation from donors with viral infections. Data-linkage can enhance current biovigilance systems. The local evidence of transmission risks generated in Chapters 4 and 5 provide much-needed data to tackle the evidence and practice gap established in Chapters 2 and 3. This allows the implementation of changes in clinical donation and transplantation practice which could safely increase donation rates. Theme 3: Recipient infections after transplantation Another finding from the research in Chapter 5 was the identification of a substantial number of new BBV infections that were unrelated to donor transmission risk. Beyond the 3 identified proven/probable transmissions reported above, an additional 68 recipients had new virus notifications, of whom 2/68 died due to HBV infection. The infections in these 68 recipients may have been undetected late transmission, reactivating HBV, or de novo infections. Substantial preventative measures are available for these conditions including vaccination and avoiding risk-exposures. Stimulated by this concern about preventable non-transmitted infection, Chapter 6 of this thesis aims to understand the burden of notifiable infections post-transplant, which often have known preventive strategies. To do this, we conducted a cohort study of all solid organ recipients transplanted in New South Wales, Australia, 2000-2015. We used data-linkage to connect transplant registers to hospital admissions, notifiable diseases database, and the death register. We then calculated standardised incidence ratios (SIR) relative to general population notification rates, accounting for age, sex, and calendar year, and identified infection-related hospitalisations and deaths. Among 4,858 solid organ recipients followed for 39,183 person-years, there were 792 notifications. Influenza was the most common infection (532 cases, incidence 1358/100,000 person-years, 95%CI:1247-1478), with the highest prevalence within 3 months post-transplant. The second most common was salmonellosis (46 cases, incidence 117/100,000 person-years, 95%CI:87-156), followed by pertussis (38 cases, incidence 97/100,000 person-years, 95%CI:71-133). Influenza and invasive pneumococcal disease (IPD) in transplant recipients showed significant excess cases compared with the general population (influenza SIR 8.5, 95%CI:7.8-9.2, IPD SIR 9.8, 95%CI:6.9-13.9), with high hospitalisation rates (47% influenza cases, 68% IPD cases) and some mortality (four influenza and one IPD deaths). By 10 years post-transplant, cumulative incidence of any vaccine-preventable disease was similar for most transplanted organs at 12%, except for lung recipients, where it was nearly 30%. Gastrointestinal diseases, tuberculosis and legionellosis had excess cases among transplant recipients, although there were few sexually transmitted infections and vector-borne diseases. Improved vaccination programs, health education, and pre-transplant donor and recipient screening have the potential to reduce preventable infections among transplant recipients. Conclusion: This thesis explores some ways in which infectious diseases impact organ donation and transplantation, and identifies areas where clinical practice could be improved. Transplant clinicians need more support to interpret hepatitis transmission risk as they have variable interpretations of suitability. Many potential donors have increased risks for BBV transmission, with otherwise favourable characteristics, and did not donate. Actual transmission risks from both Australian donors with BBV and potential donors with increased risk behaviours are, in absolute terms, low. A reconsideration of risk profiles for potential donors with increased BBV transmission risk could materially increase organ donation rates in Australia. In addition, organ transplant recipients have significant post-transplant infections, including both BBV and other notifiable infections such as vaccine-preventable and gastrointestinal infections. Important infections post-transplant could be prevented by improved vaccination uptake and optimised strategies, donor and recipient screening for latent infection, and donor recipient education.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Chronic obstructive pulmonary disease (COPD) is characterized by an expiratory flow limitation, as well as an evident reduced exercise capacity compared to that of healthy age-matched individuals. Clearly, the expiratory flow limitation plays a significant role in this exercise intolerance; however, the extent of the contributions of other systemic factors remains unclear. More specifically, there is little data thus far on the role of blood flow delivery as a possible exercise limitation in COPD, especially in light of the potential interactions between cardiac output (Qc) and pulmonary hyperinflation. Thus, the purpose of this study was to compare the slope of the Qc versus oxygen uptake (VO 2) response through several submaximal cycling loads in patients with moderately severe COPD with that of age-matched healthy control subjects (CTRL). Also examined was the possibility that ventilatory constraints such as dynamic hyperinflation contribute to an abnormal Qc response. Cardiac output was measured using the CO2-rebreathing equilibrium technique during baseline conditions and cycling at 20, 40 and 65% of peak power in 17 COPD (Age: 64 +/- 8 yrs; FEV1/FVC: 37 +/- 11%; FEV1: 41 +/- 15% predicted) and 10 age-matched CTRL subjects. Inspiratory capacity (IC) was also measured for the determination of dynamic hyperinflation during the steady state exercise bouts. The results indicate that while the absolute Qc values are lower in COPD than in CTRL during moderately intense (65% peak power) cycling (11.30 +/- 2.38 vs. 15.63 +/- 2.15 L⋅min -1, p < 0.01), likely due to their lower exercise metabolic demand, the Qc/VO2 response to increasing levels of exercise intensity is normal or hyperdynamic in COPD. Indeed, the majority of patients with COPD exhibited Qc/VO2 slopes greater than 7.0, which may be indicative of a peripheral muscle bioenergetic disturbance that may drive the need for greater oxygen delivery, and thus result in an exaggerated ce

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2016.
Type 2 diabetes mellitus (T2DM) is a metabolic disorder which is characterised by insulin resistance, defective insulin secretion or both. The chronic state of hyperglycemia in diabetes is associated with microvascular complications and macrovascular complications which account for an estimated 80% of deaths as a result of cardiovascular complications. Type 2 diabetes mellitus is an inflammatory disease and pro- inflammatory stimuli in the form of activated endothelial cells, render the vascular endothelial surface attractive for both platelets and leukocytes. Activated platelets bind to the exposed extracellular matrix (ECM) and also to each other in both physiological haemostasis and pathological thrombosis. They can also adhere to leukocytes. Platelet leukocyte interactions are divided into 3 stages; these include the initiation of the interaction, stabilization of the aggregates and amplification of leukocyte activation. This study attempts to contribute to the current knowledge of T2DM by investigating the percentage of monocytes and neutrophils forming aggregates with platelets in pre-diabetes and diabetes and comparing this to non- diabetes individuals as well as the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69). Levels of platelet activation and function will be determined by both plate monocyte aggregates (PMA) and platelet neutrophil aggregates (PNA). A total of 124 individuals were recruited from Bellville South, Cape Town, South Africa. This comprised of diabetes (DM) (n=15), pre-diabetes (pre-T2DM) (n=25) and controls (n=84). All individuals were screened for diabetes using the oral glucose tolerance test (OGTT). Platelet leukocyte measurements were performed using the Navios 8-colour flow cytometer. The median percentage of circulating platelets bound to monocytes (%PMAs) was significantly increased in the T2DM 49.04[36.78-62] and the Pre-T2DM 48.96[36.72-61.2],groups, compared to the control group 7.2[5.4-9], p<0.0001. The median %PNAs, which show interactions between neutrophils and platelets, were significantly increased in the T2DM group 13.56[10.17-16.95] compared to the control group 6.01[4.51-7.51] p<0.0001. Platelet monocyte aggregates (PMAs) were higher in both the pre-T2DM and T2DM groups when compared to the control group indicating increased interactions between platelets and monocytes. In addition to forming aggregates with leukocytes, the platelets were able to initiate activation and phenotypic change to the leukocytes by increasing the expression of CD69 and TF (CD142). This finding provides further evidence that there is a link between the inflammatory process and the prothrombotic activity evident in diabetes and pre-diabetes individuals. Furthermore, we describe elevated levels of circulating activated neutrophils which directly correlate with increased PNA formation in both the pre-T2DM and T2DM group.
South African Medical Research Council

Background Cardiovascular disease is a leading cause of mortality and morbidity worldwide. Blood pressure is the single, most important risk factor for cardiovascular disease; nearly two-thirds of all strokes and approximately half of all ischaemic heart disease events are attributable to non-optimal blood pressure. The evidence from individual randomised trials of blood pressure lowering regimens suggests that that protection from major cardiovascular events can be achieved by lowering blood pressure, even among those with so-called ‘normal’ blood pressures. However, many trials are not sufficiently powered to demonstrate modest but clinically important differences in the effects of different classes of drug on the risk of cardiovascular disease. As a result, there is considerable uncertainty about the relative effectiveness of different drug classes. Reliable and precise information about the effects of treatment is central to the management of cardiovascular risk in millions of people worldwide. The aim of the research contributing to this thesis was to generate high quality evidence about the effects of a range of blood pressure lowering regimens on major cardiovascular outcomes in important patient subgroups. Methods The research uses prospectively-planned overviews (meta-analyses) of large randomised trials to generate precise estimates of treatment effect. The trials contributing to the overviews were those participating in the Blood Pressure Lowering Treatment Trialists’ Collaboration. Data from all relevant trials were submitted to the Collaboration Secretariat based at the George Institute for International Health in Sydney, Australia for inclusion in analyses. Data were combined using standard meta-analytic techniques and reported as pooled point estimates and 95% confidence intervals for the six pre-specified primary outcomes of stroke, coronary heart disease, heart failure, cardiovascular death and total mortality. The analyses comprised three main components: (1) one main set of overview analyses to examine treatment effects in the overall study population; (2) three sets of overview analyses to examine treatment effects in subgroups of younger and older patients, men and women and patients with and without diabetes; and (3) a series of post-hoc analyses (meta-analysis and meta-regression analysis) to examine the relative contributions of blood pressure-dependent and independent effects of two main classes of blood pressure drugs, ACE-I inhibitors and angiotensin receptor blockers.

In the last three decades, autohaemotherapy with blood treated with oxygen containing low concentrations of ozone, either alone or in combination with an elevated temperature and ultraviolet irradiation (HOU), has been used in Europe for the treatment of peripheral vascular diseases. Many apparently widely differing beneficial claims have been made for this therapeutic approach. This study has provided an objective rationale as to how HOU-treated blood administered as autohaemotherapy may work as a therapeutic agent. This study has investigated the effect of the treatment of blood in vitro with HOU on the blood platelets. The results indicate that either in healthy volunteers or in patients with PVD and/or diabetes, HOU-treated blood causes a dose-dependent, reversible inhibition of blood platelet aggregation in response to ADP and collagen, calcium ionophore A23187 and thrombin. The treatment of whole blood with HOU did not result in the destruction of platelets, as indicated by a lack of increase in release of the alpha-granule component platelet-derived growth factor into the plasma, and a increase in total platelet count. The concentrations of endothelium-derived relaxing factor (EDRF, known to be closely related to or identical with nitric oxide) and prostacyclin (PGI[2]) released from immune cells in the HOU-treated blood are increased by this treatment in vitro. EDRF and PGI[2] are both well known inhibitors of platelets aggregation and vasodilators. Evidence that the observed inhibition of platelet aggregation in treated blood is at least in part caused by EDRF production is provided by the reversal of the inhibition in the presence of oxy-haemoglobin, an EDRF inhibitor. An In vivo clinical study was performed on normal healthy volunteers using autohaemotherapy with blood exposed to UV light and ozone in medical oxygen (15 mug/mL) at a temperature of 42.5 °C. These volunteers were monitored by measurement of standard haematological parameters, a clinical chemistry profile and clinical symptoms and signs. These was no evidence of harmful effects of the treatment. In vivo HOU-autohaemotherapy enhanced the expression of the activation markers IL-2R, Ber-Mac3 and HLA-DR on peripheral blood mononuclear cells demonstrating for the first time objectively -measurable systemic changes in treated individuals. There was also significant increase in plasma prostacyclin concentrations measured as its stable metabolite 6-keto-PGF[1alpha] after HOU-autohaemotherapy. Endothelial dysfunction, with a reduction in the synthesis of vasodilators, particularly nitric oxide and perhaps also prostacyclin, plays a critical role in many vascular diseases. If autohaemotherapy with HOU-treated blood can restore endothelial function, as suggested by the evidence of an increase in prostacyclin levels in treated individuals, then this therapy could represent a major advance in the treatment of a number of vascular diseases.

"May 2002" Includes bibliographical references (leaves 229-251) Describes the investigation of the alteration of ABH antigen expression on the surface of red blood cells in patients with haematological malignancies.

Thesis (M.D.)--Aberdeen University, 2008.
With: Surgical revascularisation in patients with severe limb ischaemia induces a pro-thrombotic state / P. Collins ... et al. Platelets. 2006: 17(5), 311-317. With: A preliminary study on the effects of exercising to a maximum walking distance on platelet and endothelial function in patients with intermittent claudication / P. Collins ... et. Eur. J. Vasc. Endovasc. Surg. 2006: 31, 266-273. Includes bibliographical references.

Motivated by the physiological phenomena of collapse and flow limitation for a serial pulmonary artery stenosis, we investigated the three-dimensional influence of spatial configuration on the wall motion and hemodynamic. Our numerical study focused on the effect of two geometrical parameters: the relative distance and the angular orientation between the two stenoses. The collapse of a compliant arterial stenosis may cause flow choking, which would limit the flow reserve to major vital vascular beds such as the lungs, potentially leading to a lethal ventilation-perfusion mismatch. Flow through a stenotic vessel is known to produce flow separation downstream of the throat. The eccentricity of a stenosis leads to asymmetric flow where the high velocity jets impinge on the sidewall, thereby inducing significant dissipation. The additional viscous dissipation causes a higher pressure drop for a flow through a stenotic vessel, than in a straight compliant vessel. It is likely that some particular morphology would have a higher vulnerability to the fluid induced instability of buckling (divergence), under physiological pulsatile flow. It was found that fluid pressure distribution have substantial implication for the downstream wall motion, under conditions of strong coupling between nonlinear vessel geometries, and their corresponding asymmetric flow. The three-dimensional fluid structure interaction problem is solved numerically by a finite element method based on the Arbitrary Lagrangian Eulerian formulation, a natural approach to deal with the moving interface between the flow and vessel. The findings of this investigation reveal that the closeness between stenoses is a substantial indication of wall collapse at the downstream end. Moreover, the results suggest a close link between the initial angular orientation of the distal stenosis (i.e. the constriction direction) and the subsequent wall motion at the downstream end. For cases showing evidence of preferential direction of wall motion, it was found that the constricted side underwent greater cumulative displacement than the straight side, suggestive of significant wall collapse.

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers. Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease. It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.

Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. As embryonic stem cells pose a lot of ethical issues, adult stem cells, isolated from various sources like cord blood, bone marrow or adipose tissue, are being considered as a realistic option due to their well documented therapeutic potentials. In our lab, we have standardised a method to isolate fibroblastic multipotent stem cells (PBMCs) from human peripheral blood, that are able to sustain long term in vitro culture and differentiate towards adipogenic, chondrogenic and osteogenic lineage. In this work, PBMCs were stimulated to obtain in vitro neuronal and myogenic-like cells. Moreover, their restorative potential in degenerative diseases of skeletal muscle and nervous tissue was evaluated using in vivo models. In order to test the neuronal differentiation potential, the cells were seeded (1x104) on gelatin coated dishes and cultured for 7 days in neurobasal medium with EGF and FGF followed by Retinoic acid and NGF for next 7 days. Myogenic induction was carried out using IGF and ascorbic acid for 14 days. At different time points, morphological studies were performed by SEM and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. PBMCs showed characteristic dendrite like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of PBMCs under stimulation with KCl 56 mM and the secretion of the neurotransmitter, noradrenalin, a precursor in the dopamine synthesis confirmed their ability to acquire a functional phenotype. When premarked by a cell tracker Qdot 800 and injected stearotactically into a rat brain, PBMCs showed to be migratory and proliferative as detected after 10 and 20 days of injection. No tumor mass was identified. The myogenic potential of PBMCs were confirmed by their ability to form syncitium like structures in in vitro culture and to express typical myogenic markers both at early and late phases of differentiation. PBMCs were showed to integrate within the host tissue and to take part in tissue repair as demonstrated in a bupivacaine induced muscle damage model.
Il trapianto di cellule staminali è una strategia terapeutica che sta conoscendo uno sviluppo sempre maggiore come possibile approccio clinico per il trattamento delle malattie degenerative. Considerando i problemi di carattere etico sollevati dall’impiego delle cellule staminali embrionali, le cellule staminali adulte isolate da varie fonti (sangue cordonale, midollo osseo, tessuto adiposo﴿ rappresentano una realistica alternativa, in virtù della loro potenzialità rigenerativa ben documentata. Nel nostro laboratorio è stato standardizzato un metodo per isolare cellule staminali fibroblastoidi multipotenti (Peripheral Blood Multipotent Cells, PBMC) da sangue periferico umano, che possono essere espanse in vitro durante la coltura a lungo termine e sono in grado di differenziare in senso adipogenico, condrogenico e osteogenico. Nel lavoro di tesi del Dott. Senthilkumar Rajendran, le cellule PBMC sono state stimolate per l’ottenimento in vitro di cellule simil-neuronali e -muscolari. Inoltre è stato valutato il loro potenziale rigenerativo nel trattamento di malattie degenerative del muscolo scheletrico e del tessuto nervoso attraverso la sperimentazione in vivo su modelli animali. Al fine di testare il potenziale di differenziazione neuronale, le cellule sono state seminate (1x104) su coating di gelatina e coltivate per i primi 7 giorni in Neurobasal medium addizionato con EGF e FGF, e per i 7 giorni successivi in terreno basale contenente acido retinoico e NGF. L’induzione miogenica è stata effettuata utilizzando IGF e acido ascorbico per 14 giorni. Ad ogni time point, sono stati realizzati studi morfologici mediante SEM e analisi di espressione di specifici marcatori neuronali e miogenici mediante RT-PCR, citofluorimetria e western blot. Le cellule PBMC hanno mostrato una caratteristica morfologia simil-dendritica e l’espressione di specifici marcatori neuronali a livello sia di mRNA che di proteine. Lo studio del flusso del calcio dopo stimolazione con KCl 56 mM e l’attività di secrezione del neurotrasmettitore noradrenalina, precursore nella sintesi della dopamina, hanno confermato la capacità delle cellule PBMC di acquisire un fenotipo funzionale. Dopo marcatura con il tracker cellulare Qdot 800 e iniezione per stereotassi in un cervello di ratto, le PBMC hanno dimostrato un elevato potenziale migratorio e proliferativo dopo 10 e 20 giorni dall'impianto. Non è stata identificata alcuna massa tumorale. Il potenziale miogenico delle popolazioni isolate è stato confermato dalla loro capacità di formare strutture simil-sinciziali durante la coltura in vitro e di esprimere marcatori tipici della linea miogenica, sia a tempi precoci che nelle fasi tardive del differenziamento. Infine, testate in un modello animale di danno muscolare indotto con bupivacaina, le cellule PBMC sono state in grado di integrarsi all'interno del tessuto ospite e di prendere parte nella riparazione dei tessuti.

This study explored the narratives people living with sickle cell disorder construct to explain how the experience of employment influences their identity and subsequently their quality of life. Nine individuals with sickle cell disorder were interviewed and gave detailed autobiographical narratives which were transcribed and processed using narratives analysis. Participants were 4 men and 5 women aged between 22 and 60 years. Participants' passion for their job roles was illustrated by their positive work identities. Earlier experiences were felt to be influential on drive and resilience to work challenges. In the narratives, poor understanding of sickle cell disorder by organisations and reduced ability to manage their condition impacted physical health; psychological and emotional wellbeing; social and cultural experiences. This study has implications for the clinical practice and future research of adults living with sickle cell disorder, contributing to the broadening general understanding of sickle cell disorder.

This thesis describes studies into the pathogenesis of Cholesterol—related early vascular injury. In addition, this thesis examines the potential reversibility of this injury with 2 agents: a) simvastatin, an HMGCoA reductase inhibitor, in a model independent of any reduction in lipids and 2) high dose aspirin. Hypercholesterolaemia led to a decrease in NO bioavailability, in association with a decrease in the enzyme, endothelial nitric oxide synthase and increased oxidative stress. In addition, there was an increase in the pro—inflammatory transcription factor, NF—KB, in the intima of the epicardial coronary arteries. Moreover, activated NF—KB was present in macrophages, foam cells and vascular smooth muscle cells in coronary atheromatous plaque and its expression increased in unstable coronary syndromes. These data support a role for NF—KB in the pathogenesis of early atherosclerosis and the development of unstable coronary syndromes. In addition, this thesis demonstrated for the first time that simvastatin, an HMGCoA reductase inhibitor, preserves endothelium—dependent vasorelaxation in both large and small coronary vessels in porcine experimental HC, despite no reduction in plasma lipids. This effect was associated with normalisation of eNOS protein levels. Furthermore, in vivo plasma markers of oxidative stress were attenuated by treatment with simvastatin. However, there was no attenuation in the activation of the proinflammatory transcription factor, NF—KB. These studies suggest a role for the HMGCoA reductase inhibitors in reducing cardiac morbidity and mortality, beyond their effect on cholesterol levels. The current studies also demonstrated that high dose aspirin therapy preserved endothelial function in large coronary vessels. This alteration in the generation of prostanoids in favour of vasodilatation may be an important component of the therapeutic benefit of aspirin in HC-induced atherosclerosis. In summary, results of studies described in this thesis provide insights into the molecular mechanisms that may be responsible for early vascular injury in hypercholesterolaemia and its reversibility with the therapeutic agents, simvastatin and high dose aspirin.

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Tese (Doutoramento)
IPEN/D
Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP