Orientador: Maria Jaqueline Mamprim / Banca: Luiz Henrique de Araújo Machado / Banca: Luciana Del Rio Pinoti Ciarlini / Banca: Tilde Rodrigues Froes / Banca: Fabiano Séllos Costa / Resumo: O objetivo do estudo foi avaliar as alterações parenquimatosas difusas hepáticas pela ultrassonografia em modo B e, quantitativamente, através do histograma, assim como as alterações vasculares, através da ferramenta Doppler, a fim de correlacionar os resultados obtidos às afecções hepáticas diagnosticadas pelo exame histopatológico. Foram submetidos à biopsia percutânea 41 cães com alterações hepáticas difusas detectadas pela ultrassonografia. O trabalho foi divido em três etapas. No primeiro estudo 25 cães com hepatite crônica, agrupados segundo o grau de fibrose, foram avaliados pela ultrassonografia em modo B. Quatro padrões de ecotextura foram descritos, concomitante ao aumento difuso da ecogenicidade hepática, sendo possível associar três destes padrões ao grau da fibrose hepática. Em um segundo estudo 25 cães com hepatite crônica e 11 cães com neoplasia hepática foram avaliados através da ultrassonografia Doppler. Mapeamento vascular do parênquima hepático foi realizado através do Doppler colorido e mensurações quantitativas pelo Doppler espectral. As alterações hemodinâmicas observadas na doença hepática crônica como portalização do fluxo da veia hepática, dilatação da artéria hepática, circulação portal colateral adquirida e fluxo portal hepatofugal, tornaram-se mais evidentes à medida que o grau de fibrose se agravou, permitindo a associação de alterações hemodinâmicas significativas com o estágio avançado da doença, conferindo, assim, um valor prognóstico à avaliação Doppler na doença hepática crônica dos cães. O estudo Doppler também auxiliou a diferenciar as hepatites crônicas das neoplasias hepáticas difusas, através da detecção de alterações vasculares sugestivas de malignidade, como hipervascularização caótica do parênquima hepático. No terceiro estudo uma... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to assess diffuse hepatic parenchymal changes, subjectively, by B mode ultrasonography and, quantitatively, by histogram, as well as the vascular changes, by Doppler ultrasound, in order to correlate the results to hepatic disorders, diagnosed by histopathology. A total of 41 dogs with diffuse liver disorders detected by ultrasonography underwent percutaneous hepatic biopsy. In the first study 25 dogs with chronic hepatitis, grouped based on the severity of fibrosis, were evaluated by B mode ultrasonography. Diffuse increase in hepatic echogenicity and four different echotexture patterns were described, and in 3 cases, associated with the degree of fibrosis. In the second study 25 dogs with chronic hepatic disease, grouped based on the severity of fibrosis, and 11 dogs with diffuse neoplasia, were assessed by Doppler ultrasonography. Vascular mapping of the hepatic parenchyma was performed using color Doppler, and quantitative measurements by spectral Doppler. Hemodynamic changes detected in chronic liver disease such as hepatic vein flow portalization, hepatic artery dilatation, acquired portal collateral circulation and hepatofugal flow in the portal vein were more evident as the degree of fibrosis became worse, thus significant hemodynamic changes were associated with the advanced stage of disease in dogs. Doppler studies also aided to differentiated chronic hepatitis and diffuse hepatic neoplasia, detecting vascular changes suggestive of malignance, such as chaotic hypervascularity of liver parenchyma. In the third study quantitative assessment of liver echogenicity and echotexture by histogram was performed in 10 healthy dogs, 5 dogs with hepatic fatty degeneration and 25 dogs with chronic hepatitis. It was observed quantitatively increased echogenicity in dogs with hepatic fatty... (Complete abstract click electronic access below) / Doutor

Orientador: Maria Inês de Moura Campos Pardini / Coorientador: Rejane Maria Tommasini Grotto / Banca: Giovanni Faria Silva / Banca: Alexandre Neime Barbosa / Banca: Liliana Moura Massis / Banca: Paulo Inácio da Costa / Resumo: Não disponível / Abstract: Not available / Doutor

Introdução: O envolvimento hepático na doença celíaca (DC) é amplamente reconhecido e atualmente é uma das manifestações extra-intestinais mais freqüentes. Com o advento de marcadores sorológicos de elevada especificidade e sensibilidade, sobretudo o anticorpo antiendomísio (EMA), a DC tem sido descrita em associação a várias hepatopatias. Objetivos: caracterizar as formas de triagem de DC em portadores de hepatopatia crônica; caracterizar e estudar os pacientes cujo diagnóstico de DC foi realizado durante a investigação de uma doença hepática; pesquisar a reatividade do antiendomísio em pacientes com hepatite auto-imune, cirrose biliar primária, colangite esclerosante primária e hipertensão portal não cirrótica; avaliar o comportamento da doença hepática na vigência de dieta sem glúten. Métodos: Os pacientes foram triados pela detecção dos anticorpos anti-reticulina e anticorpo antimatriz extracelular durante a rotina de imunofluorescência de pesquisa dos auto-anticorpos hepáticos; pela presença de manifestações de DC em hepatopatas crônicos, pelo aspecto endoscópico sugestivo de DC e pela pesquisa sistemática do EMA nas patologias referidas anteriormente. Todos os pacientes foram submetidos à pesquisa do EMA, anti-reticulina IgG ou antimatriz de fibroblastos IgG na presença de deficiência de IgA. Em caso de positividade desses marcadores, foram submetidos à endoscopia digestiva alta para biópsia intestinal e caracterizados do ponto de vista clínico, laboratorial e histopatológico. A evolução desses dados permitiu a caracterização da evolução da doença hepática e da doença celíaca a partir da introdução da dieta sem glúten. Resultados: Foram identificados 43 pacientes com auto-anticorpos relacionados à DC (em 42 o EMA IgA e em um o antimatriz extracelular IgG em associação com deficiência de IgA). A rotina de pesquisa de auto-anticorpos hepáticos identificou 31 pacientes; seis apresentavam hepatopatia crônica e manifestação de DC; em três o exame endoscópico foi sugestivo de DC e a pesquisa sistemática do EMA foi positiva em três casos. O diagnóstico de DC foi confirmado em 37 de 40 pacientes (92,5%) em que a biópsia intestinal foi realizada. A idade dos pacientes variou de 2 a 68 anos, com mediana de 35 anos. Houve maior prevalência de acometimento no sexo feminino (65%). A DC foi mais prevalente na raça branca (87%), mais foi identificada em quatro mulatos e um negro. As doenças hepáticas mais freqüentes foram hipertransaminasemia criptogênica, hepatite auto-imune, hiperplasia nodular regenerativa e hepatite pelo vírus C. Conclusões: 1) A reatividade do anti-reticulina, a presença de diarréia inexplicada e a análise endoscópica da mucosa duodenal foram as formas de seleção mais efetivas de se identificar a DC em hepatopatas crônicos. 2) A ausência de manifestações clínicas de DC nesse grupo de pacientes foi bastante expressiva. 3) A pesquisa sistemática do EMA em cirrose biliar primaria, hepatite auto-imune, colangite esclerosante primária não contribuiu para o diagnóstico de DC em um número significativo de pacientes, ao contrário do observado no grupo de hipertensão portal não cirrótica, especialmente hiperplasia nodular regenerativa 4) As doenças hepáticas em que mais freqüentemente foi diagnosticada a DC foram a hiperplasia nodular regenerativa, hepatite auto-imune, hipertransaminasemia criptogênica, hepatite pelo vírus C e cirrose biliar primária antimitocôndria negativo. 5) A retirada do glúten da dieta contribuiu de maneira efetiva para normalização das enzimas hepáticas nos casos de hipertransaminasemia criptogênica. Nos grupos de hiperplasia nodular regenerativa, hepatite B e C, cirrose biliar primária, álcool e hepatite auto-imune, o papel da dieta foi de difícil avaliação em razão da interferência da instituição do tratamento específico e da evolução natural da doença hepática de base. / Introduction: The hepatic involvement in Celiac Disease (CD) is well known and widely regarded as a frequent extra-intestinal manifestation. With the advent of highly specific and sensitive serological markers, especially the antiendomysial antibody (EMA), CD has been described in association with several liver conditions. Objectives: To characterize ways for screening patients with chronic liver conditions in order to diagnose CD, to characterize and study patients whose CD diagnoses were performed when investigating hepatic diseases, to test the reactivity of EMA in patients with autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis and non-cirrhotic portal hypertension, to evaluate the course of the hepatic disease in gluten-free diet. Methods: Patients were selected by the detection of antireticulin and anti-extracellular matrix antibodies during routine immunoflourescence determination for hepatic autoantibodies, by the presence of CD manifestations in chronic patients with liver diseases, by the endoscopic aspects suggestive of CD and by systematic search for EMA in the above mentioned pathologies. All patients were submitted to tests for EMA, antireticulin IgG or antimatrix of IgG fibroblasts in IgA deficiency. When testing positive for these markers, patients were submitted to upper digestive endoscopy for intestinal biopsy, and were also characterized from the clinical, laboratorial and histological point of view. The assessment of these data enabled the characterization of the hepatic condition as well as the CD from the onset of a gluten-free diet. Results: 43 patients with autoantibodies related to CD were identified (42 tested positive for IgA EMA and 1 for IgG extracellular antimatrix in the presence of IgA deficiency). Routine determination of hepatic autoantibodies identified 31 patients. Of those, 6 presented chronic liver diseases and CD manifestations. In 3 patients, the endoscopic exam was suggestive of CD; systematic EMA determination was positive in all three cases. The CD diagnosis was confirmed in 37 out of 40 patients (92.5%) that performed intestinal biopsy. Patients aged between 2 and 68 years (median: 35 years). Female patients were most affected (65%). CD was more prevalent in white patients (87%), but was also found in four mulattoes and 1 black person. The most common liver disorders were cryptogenic hypertransaminasemia, autoimmune hepatitis, nodular regenerative hyperplasia and chronic hepatitis C. Conclusions: 1) The reactivity of antireticulin, the presence of unexplained diarrhea and the endoscopic analysis of the duodenal mucous were the most effective ways to identify CD in chronic liver diseases. 2) The absence of CD clinical manifestations in this group of patients was impressive. 3) Contrary to what was observed in the group with non-cirrhotic portal hypertension, especially regenerative nodular hyperplasia, the systematic determination of EMA in primary biliary cirrhosis, autoimmune hepatitis and primary sclerosing cholangitis did not contribute to CD diagnosis in a significant number of patients. 4) CD was most frequently diagnosed in the following liver diseases: cryptogenic hypertransaminasemia, autoimmune hepatitis, nodular regenerative hyperplasia and chronic hepatitis C and negative antimitochondrial primary biliary cirrhosis. 5) The removal of gluten from the diet contributed effectively to bring the hepatic enzymes levels back to normal in cases of cryptogenic hypertransaminasemia. However, the role of diet was difficult to evaluate in nodular regenerative hyperplasia, autoimmune hepatitis, alcohol disease and primary biliary cirrhosis groups due to the nature of the specific treatments and the natural course of the hepatic conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / In present study, single quantum (SQ) and triple quantum filtered (TQF) 23Na magnetic resonance imaging (MRI) was used to monitor the severity and progression of CCl4 induced acute and chronic liver damage in rat model. SQ 23Na MRI was proposed to measure the 23Na signal intensity (SI) of total tissue sodium ions, and TQF 23Na MRI was proposed to measure the SI of intracellular sodium ions. In addition, shift reagent aided 23Na and 31P magnetic resonance spectroscopy (MRS) was used to measure in vivo intracellular sodium concentration ([Na+i]), total tissue sodium concentration ([Na+t]) and relative extracellular space (rECS) of liver in the same model. In acute high dose CCl4 intoxication, 24 hours after single dose of CCl4 in 5ml per kg body weight of mixture of CCl4 and oil in 1:1 ratio, SQ 23Na SI increased by 83% and TQF 23Na SI increased by 174% compared to the baseline level. According to SR-aided 23Na and 31P MRS, [Na+i] increased by 188% and [Na+t] increased by 43%. In addition, there was significant decrease in cellular energetic level, represented by ATP/Pi ratio. Histology examination showed pronounced inflammatory response in centrilobular regions, with neutrophiles infiltration, fatty accumulation and swollen hepatocytes. In chronic 8-week experiment, chronic damage was induced by biweekly administration of CCl4 in a dosage of 0.5 ml per kg body weight. From week 1 to week 6, SQ 23Na SI remained relatively constant, and then increased by 15% from week 6 to week 8. TQF 23Na SI progressively increased from week 1 to week 8, totally by 56%. Both SQ and TQF 23Na SI showed significant difference between treated group and control at every week. SR-aided 23Na and 31P MRS experiment showed that, at the end of 8-week CCl4 intoxication, both [Na+t] and rECS were higher than control, by 49% and 47% respectively; however, there was no significant difference for [Na+i] between two groups. Histology examination showed excessive deposition of extracellular matrix. In conclusion, SQ and TQF 23Na MRI appears valuable in the functional assessment of liver in noninvasive approach, and could be a promising diagnostic modality for liver diseases in clinical area.

Background: Improvement in neonatal care has changed the features of severe hyperbilirubinemia and reduced the number of babies who need exchange transfusion (ET) to avoid bilirubin-induced neurological dysfunction. We conducted this study to determine the demographic and clinical characteristics of the exchanged babies, in order to identify their risk factors, and to determine the adverse effects and outcomes associated with ET. Methodology: This was a retrospective descriptive study, reviewing folders of infants who required ET at CMJAH from June 2006 to December 2011. Results: There were 63 patients who underwent 66 exchange transfusions. Patients exchanged in the neonatal unit accounted for 60.3%, with the rest of the patients (39.7%) being exchanged in the general ward. Preterm babies accounted for 45.7%, and the majority were inborn (44%). The majority were male (58.7%), term (54.3%), and the mean birth weight was 2.29 Kg (±0.89). The median age at exchange was 5 days (mean 4.5 days ±2.1 SD). The cause of jaundice was undetermined in most patients (84.1%), while ABO incompatibility and Rhesus disease accounted for 7.9% and 6.3%, respectively. Seven babies (11.1%) had an abnormal neurological examination before exchange and five (7.9%) were labelled as kernicterus. The mean bilirubin before exchange was 325 mmol/l ±118. The complications of ET were seen in 22.2% of patients. These were Necrotising Enterocolitis (NEC) (1.58%); seizure (1.58%); apnoea (4.76%); bleeding (3.1%); renal failure (3.1%); hypoglycaemia (4.76%); thrombocytopenia (67.6%); and hypercalcemia (85%). We had three deaths, of which two were due to neonatal sepsis acquired prior to exchange, with one case of perforated NEC in an infant with other comorbidities. Hence, the mortality associated with ET in our study was 1.5 percent. At discharge, three infants remained with signs of kernicterus (4.7%). Conclusion: Kernicterus remains a cause of concern in our settings, and mechanisms ought to be put in place to detect severe jaundice in discharged term babies who may benefit from early phototherapy (PTT) and ET; as this is shown to be a relatively safe procedure in our settings, especially in infants without other severe comorbidities. ACKNOWLEDGEMENTS I would like to take this opportunity to thank Prof Daynia Ballot, my research supervisor, who has been an inspiration for research and accepted the task of guiding me through the challenging journey of conducting and writing this review.

No description available.

In my dissertation, I have worked on two distinct projects related to the immune system. The abstracts for the two projects that make up my dissertation work are below. In the first project (presented in Chapter 2), we study regulatory T (Treg) cells in chronic liver disease. Current dogma holds that Treg cells preserve tissue function in settings of inflammation and damage. Consistent with this, herein we observe that Treg cells – in particular those producing the epidermal growth factor receptor (EGFR)-ligand amphiregulin (Areg) – are enriched in the livers of mice and humans with non-alcoholic steatohepatitis (NASH). Mouse and human Treg cells undergo substantial transcriptional changes in chronic liver damage, reflecting their increased activation; however, rather than playing a protective role, we find that Treg cell–derived Areg promotes NASH-induced liver fibrosis, the key prognostic factor for patients – through the direct activation of EGFR on hepatic stellate cells. Clinically, NASH is closely linked to insulin resistance, but the mechanistic contributions of NASH-induced disease processes to insulin resistance has hitherto been unclear. We further observe that Treg cell–derived Areg promotes glucose intolerance in a NASH-dependent manner, also mediated through EGFR signaling on hepatic stellate cells. Mechanistically, in the setting of NASH, we find that Areg from Treg cells promotes hepatocyte gluconeogenesis – through hepatocyte detection of fibrosis development and soluble mediators, including IL-6, produced by activated hepatic stellate cells – offering new insight into the cellular interplay of how chronic liver disease promotes insulin resistance. Taken together, we provide the first evidence that Treg cells mediate a maladaptive role in tissue injury, finding that their production of a growth factor plays a central role in liver disease and promotes liver fibrosis and glucose intolerance in NASH. In the second project (presented in Chapter 3), we use engineered bacteria to produce chemokines in the tumor to promote anti-tumor immunity. Tumors employ multiple mechanisms to actively exclude immune cells involved in anti-tumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models – an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells – using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive anti-tumor immune responses, offering a new cancer immunotherapy strategy.

Eighteen healthy dogs were divided into three equal groups. All dogs were evaluated at the beginning of the experiment with complete physical examination, complete blood count, serum alanine aminotransferase, serum alkaline phosphatase, serum bilirubin, serum albumin, sulfobromophthalein. excretion test, ammonia tolerance test, glucagon response test, portal and intraparenchymal pressures, operative mesenteric portography, and histologic assessment of hepatic tissue. The left hepatic vein was ligated in the chronic and acute dogs. The dogs had a ligature placed loosely around the left hepatic vein. Acute and control dogs were evaluated 24 hours postoperatively with the hematologic and biochemical tests listed above. Acute dogs were evaluated with portal and intraparenchymal pressure, operative mesenteric portography and histologic evaluation of hepatic tissue at 48 hours postoperatively. Chronic and control dogs were evaluated at 4 weeks postoperatively with all of the tests listed above. The results of all tests performed supported a transient hepatic congestion which resolved bv the fourth postoperative week. No longstanding effect on hepatic function was found. The conclusion of this experiment was that, in normal dogs, left hepatic vein ligation does not cause severe or permanent liver damage. These findings support a clinical trial of this procedure in patients with patent ductus venosus. / Master of Science

In the growing landscape of innovative non-viral delivery vehicles, polymeric and lipid nanoparticles remain at the forefront for their versatility in encapsulating a variety of therapeutic payloads. This thesis investigates their potential for facilitating the transport of nucleic acid components into cells, with a focus on targeted delivery to the liver and brain. To achieve this, we address key considerations including the composition of the delivery vector, the nature of the therapeutic cargo, and the chosen delivery route. The challenge of targeted delivery to specific organs or cell types, i.e. hepatocytes or neurons, is addressed through rational design and development of libraries of nanoparticulate systems tailored for nucleic acid therapeutics. Although liver gene editing using non-viral systems has been extensively studied, oral delivery for liver targeting remains challenging due to the mucosal barrier. To that end, we explore intraduodenal delivery as a strategy to bypass the mucosal barrier and target the liver. Furthermore, insights from collaborative research with the Mao lab at Johns Hopkins University reveal that tuning the composition of lipid nanoparticles (LNPs) can influence their preferential targeting of specific cell types. Leveraging this, we employed an in vitro library screening and machine learning approach to identify populations of LNPs capable of preferentially transfecting hepatocytes. The efficacy of these LNPs in liver gene editing is then evaluated through “cluster-mode” screening in vivo, and therapeutic efficacy was demonstrated using a proof-of-concept in vivo model for PCSK9 and ANGPTL3 knockdown, resulting in 27% serum cholesterol knockdown. In addition to liver-targeted gene delivery, this thesis also investigates the potential of polymeric and lipid nanoparticles for delivering nucleic acid therapeutics to the brain. However, overcoming the blood-brain barrier (BBB) is crucial for systemic delivery to the brain. To circumvent the BBB, we explored two methods: intracranial injection and theranostic ultrasound (THUS)-mediated temporary opening of the BBB. While intracranial injection achieves localized gene editing, THUS offers a non-invasive approach for transient and widespread BBB opening. Utilizing the previously validated in vitro screening and machine learning approaches for chitosan-grafted bPEI (CS-PEI) and lipid nanoparticle (LNP) carriers with tunable compositions, we assessed their efficacy in systemic gene delivery to the brain, and specifically their capability in preferentially transfecting neuronal cells over hepatocytes. Subsequently, we validated their efficiency via intracranial administration using the Ai14 reporter mouse model and observed up to 20% gene editing of the targeted cross-sectional area of the brain hemisphere using the top-performing cluster. Through comprehensive investigations into both brain and liver gene delivery, this thesis aims to contribute to the advancement of non-viral nanoparticle-based gene therapy strategies for treating a range of cholestatic liver diseases and hereditary neurodegenerative diseases.

This thesis addresses the genetic basis of a rare autosomal recessive primary immunodeficiency disorder with the characteristic additional feature of venoocclusive disease of the liver (VODI). The interest in this condition was stimulated both by the potential to identify the genetic basis of a rare immunodeficiency and the opportunity to gain an insight into the biological basis of hepatic veno-occlusive disease, a poorly understood condition that is encountered most frequently in Australia as a consequence of bone marrow transplantation. The gene responsible for VODI was identified by homozygosity mapping and DNA sequence analysis of positional candidates and was shown to be the PML Nuclear Body expressed protein Sp110. This is the first time a PML Nuclear Body protein has been shown to be involved in immunodeficiency disorder. Subsequent immunofluorescence studies of affected patient cell lines showed absence of Sp110 in patient B cells. The role of SP110 alleles in the susceptibility of bone marrow transplant patients to hepatic veno-occlusive disease was investigated using a cohort of patients from the Fred Hutchinson Cancer Center, Seattle. A SNP association study identified initial evidence for an association, but the study lacked sufficient power after correction for multiple testing. Contemporaneously, Dr Igor Kramnik published a report that the murine homologue of Sp110, Ifi75 (also termed Ipr1) was deleted in mice that were supersusceptible to infection with Mycobacterium tuberculosis. A further SNP association study was therefore performed utilising a NSW cohort of Mantouxpositive South East Asian migrants, which detected evidence that alleles of SP110 may be associated with progression of M. tuberculosis infection. Again, the limited size of this cohort precluded definitive findings.