Nina Varah MSc Thesis / BACKGROUND: Brown adipose tissue (BAT) has emerged as an attractive target to address the dramatic rise in obesity and non-alcoholic fatty liver disease (NAFLD) in adults and children due to its ability to clear lipids through thermogenesis when activated with cold stimulation. Cross-sectional studies have identified an inverse relationship between BAT and NAFLD in adults, although no linking mechanism or relevance in children is known. Metabolomics provides a non-invasive platform to investigate BAT physiology and its relationship with hepatic fat in an effort to identify potential targets for further investigation. PROJECT OBJECTIVES: 1) To explore the associations between the plasma metabolome and BAT in adults and children. 2) To explore the associations between the plasma metabolome and hepatic fat in adults and children. 3) To identify metabolites associated with both BAT and hepatic fat as potential linking mechanisms for further study. METHODOLOGY: We recruited 63 male and female adults aged 18 to 57 years and 25 healthy male children aged 8 to 10 years into this cross-sectional study. Study participants underwent blood work, body composition measurement (dual energy X-ray absorptiometry; DXA) and magnetic resonance imaging (MRI) - proton density fat fraction (PDFF) measurements of whole liver hepatic fat, pre- and post-cold supraclavicular fat. BAT activity was calculated as the percent change between post and pre-cold BAT PDFF with the cold stimulus consisting of a water-perfused suit maintained at 18°C for 3-hours (adult) or 1-hour (pediatric). Targeted liquid-chromatography/mass spectrometry metabolomics of 102 metabolites was conducted on fasted plasma and multivariate linear regression with multiple testing correction was used to examine metabolite predictors of BAT measures and hepatic fat. RESULTS: In the adult cohort (n=63, median age 25.9 years, median body mass index (BMI) 25.4 kg/m2), five metabolites were associated with baseline BAT lipid content, where an elevated lipid content may indicate a whiter adipose tissue-like phenotype. Aconitate and creatine commonly predict increased baseline BAT lipid content (β=0.420, P=0.001 and β=0.408, P=0.001, respectively), and reduced BAT activity (β=-0.462, P=0.002 and (β=-0.402, P=0.002, respectively). Alanine and two acyl-carnitines also predicted reduced BAT activity. Glutamic acid was similarly related to higher baseline BAT (β=0.480, P<0.001) and hepatic lipid content independent of age and sex (β=0.392, P=0.002). Three other metabolites were directly related to hepatic fat, and serine inversely. In children (n=25, median age 9.89 years, mean BMI Z-score 1.25), cysteine and cystine were trending towards a significant relationship with higher baseline BAT lipid content, and were both related to elevated hepatic fat independent of adiposity (cysteine: quadratic β=-0.714, p<0.001 and cystine: quadratic β=0.592, p<0.001). Two hydroxy-proline isomers and L-carnitine were associated with reduced BAT activity. CONCLUSION: In adults, several metabolites were associated with reduced BAT activity and with a higher baseline BAT lipid content in the non-stimulated state – aconitate and creatine were related to both. Acylcarnitines or their metabolites related to BAT in both children and adults, which may suggest areas for subsequent investigation of BAT metabolism. Glutamic acid in adults and cysteine and cystine in children were weakly related to elevated baseline BAT and hepatic fat content. Further, amino acids such as glutamic acid and cysteine may be markers of increased ectopic fat accumulation – and are also associated with a whiter ambient BAT phenotype. Cumulatively, these findings highlight targets for further investigation into BAT physiology and the link to the liver. / Thesis / Master of Science (MSc)

Obesity and its comorbidities have emerged as serious healthcare concerns in the western world due to increased prevalence of nutritional overabundance and decreased physical activity. Due to the significant population affected and economic burden placed on national healthcare systems, there is a demonstrated need for effective weight management therapeutics. Obesity presents clinically diverse phenotypes that increase a person's susceptibility to comorbidities that commonly result in deteriorated health (cardiovascular disease, diabetes mellitus, hypertension, etc). A comorbidity of specific relevance is non-alcoholic fatty liver disease (NAFLD) and its advanced disease state known as non-alcoholic steatohepatitis (NASH), as it has had a documented rise in prevalence parallel to that observed with obesity. Currently there are no FDA approved therapeutics for NAFLD or NASH, with the majority in clinical development aiming to mitigate the effects caused by accumulation of adipose tissue in the liver known as steatosis. An alternative therapeutic approach is to use small molecules to uncouple oxidative phosphorylation in the mitochondria by passively shuttling protons from the mitochondrial inner membrane space into the mitochondrial matrix. Mitochondrial uncoupling results in the disruption of the proton motive force leading to an upregulation of metabolism (i.e., decrease in steatosis). Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating the advanced disease state of NASH. In this study, we report the structure-activity relationship (SAR) profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrated that a wide array of substituents are tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a read-out. In particular, compound SHS4121705 (2.12i) displayed an EC50 of 4.3 M in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the preclinical stelic animal model (STAM) mouse model of NASH, administration of 2.12i at 25 mg kg-1day-1 resulted in decreased liver triglyceride levels and improved liver enzymes, NAFLD activity score, and fibrosis without affecting body temperature or food intake. Overall, our initial studies showcased the promise of mitochondrial uncouplers toward the treatment of NASH. While initial results were promising, the lead compound 2.12i had reduced potency compared to the alkyl derivatives reported in the SAR, unfortunately alkyl derivatives suffered from poor physiochemical properties, possibly due to metabolism of the alkyl chain. We hypothesized that addressing metabolic liabilities of these compounds could lead to increased potency with maintained efficacy in the STAM mouse model of NASH. Herein, we detail the SAR profiling of a 6-amino-[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core derivatized with 1,1'-biphenyl anilines capable of eliciting mild mitochondrial uncoupling. A wide array of substituents are tolerated, and demonstrated sustained and stable increases in ¬cellular oxygen consumption rates over a broad concentration range. In particular, compound SHS4091862 (3.9b) displayed an EC50 of 2.0 μM in L6 myoblast cells with a pharmacokinetic profile of Cmax = 46 μM and t1/2 = 4.7 h indicating excellent oral bioavailability. Administration of 3.9b at 60 mg kg-1 day-1 in the STAM mouse model of NASH decreased fibrosis, steatosis, and hepatocellular ballooning to result in a 1.9-point decrease in NAFLD activity score (NAS) compared to vehicle. No changes in food intake, body weight, alanine transaminase (ALT) or aspartate transaminase (AST) levels were observed with 3.9b. Positive control Resmetirom afforded a 1.2-point decrease in NAS score, but increased ALT levels. Cumulatively, our work demonstrates the therapeutic potential of small molecule mitochondrial uncouplers to address metabolic diseases, namely NAFLD. / Doctor of Philosophy / There has been a significant increase in the population suffering from metabolic diseases in the western world. Among the most concerning metabolic diseases are obesity and nonalcoholic fatty liver disease, which have been shown to arise from excessive consumption of calorie dense food and limited physical activity. A novel approach to combat these diseases is to use mitochondrial uncouplers that disrupt the body's natural process for ATP production, causing an increase in metabolism. This increase in the metabolic rate results in the reduction of fat mass including in organs such as the liver. This work describes the design, development, and biological study of mitochondrial uncouplers capable of producing an increase in metabolism; specifically, SHS4121705 (2.12i) and SHS4091862 (3.9b) were shown to be potent uncouplers in vitro and were active in mouse models of fatty liver disease.

<p dir="ltr">In the three conducted studies, we investigated the role of serum albumin in metabolic processes, particularly in lipid metabolism and glucoregulation. The first study explored how disrupting the binding of free fatty acids (FFA) to circulating albumin affects lipid metabolism and glucose control. Male and female albumin knockout mice exhibited significantly reduced plasma FFA levels, hepatic lipid content, and blood glucose during tolerance tests compared to wild-type mice. Additionally, albumin deficiency led to changes in adipose tissue gene expression, indicating the importance of albumin and plasma FFA concentration in metabolic regulation. In the second study, the focus was on determining if impeding serum albumin's function in transporting FFAs could prevent hepatic steatosis and metabolic dysfunction in obesity. Albumin knockout mice, despite being obese due to a high-fat diet, showed lower plasma FFA levels, improved glucose tolerance, and reduced hepatic lipid accumulation compared to wild-type mice. Elevated gene expression in liver and adipose tissues suggested albumin's involvement in hepatic lipid accumulation and glucose metabolism in obesity. Lastly, in the third study, we examined the phenotype of heterozygous albumin knockout mice and compared it to wild-type and homozygous knockout mice. While homozygous knockout mice exhibited improved glucoregulation and reduced plasma FFA concentration, heterozygous knockout mice did not show significant improvements compared to wild-type mice. The findings imply that a minor suppression of albumin expression may not be adequate to enhance glucoregulation. In summary, the studies emphasize the crucial role of serum albumin in metabolic processes, illustrating how disrupting FFA binding to albumin leads to improved glucose control and reduced hepatic lipid accumulation. However, minor suppression of albumin expression may not effectively enhance metabolic health. These findings provide valuable insights into potential therapeutic interventions targeting the albumin-FFA pathway to improve metabolic outcomes.</p><p dir="ltr"><br></p>

The unfolded protein response (UPR) is composed of three highly conserved pathways (ATF6, IRE1, PERK). Cellular stressors induce protein misfolding and aggregation in the endoplasmic reticulum (ER). This signaling pathway maintains protein homeostasis when there is stress in the ER. When the UPR is activated, the eukaryotic initiation factor 2 alpha (eIF2α) becomes phosphorylated, which inhibits global mRNA translation. If ER stress remains chronically unmitigated, the UPR induces apoptosis. GADD34 and CReP shift in expression when the UPR is activated and work as phosphatases and dephosphorylate eIF2α in a feedback loop, allowing protein synthesis to resume. Several human diseases, including fatty liver disease (FLD) are affected by cell stress from improper protein folding and accumulation, making the UPR a therapeutic target. Previous studies have indicated the UPR to both cause or become activated by FLD, depending on the duration of cellular stress. At least 25% of humans worldwide have steatosis, and zebrafish are a powerful model organism for FLD studies. Their embryos are easily obtained, and the liver develops quickly in their transparent larvae, which allows us to visualize the development of fat in the liver. It is unknown how exactly the UPR is involved in inducing lipogenesis in hepatocytes. We sought to better understand the link between UPR activation and steatosis. Pharmacological treatments with various drugs, some of which induce ER stress, were administered over different durations in zebrafish embryos and subsequently the expression of UPR network and lipogenesis genes were quantified through RT-qPCR. To visualize whether these drugs induced steatosis, zebrafish livers were stained with Oil Red O and imaged. Our results indicate that all chronic durations of pharmacological treatments resulted in fatty liver, and the expression of atf6 decreased in response to treatment that prevents the dephosphorylation of eIF2α. This data provides insight pertaining to the activity of the UPR network during FLD in zebrafish models.

To compare ultrasound-based acoustic structure quantification (ASQ) with established non-invasive techniques for grading and staging fatty liver disease.

Contexto: O tratamento da hepatite autoimune (HAI) composto por prednisona e azatioprina proporciona melhora clínico-laboratorial em até 90% dos pacientes. Entretanto, a remissão completa não é alcançável na maioria dos casos. Cloroquina é um antimalárico utilizado no tratamento de doenças reumatológicas autoimunes e em estudo aberto de manutenção da remissão da HAI foi sugerido menor risco de recidiva da doença com o uso da droga. Objetivos: Avaliar o uso da cloroquina em associação à prednisona no tratamento da HAI em estudo randomizado. Métodos: 57 pacientes com indicação de tratamento da HAI foram randomizados para receber azatioprina ou cloroquina associadas à prednisona, de 2003 a 2012. Para os que mantiveram normalização das transaminases por 18 meses, biópsia hepática foi realizada para avaliação histológica. O desfecho primário foi a remissão completa ao tratamento, composta por remissão bioquímica e histológica da doença. O valor de p < 0,05 foi considerado estatisticamente significativo. Resultados: Não houve diferença entre os grupos quanto às características clínicas, sorológicas, histológicas e de tratamento prévio, ao início do estudo. A idade média foi de 37,2 ± 16,84 anos, 43,8% com fibrose avançada (F3/4) no início do estudo. Não houve diferença estatística na taxa de resposta bioquímica (67% vs. 53,8%, p=0,413) ou histológica (32,2% vs. 15,4%, p=0,21), assim como na dose média de prednisona utilizada. Os pacientes que não atingiram remissão completa no estudo tiveram seguimento com nova terapia. Entre eles, quatro obtiveram remissão histológica com a associação de azatioprina, cloroquina e prednisona. Em relação aos efeitos adversos, houve maior taxa no grupo da cloroquina, porém com tendência a menor prevalência de comorbidades neste grupo. Conclusão: Quando bem toleradas, cloroquina e prednisona proporcionaram resposta completa em pacientes com AIH, sem diferença estatística em relação à terapia padrão. (ClinicalTrials.gov NCT 02463331) / Background: The treatment of autoimmune hepatitis (AIH) with prednisone and azathioprine provides disease remission. However, a complete biochemical and histological response is unreachable in most patients. Chloroquine is an antimalarial drug used for treating rheumatological diseases. It was studied as a single drug for the maintenance of AIH remission in an open study, which suggested a lower risk of relapse in the chloroquine group. Aims: To evaluate a possible role of chloroquine and prednisone for AIH treatment in a randomized study. Methods: 57 AIH adult patients with indication of treatment were enrolled to receive azathioprine or chloroquine, both with varying doses of prednisone, from 2003 to 2012. For those who had maintained biochemical remission for 18 months, liver biopsy was performed to evaluate histological remission. The primary outcome was the achievement of complete response to treatment. A p-value < 0.05 was considered statistically significant. Results: There were no significant differences between the groups concerning clinical, serological, histological, and treatment features at baseline. The average age was 37.2 ± 16.84 years, 43.8% with advanced fibrosis (F3/4) at baseline. There was no statistical differences in biochemical (67.7% vs. 53.8%, p=0.41) or histological response rate (32.26% vs. 15.38%, p = 0.217), as well as in the mean prednisone dose. There was a higher rate of adverse effects in the chloroquine group, but a lower frequency of comorbidities in this group. Conclusion: When well tolerated, chloroquine with prednisone provided a complete therapeutic response in AIH patients with no statistical difference when compared to the standard treatment. (ClinicalTrials.gov NCT 02463331)

INTRODUÇÃO: A cirrose é a oitava causa de mortalidade no mundo, e sua progressão e estadiamento são de extrema importância nos pacientes com doença terminal do fígado. A presença de cirrose é reconhecida como risco aumentado de carcinoma hepatocelular (CHC) e o seu aparecimento está diretamente relacionado ao grau de fibrose do fígado. Na última década, notou-se o desenvolvimento e aperfeiçoamento dos métodos de predição do grau de fibrose e cirrose, através de métodos não-invasivos, com o objetivo de substituir a biópsia hepática. A população em lista de espera para transplante de fígado apresenta graus diferentes de fibrose hepática, que pode não estar diretamente relacionada ao MELD. Além disso, esses pacientes apresentam CHC no momento da triagem para transplante de fígado. Não existe avaliação desta população por elastografia. OBJETIVO: O objetivo deste trabalho foi avaliar os pacientes em triagem para transplante de fígado, com e sem carcinoma hepatocelular, pela elastografia hepática com Fibroscan® e ARFI. MÉTODO: Foram estudados 103 pacientes adultos do ambulatório de triagem da Disciplina de Transplante de Órgãos do Aparelho Digestivo HC/FMUSP, no período de outubro de 2012 à dezembro de 2013. A amostragem foi por conveniência e foram avaliados dados clínicos, epidemiográficos, laboratoriais, imagem, elastográficos e o desfecho. Análise de elastografia transitória (ET) foi feita pelo Fibroscan® TM (Echosens, França) e força impulso por radiação acústica (ARFI) (Siemens Acuson S2000, Alemanha) nos grupos com e sem CHC comprovados de acordo com orientação de diagnóstico pelas diretrizes européias (EASL) e americanas (AASLD). Para a análise estatística foi realizado o teste de Mann-Whitney, teste não paramétrico aplicado para duas amostras independentes; o teste de Fisher e o método ANOVA através do teste de Kruskal-Wallis ou teste de Tukey para comparações múltiplas. Foi realizado também a curva ROC para avaliação dos testes diagnósticos e ponto de corte. O valor considerado de p significativo foi <0,05. RESULTADOS: Entre os pacientes avaliados, a maioria foi de homens (68%), com idade média de 53 ± 11,5 anos. A etiologia mais comum foi o vírus da hepatite C (VHC) em 34,9%. A classificação pelo escore Child-Turcotte-Pugh (CTP) mostrou: pacientes classe A em 38,4%, classe B em 47,2% e classe C em 14,2%. O valor do MELD médio dos pacientes foi de 14,75 (± 6,45) e a mediana de 14 (variando, 6 - 32). Na população estudada de 103 pacientes, a ET (Fibroscan®) foi realizada com sucesso em 75 de 103 pacientes e ARFI em 78 de 78 pacientes. A etiologia VHC e elevados valores de alfa-feto proteína foram fatores de risco para a presença de CHC. Os valores de MELD mais elevados foram significativos nos pacientes que evoluíram a óbito. A curva ROC mostrou respectivamente sensibilidade e especificidade para a AFP de 50% e 86% (valor de corte 9,1); ET (valor de corte 9 kPa) 92% e 17%; e ARFI 21% e 92% (valor de corte 2,56 m/s). O valor médio da ET nos pacientes com CHC foi de 30,4 ± 21,0 kPa, do ARFI do parênquima hepático foi de 1,97 ± 0,64 e ARFI do nódulo hepático foi de 1,89 ± 0,74. CONCLUSÃO: Os pacientes em triagem para transplante de fígado com carcinoma hepatocelular apresentam valores elevados de elastografia tanto pelo Fibroscan® quanto pelo ARFI®. A elastografia apresenta-se como uma importante ferramenta não invasiva para o acompanhamento de cirróticos graves podendo ajudar no manejo do carcinoma hepatocelular / INTRODUCTION: Cirrhosis is the eighth leading cause of mortality worldwide and its progression and staging are extremely important in patients with end liver disease. The presence of cirrhosis is recognized as an increased risk of hepatocellular carcinoma (HCC) and its incidence is directly related to the degree of liver fibrosis. In the last decade, was noted the development and improvement of methods for predicting the degree of fibrosis and cirrhosis using non-invasive methods, aiming to replace the liver biopsy. The population on the liver transplant waiting list presents different degrees of liver fibrosis, which may not be directly related to MELD. In addition, these patients have HCC at the time of screening for liver transplantation. There is no evaluation of this population by elastography. OBJECTIVE: The aim of this study was to evaluate patients on screened to the list for liver transplantation, with and without hepatocellular carcinoma, by liver elastography with Fibroscan and ARFI. METHOD: Were studied 103 adult patients from the screening for liver transplantation waiting list on the Liver and Gastrointestinal Transplant Division HC/FMUSP from October 2012 to December 2013. Sampling for convenience and evaluation clinical data, epidemiological, laboratory, imaging, elastography findings and outcome. Analysis of transient elastography (TE) by Fibroscan TM (Echosens, France) and Acoustic Radiation Force Impulse (ARFI) by (Siemens Acuson S2000, Germany) in patients with and without HCC proven in accordance with guidelines of diagnosis EASL/AASLD. Fisher\'s ANOVA or Kruskal-Wallis tests Whitney-Mann Test were performed. Tukey and define cut-of for examinations with ROC curves. The p value considered was < 0.05. RESULTS: Among the patients, the majority were men (68%), mean age 53 ± 11.5 years. This is the most common cause of hepatitis C virus (HCV) 34.9%. The classification by Child-Turcotte-Pugh score (CTP) showed: class A patients in 38.4%, 47.2% in class B and class C in 14.2%. The average value of MELD patients was 14.75 (± 6.45) and a median of 14 (range, 6-32). In the study population of 103 patients, the ET (Fibroscan) was successfully performed in 75 of 103 patients and ARFI in 78 of 78 patients. The HCV etiology and high levels of alpha-fetoprotein were risk factors for the presence of HCC. MELD values were significant higher in patients who died. The ROC curve shown respectively sensitivity and specificity for AFP of 50% and 86% (cutoff 9.1); ET (9 cutoff kPa) 92% to 17%; and ARFI 21% and 92% (cut-off 2.56 m / s). The average value of ET in HCC patients was 30.4 ± 21.0 kPa, the ARFI parenchymal liver was 1.97 ± 0.64 and ARFI liver nodules was 1.89 ± 0.74. CONCLUSION: Patients in screening for liver transplantation with hepatocellular carcinoma have elevated values of both elastography by Fibroscan as the ARFI®. Elastography is presented as an important non-invasive tool for monitoring severe cirrhosis may help in management of hepatocellular carcinoma

A esteatose hepática é caracterizada histologicamente pelo acúmulo de triglicerídeos no citoplasma dos hepatócitos. A biópsia ainda é o padrão ouro para diagnóstico e avaliação da gravidade, no entanto, é um método invasivo e sujeita a erro de amostragem. Com os avanços dos métodos diagnósticos por imagem, a RM se tornou um método bem estabelecido para detecção e quantificação da esteatose. A maneira mais simples é a obtenção do cálculo da fração de gordura pela técnica gradiente-eco desvio químico. Todavia, não há estudos demonstrando a melhor forma de medir a intensidade de sinal para esse cálculo. Em nosso estudo, através da revisão de exames de RM, avaliamos três diferentes estratégias de medidas para quantificação com amostra de conveniência com 74 exames apresentando esteatose pareados em dois grupos, esteatose homogênea (n=37) e heterogênea (n=37). No grupo de esteatose heterogênea, o uso de ROI de 1cm² para medir a intensidade de sinal na área mais alterada apresentou variações significativas na quantificação, enquanto a média de quatro ROIs de 1cm² ou a segmentação de área representativa em corte axial não apresentaram variações significativas. Na esteatose hepática homogênea, qualquer estratégia utilizada não demonstrou diferença significativa. O coeficiente de correlação intraclasse variou entre 0,96 e 0,99, com IC 95% de 0,93-0,99. Assim, a quantificação da gordura hepática por RM utilizando apenas um ROI é menos representativa, principalmente na esteatose heterogênea. Não houve diferença significativa entre a obtenção da média de 4 ROIs e a segmentação de área representativa do parênquima. / Hepatic steatosis is characterized histologically by the triglyceride accumulation in the cytoplasm of hepatocytes. A biopsy is still considered the gold standard for diagnosis and assessment of severity, however, is an invasive and subject to sampling error method. With the improvement of diagnostic imaging methods, MRI has become a well-established method for the detection and quantification of liver fat. The easiest way is to obtain the calculation of the fat fraction by GRE technique with chemical shift technique. However, there are no studies demonstrating the best way to measure the signal intensity for this calculation. In our study by MRI review, we evaluate three different strategies for measuring the signal intensity with a convenience sample of 74 exams showing steatosis paired into two groups, diffuse steatosis (n = 37) and heterogeneous (n = 37). In heterogeneous steatosis group, the strategy with a ROI of 1 cm² to measure the signal intensity in the most altered area showed significant variations in the quantification, while the average of four ROIs of 1cm² or representative target area in axial section did not vary significant. In diffuse hepatic steatosis, any strategy used showed no significant difference. The intraclass correlation coefficient ranged between 0.96 and 0.99, with 95% of 0.93-0.99. Thus, the quantification of fat liver by MRI using only ROI is less representative, especially in heterogeneous steatosis. There was no significant difference between the average of 4 ROIs strategy and the strategy of representative segmentation area of parenchyma.

There are multiple immune mechanisms identified for persistence of hepatitis B virus (HBV) infection. This thesis considers the vital role that inhibitory receptors play in contributing to impairment of the adaptive immune system in chronic hepatitis B (CHB), and the potential role they play in the innate immune system, focusing on the inhibitory receptor leucocyte-associated immunoglobulin-like receptor (LAIR)-1. The unique aspect of this work is that for the first time LAIR-1 expression has been investigated on natural killer (NK) cells in CHB. Our striking findings of increased LAIR-1 expression on peripheral NK cells in CHB and an inverse correlation between expression and effector function suggest this inhibitory receptor could have a potential role in exhaustion of NK cells in CHB. We therefore additionally explored the expression of LAIR-1 on circulating NK cells from patients with hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). The particular relevance of LAIR-1 to liver disease is that one of its major ligands is collagen. We demonstrated a downregulation of LAIR-1 expression on intrahepatic NK cells, which we postulate might occur following repetitive engagement with abundant collagen within the liver. In line with this, intrahepatic NK cells with a liver-resident (CXCR6+) phenotype had even lower LAIR-1 expression than liver infiltrating (non-resident, CXCR6-) NK cells. Furthermore, preliminary experiments display attenuation of the cytotoxic degranulation capacity (CD107a) by circulating NK cells from CHB patients upon exposure to plate-bound collagen. We demonstrate differential expression of LAIR-1 on NK cells in viral hepatitis, HCC and NAFLD and between peripheral and intrahepatic NK cells. Preliminary experiments demonstrate a role in inhibiting NK cell function suggesting this as a novel therapeutic target to harness the capacity of NK cells to control chronic infection and cancer.

Contexto: O tratamento da hepatite autoimune (HAI) composto por prednisona e azatioprina proporciona melhora clínico-laboratorial em até 90% dos pacientes. Entretanto, a remissão completa não é alcançável na maioria dos casos. Cloroquina é um antimalárico utilizado no tratamento de doenças reumatológicas autoimunes e em estudo aberto de manutenção da remissão da HAI foi sugerido menor risco de recidiva da doença com o uso da droga. Objetivos: Avaliar o uso da cloroquina em associação à prednisona no tratamento da HAI em estudo randomizado. Métodos: 57 pacientes com indicação de tratamento da HAI foram randomizados para receber azatioprina ou cloroquina associadas à prednisona, de 2003 a 2012. Para os que mantiveram normalização das transaminases por 18 meses, biópsia hepática foi realizada para avaliação histológica. O desfecho primário foi a remissão completa ao tratamento, composta por remissão bioquímica e histológica da doença. O valor de p < 0,05 foi considerado estatisticamente significativo. Resultados: Não houve diferença entre os grupos quanto às características clínicas, sorológicas, histológicas e de tratamento prévio, ao início do estudo. A idade média foi de 37,2 ± 16,84 anos, 43,8% com fibrose avançada (F3/4) no início do estudo. Não houve diferença estatística na taxa de resposta bioquímica (67% vs. 53,8%, p=0,413) ou histológica (32,2% vs. 15,4%, p=0,21), assim como na dose média de prednisona utilizada. Os pacientes que não atingiram remissão completa no estudo tiveram seguimento com nova terapia. Entre eles, quatro obtiveram remissão histológica com a associação de azatioprina, cloroquina e prednisona. Em relação aos efeitos adversos, houve maior taxa no grupo da cloroquina, porém com tendência a menor prevalência de comorbidades neste grupo. Conclusão: Quando bem toleradas, cloroquina e prednisona proporcionaram resposta completa em pacientes com AIH, sem diferença estatística em relação à terapia padrão. (ClinicalTrials.gov NCT 02463331) / Background: The treatment of autoimmune hepatitis (AIH) with prednisone and azathioprine provides disease remission. However, a complete biochemical and histological response is unreachable in most patients. Chloroquine is an antimalarial drug used for treating rheumatological diseases. It was studied as a single drug for the maintenance of AIH remission in an open study, which suggested a lower risk of relapse in the chloroquine group. Aims: To evaluate a possible role of chloroquine and prednisone for AIH treatment in a randomized study. Methods: 57 AIH adult patients with indication of treatment were enrolled to receive azathioprine or chloroquine, both with varying doses of prednisone, from 2003 to 2012. For those who had maintained biochemical remission for 18 months, liver biopsy was performed to evaluate histological remission. The primary outcome was the achievement of complete response to treatment. A p-value < 0.05 was considered statistically significant. Results: There were no significant differences between the groups concerning clinical, serological, histological, and treatment features at baseline. The average age was 37.2 ± 16.84 years, 43.8% with advanced fibrosis (F3/4) at baseline. There was no statistical differences in biochemical (67.7% vs. 53.8%, p=0.41) or histological response rate (32.26% vs. 15.38%, p = 0.217), as well as in the mean prednisone dose. There was a higher rate of adverse effects in the chloroquine group, but a lower frequency of comorbidities in this group. Conclusion: When well tolerated, chloroquine with prednisone provided a complete therapeutic response in AIH patients with no statistical difference when compared to the standard treatment. (ClinicalTrials.gov NCT 02463331)