C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects. Our initial work identified CTRP3’s promise as an effective treatment for Nonalcoholic fatty liver disease (NAFLD). Specifically, we demonstrated that mice fed a high fat diet failed to develop NAFLD when treated with CTRP3. The purpose of this current project is to identify putative receptors which mediate the hepatic actions of CTRP3. Methods We used Ligand-receptor glycocapture technology with TriCEPS™-based ligand-receptor capture (LRC-TriCEPS; Dualsystems Biotech AG). The LRC-TriCEPS experiment with CTRP3-FLAG protein as ligand and insulin as a control ligand was performed on the H4IIE rat hepatoma cell line. Results Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis (FACS) demonstrated successful oxidation and crosslinking of CTRP3-TriCEPS and Insulin-TriCEPS complexes to cell surface glycans. Demonstrating the utility of TriCEPS under these conditions, the insulin receptor was identified in the control dataset. In the CTRP3 treated cells a total enrichment of 261 peptides was observed. From these experiments 5 putative receptors for CTRP3 were identified with two reaching statistically significance: Lysosomal-associated membrane protein 1 (LAMP-1) and Lysosome membrane protein 2 (LIMP II). Follow-up Co-immunoprecipitation analysis confirmed the association between LAMP1 and CTRP3 and further testing using a polyclonal antibody to block potential binding sites of LAMP1 prevented CTRP3 binding to the cells Conclusion The LRC-TriCEPS methodology was successful in identifying potential novel receptors for CTRP3. Relevance The identification of the receptors for CTRP3 are important prerequisites for the development of small molecule drug candidates, of which none currently exist, for the treatment NAFLD.

Adipose tissue inflammation has an impact on liver health and it has been demonstrated that chronic alcohol consumption leads to the expression of pro-inflammatory markers in the adipose tissue. A thorough characterization of alcohol-induced adipose inflammation is lacking, and is important to understand in order to identify immune-related mechanisms that drive this phenomenon. Current therapeutic regimens for alcoholic liver disease are ineffective. It is critical to understand how other organs influence liver injury in this disease when developing novel and effective therapies in the future. Alcoholic liver disease exhibits a sexual dimorphism; women are more susceptible to liver injury than men and the same paradigm exists in rodent models. Here, I demonstrate that female mice have greater alcohol-induced adipose tissue inflammation than male mice, evidenced by greater expression of pro-inflammatory cytokines and cell markers. Further, female mice also exhibit higher expression of toll-like receptor genes in the adipose tissue, suggesting a potential role for the innate immune system in alcohol-induced adipose inflammation. Toll-like receptor 4 (TLR4) has been demonstrated to drive inflammation in both the liver and adipose tissue. I used both germline and conditional knockouts of Tlr4 to characterize alcohol-induced changes in the immune cell composition of adipose tissue. Alcohol increased the number of pro-inflammatory adipose tissue macrophages. This macrophage phenotype switching is partially dependent on TLR4; germline, but not myeloid-specific, Tlr4-deletion prevents macrophage phenotype switching. Overall, my work demonstrates that alcohol-induced adipose tissue inflammation is related to liver injury and that TLR4 contributes to adipose macrophage phenotype switching.

Patients with type 2 diabetes (T2D) are at risk for non-alcoholic fatty liver disease (NAFLD) and associated complications. This study evaluated the performance of international (EASL-EASD-EASO) and national (DGVS) guidelines for NAFLD risk stratification. Patients with T2D prospectively underwent ultrasound, liver stiffness measurement (LSM) and serum-based fibrosis markers. Guideline-based risk classification and referral rates for different screening approaches were compared and the diagnostic properties of simplified algorithms, genetic markers and a new NASH surrogate (FAST score) were evaluated. NAFLD risk was present in 184 of 204 screened patients (age 64.2 ± 10.7 years; BMI 32.6 ± 7.6 kg/m2). EASL-EASD-EASO recommended specialist referral for 60–77% depending on the fibrosis score used, only 6% were classified as low risk. The DGVS algorithm required LSM for 76%; 25% were referred for specialised care. The sensitivities of the diagnostic pathways were 47–96%. A simplified referral strategy revealed a sensitivity/specificity of 46/88% for fibrosis risk. Application of the FAST score reduced the referral rate to 35%. This study (a) underlines the high prevalence of fibrosis risk in T2D, (b) demonstrates very high referral rates for in-depth hepatological work-up, and (c) indicates that simpler referral algorithms may produce comparably good results and could facilitate NAFLD screening.

Mesenchymal stem cells and their regenerative and immunomodulatory potential Abstract Mesenchymal stem cells (MSCs) possess multidirectional regenerative ability, which, together with their immunomodulatory potential, makes them promising cell type for therapy of wide variety of diseases. Despite ongoing research, which proved MSCs application to be safe, reported effect of MSCs administration on patients is not convincingly beneficial yet. In our work we focused on elucidation of MSCs role in regeneration of vital organs, heart and liver, where a large damage is life threatening for patients and any improvement in therapy would save many lives. Similar situation is in Graft versus host disease (GVHD), where MSCs immunomodulatory properties could be beneficial. Role of MSCs in heart regeneration was examined in vitro. Primary adult swine cardiomyocytes (CMCs) were co-cultured with or without swine MSCs for 3 days and morphological and functional parameters (contractions, current, respiration) of CMCs were measured. MSCs showed supportive effect on CMCs survival, especially at day 3 of the experiment, where in co-culture was significantly higher number of viable CMCs with physiological morphology and maintained function. Effect of MSCs on liver regeneration was observed in swine model of chronic liver...

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Background: Dogs with acquired chronic liver disease often have hemostatic derangements. It is currently unknown whether dogs with acquired chronic liver disease have decreased platelet function and alterations in von Willebrand factor (vWF) that may contribute to hemostatic abnormalities. Hypothesis: Dogs with chronic liver disease have prolonged platelet closure time (CT), assessed with the PFA-100®, and buccal mucosal bleeding time (BMBT), and increased vWF concentration compared to healthy dogs. Animals: Eighteen dogs with chronic acquired liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy and eighteen healthy age-matched control dogs. Methods: Prospective study. BMBT, CT using the PFA-100®, and vWF antigen were measured in dogs with chronic liver enzyme elevation undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy. After undergoing ultrasound-guided needle biopsy, dogs were monitored for hemorrhage with serial packed cell volume measurements and focused assessment with sonography. An unpaired t-test was used for normally distributed data and the Mann-Whitney test was used when non-Gaussian distribution was present. The level of significance was set at P <0.05. Results: The CT was not different between the two groups (P = 0.27). The BMBT was significantly longer in the liver disease group compared to the control group (P = 0.019). There was no difference in the mean vWF antigen of the two groups (P = 0.077). Conclusions and clinical relevance: These results demonstrate mild impairment of primary hemostasis in dogs with chronic liver disease based on prolongation of BMBT. / Master of Science / Background: Dogs with chronic liver disease often have abnormal blood clotting activity. It is currently unknown whether dogs with chronic liver disease have decreased platelet function and alterations in von Willebrand factor (vWF) that may contribute to blood clotting abnormalities. Platelet function can be assessed using the PFA-100®, which measures platelet closure time (CT), and buccal mucosal bleeding time (BMBT). The PFA-100 simulates blood in circulation to assess platelet function. BMBT is a crude but readily available test to assess platelet function in practices without sophisticated methods of assessing platelet function. Hypothesis: Dogs with chronic liver disease have prolonged CT and BMBT, which both suggest platelet dysfunction. Additionally, dogs with chronic liver disease have increased vWF concentration compared to healthy dogs. Animals: Eighteen dogs with chronic acquired liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy and eighteen healthy age-matched control dogs. Methods: Prospective study. BMBT, CT, and vWF antigen were measured in dogs with chronic liver disease undergoing ultrasound-guided needle biopsy of the liver or laparoscopic liver biopsy. After undergoing ultrasound-guided needle biopsy, dogs were monitored for hemorrhage. Results: The CT was not different between the two groups but the BMBT was significantly longer in the liver disease group compared to healthy dogs. There was no difference in the mean vWF antigen between the two groups. Conclusions and clinical relevance: These results demonstrate mild impairment of blood clotting activity in dogs with chronic liver disease based on prolongation of BMBT compared to healthy dogs. Prolongation of BMBT compared to healthy dogs is suggestive of endothelial dysfunction and/or platelet dysfunction in dogs with chronic liver disease.

Kroniska leversjukdomar utgör en stor utmaning för sjukvårdssystemen som idag inte kan möta det ökande behovet av levertransplantationer hos patienter som befinner sig i sjukdomens slutskedet. Nationella screeningsprogram för att tidigt upptäcka leversjukdomar har visat sig svåra att införa på grund av specificitetsproblem i kombination med en tidig och ofta asymptomatisk sjukdomsbild vilket gör leversjukdomar svårupptäckta. Detta i kombination med stora risker och kostnader som är förknippade med diagnostiska och prognostiska standardmetoder, såsom leverbiopsi, har gjort det komplicerat att upptäcka sjukdomen tidigt. Utvecklingen av tillförlitliga och minimalt invasiva metoder bör därför prioriteras för att minska transplantationsbehovet av leversjukdomspatienter. Denna studie syftar till att bidra till detta arbete genom att undersöka optimala biomarkörer för leversjukdom genom robusta kvantifieringsmetoder med klinisk potential. I detta examensarbete används riktad masspektrometri och tunga rekombinanta proteinstandarder för att identifiera och absolutkvantifiera 108 proteiner i 2 μL plasma från 245 patienter med leversjukdom samt friska individer. Diagnoserna är spridda över olika stadier och patienterna har olika sjukdomsprogression. Genom differentierad expressionsanalys av proteinnivåerna och vägledd maskininlärning har biomarkörpaneler för stratifiering av leverfibros tagits fram. Detta som en del av The Human Disease Blood Atlas, vars målsättning är att utforska sjukdomsassocierade proteinprofiler och bygga en öppen databas tillgänglig för forskare världen över. Dessa resultat erbjuder lovande vägar för förbättrad diagnostik och personligt anpassade behandlingsstrategier inom leversjukdomshantering. Strategin är kompatibel med teknologier som underlättar biomarkörupptäckt och minimalt invasiv provtagning. / Advanced liver disease poses significant challenges to healthcare systems, which cannot meet the needs of liver transplantation for end-stage liver disease patients. This can be mitigated by the early detection of chronic conditions. However, chronic liver disease tends to be asymptomatic in its early stages and current diagnostic and prognostic gold standard methods, such as liver biopsy, can be expensive and risky, discouraging population-wide screenings. Thus, the development of reliable and minimally invasive methods should be a priority to enhance liver disease patient outcomes. This study aims to contribute to this effort by investigating optimal liver disease biomarkers through robust quantification methods with translational potential. Here, targeted mass spectrometry and heavy recombinant peptide isotopes (SIS-PrEST) are employed for the identification and absolute quantification of more than 100 proteins in small amounts of plasma from over 300 liver disease patients, whose conditions have different etiologies and stages of disease progression. Through differential expression analysis and supervised machine learning, potential biomarker panels for liver disease etiologies and fibrosis levels are identified as part of the Human Disease Blood Atlas’s effort to explore disease-specific protein profiles and build an open-source database accessible to scientists worldwide. These findings offer promising avenues for improved diagnostics and personalized treatment strategies in liver disease management and the strategy is compatible with technologies that facilitate biomarker discovery and minimally disruptive sampling.

Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores. / AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer. Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit. ‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik. Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.

Title: Using of alternative therapeutic techniques to vertebrovisceral links of liver and gallbladder diseases Objectives: The aim of this thesis is to find out a sufficient amount of information on the subject of functional liver and gallbladder diseases, possibilities of conventional treatment and alternative treatment options. Another goal of this thesis is to compile an assessment protocol, based on the theoretical background, according to which it is possible to diagnose patients with functional liver and gallbladder diseases. An additional goal of this thesis is to create a treatment concept for patients with functional liver and gallbladder diseases. This treatment concept will be based on the knowledge of yoga and traditional Chinese medicine. Last goal is to compare conventional physiotherapy with the concept created. Methods: The theoretical part of this thesis focuses on the issue of functional disease of liver and gallbladder. In takes into account the current research and data found in Czech and foreign literature. The practical part looks into the effects of complex therapy methods using experiments. The therapy itself includes changes of the patients' lifestyles, which are based on traditional Chinese medicine and yoga asanas, which were compiled for the purposes of this thesis for...