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Yellow fever (YF) is a viral hemorrhagic fever that typically involves the liver. Brazil recently experienced its largest recorded YF outbreak, and the disease was fatal in more than a third of affected individuals, mostly because of acute liver failure. Affected individuals are generally treated only supportively, but during the recent Brazilian outbreak, selected patients were treated with liver transplant. We took advantage of this clinical experience to better characterize the clinical and pathological features of YF-induced liver failure and to examine the mechanism of hepatocellular injury in YF, to identify targets that would be amenable to therapeutic intervention in preventing progression to liver failure and death. Patients with YF liver failure rapidly developed massive transaminase elevations, with jaundice, coagulopathy, thrombocytopenia, and usually hepatic encephalopathy, along with pathological findings that included microvesicular steatosis and lytic necrosis. Hepatocytes began to express the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular calcium (Ca2+) channel that is not normally expressed in hepatocytes. Experiments in an animal model, isolated hepatocytes, and liver-derived cell lines showed that this new expression of ITPR3 was associated with increased nuclear Ca2+ signaling and hepatocyte proliferation, and reduced steatosis and cell death induced by the YF virus. Conclusion: Yellow fever often induces liver failure characterized by massive hepatocellular damage plus steatosis. New expression of ITPR3 also occurs in YF-infected hepatocytes, which may represent an endogenous protective mechanism that could suggest approaches to treat affected individuals before they progress to liver failure, thereby decreasing the mortality of this disease in a way that does not rely on the costly and limited resource of liver transplantation.

The expression of the inositol 1,4,5-trisphosphate receptor type 3 (ITRP3) in hepatocytes is a common event in the pathogenesis of hepatocellular carcinoma (HCC), regardless of the type of underlying liver disease. However, it is not known whether ITPR3 expression in hepatocytes is involved in tumor maintenance. The aim of the present study was to determine whether there is an association between ITPR3 expression and clinical and morphological parameters using HCC samples obtained from liver explants from patients (n=53) with different etiologies of underlying chronic liver disease (CLD). ITPR3 expression, mitosis and apoptosis were analyzed in human liver samples by immunohistochemistry. Clinical and event-free survival data were combined to assess the relationship between ITPR3 and liver cancer growth in patients. RNA sequencing analysis was performed to identify apoptotic genes altered by ITPR3 expression in a liver tumor cell line. ITPR3 was highly expressed in HCC tumor cells relative to adjacent CLD tissue and healthy livers. There was an inverse correlation between ITPR3 expression and mitotic and apoptotic indices in HCC, suggesting that ITPR3 contributed to the maintenance of HCC by promoting resistance to apoptosis. This was confirmed by the upregulation of CTSB, CHOP and GADD45, genes involved in the apoptotic pathway in HCC. The expression of ITPR3 in the liver may be a promising prognostic marker of HCC.

Long-term infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a challenge to virus dispersion and the control of coronavirus disease 2019 (COVID-19) pandemic. The reason why some people have prolonged infection and how the virus persists for so long are still not fully understood. Recent studies suggested that the accumulation of intra-host single nucleotide variants (iSNVs) over the course of the infection might play an important role in persistence as well as emergence of mutations of concern. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during prolonged infection. Thirty-three patients who remained reverse transcription polymerase chain reaction (RT-PCR) positive in the nasopharynx for on average 18 days from the symptoms onset were included in this study. Whole-genome sequences were obtained for each patient at two different time points. Phylogenetic, populational, and computational analyses of viral sequences were consistent with prolonged infection without evidence of coinfection in our cohort. We observed an elevated within-host genomic diversity at the second time point samples positively correlated with cycle threshold (Ct) values (lower viral load). Direct transmission was also confirmed in a small cluster of healthcare professionals that shared the same workplace by the presence of common iSNVs. A differential accumulation of missense variants between the time points was detected targeting crucial structural and non-structural proteins such as Spike and helicase. Interestingly, longitudinal acquisition of iSNVs in Spike protein coincided in many cases with SARS-CoV-2 reactive and predicted T cell epitopes. We observed a distinguishing pattern of mutations over the course of the infection mainly driven by increasing A→U and decreasing G→A signatures. G→A mutations may be associated with RNA-editing enzyme activities; therefore, the mutational profiles observed in our analysis were suggestive of innate immune mechanisms of the host cell defense. Therefore, we unveiled a dynamic and complex landscape of host and pathogen interaction during prolonged infection of SARS-CoV-2, suggesting that the host’s innate immunity shapes the increase of intra-host diversity. Our findings may also shed light on possible mechanisms underlying the emergence and spread of new variants resistant to the host immune response as recently observed in COVID-19 pandemic.

Background & objectivesHepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+release channels in HCC.DesignExpression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects ofde novoITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice.ResultsITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. TheITPR3gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion ofITPR3from human HCC cells enhanced apoptosis.ConclusionsThese results provide evidence thatde novoexpression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.

Prolonged infection of SARS-CoV-2 represents a challenge to the development of effective public health policies to control the COVID-19 pandemic. The reason why some people have persistent infection and how the virus survives for so long are still not fully understood. For this reason, we aimed to investigate the intra-host evolution of SARS-CoV-2 during persistent infection. Thirty-three patients who remained RT-PCR positive in the nasopharynx for at least 16 days were included in this study. Complete SARS-CoV-2 sequences were obtained for each patient at two time points. Phylogenetic, populational, and computational analysis of viral sequences confirmed persistent infection with evidence for a transmission cluster in health care professionals that shared the same workplace. A high number of missense variants targeting crucial structural and non-structural proteins such as Spike and Helicase was found. Interestingly, longitudinal acquisition of substitutions in Spike protein mapped many SARS-CoV-2 predicted T cell epitopes. Furthermore, the mutational profiles observed were suggestive of RNA editing enzyme activities, indicating innate immune mechanisms of the host cell. Viral quasispecies analysis corroborates persistent infection mainly by increasing richness and nucleotide diversity over time. Altogether, our findings highlight a dynamic and complex landscape of host and pathogen interaction during persistent infection suggesting that the host’s innate immunity shapes the increase of intra-host diversity with possible implications for therapeutic strategies and public health decisions during the COVID-19 pandemic.

Brazil currently has one of the fastest growing SARS-CoV-2 epidemics in the world. Due to limited available data, assessments of the impact of non-pharmaceutical interventions (NPIs) on virus transmission and epidemic spread remain challenging. We investigate the impact of NPIs in Brazil using epidemiological, mobility and genomic data. Mobility-driven transmission models for São Paulo and Rio de Janeiro cities show that the reproduction number (Rt) reached below 1 following NPIs but slowly increased to values between 1 to 1.3 (1.0–1.6). Genome sequencing of 427 new genomes and analysis of a geographically representative genomic dataset from 21 of the 27 Brazilian states identified >100 international introductions of SARS-CoV-2 in Brazil. We estimate that three clades introduced from Europe emerged between 22 and 27 February 2020, and were already well-established before the implementation of NPIs and travel bans. During this first phase of the epidemic establishment of SARS-CoV-2 in Brazil, we find that the virus spread mostly locally and within-state borders. Despite sharp decreases in national air travel during this period, we detected a 25% increase in the average distance travelled by air passengers during this time period. This coincided with the spread of SARS-CoV-2 from large urban centers to the rest of the country. In conclusion, our results shed light on the role of large and highly connected populated centres in the rapid ignition and establishment of SARS-CoV-2, and provide evidence that current interventions remain insufficient to keep virus transmission under control in Brazil.One Sentence SummaryJoint analysis of genomic, mobility and epidemiological novel data provide unique insight into the spread and transmission of the rapidly evolving epidemic of SARS-CoV-2 in Brazil.

Clinical conditions that result in endotoxemia, such as sepsis and alcoholic hepatitis (AH), often are accompanied by cholestasis. Although hepatocellular changes in response to lipopolysaccharide (LPS) have been well characterized, less is known about whether and how cholangiocytes contribute to this form of cholestasis. We examined effects of endotoxin on expression and function of the type 3 inositol trisphosphate receptor (ITPR3), because this is the main intracellular Ca2+ release channel in cholangiocytes, and loss of it impairs ductular bicarbonate secretion. Bile duct cells expressed the LPS receptor, Toll-like receptor 4 (TLR4), which links to activation of nuclear factor-κB (NF-κB). Analysis of the human ITPR3 promoter revealed five putative response elements to NF-κB, and promoter activity was inhibited by p65/p50. Nested 0.5- and 1.0-kilobase (kb) deletion fragments of the ITPR3 promoter were inhibited by NF-κB subunits. Chromatin immunoprecipitation (ChIP) assay showed that NF-κB interacts with the ITPR3 promoter, with an associated increase in H3K9 methylation. LPS decreased ITPR3 mRNA and protein expression and also decreased sensitivity of bile duct cells to calcium agonist stimuli. This reduction was reversed by inhibition of TLR4. ITPR3 expression was decreased or absent in cholangiocytes from patients with cholestasis of sepsis and from those with severe AH. Conclusion: Stimulation of TLR4 by LPS activates NF-κB to down-regulate ITPR3 expression in human cholangiocytes. This may contribute to the cholestasis that can be observed in conditions such as sepsis or AH.

Introduction: Endothelial dysfunction may contribute to hypercoagulable and inflammation states presents in renal transplant, chronic kidney disease (CKD) and its causes. These disorders can be evaluated by markers, such as thrombomodulin (TM), von Willebrand factor (vWF) and interleukin 6 (IL-6). Objectives: The aim of this study was to assess TM, vWF and IL-6 in renal transplant recipients (RTR) and associate their plasma levels with primary cause of end-stage renal disease (ESRD) and allograft function. Methods: 160 RTR were grouped according to the primary cause of CKD (G1: glomerulopathy; G2: hypertensive nephrosclerosis; G3: diabetic nephropathy; and G4: other causes/unknown etiology); creatinine plasma levels (C1 < 1.4 and C2 ≥ 1.4 mg/dl); and the estimated glomerular filtration rate (eGFR) (R1< 60 and R2 ≥ 60 ml/min/1.73 m2). TM and vWF were determined by the enzyme-linked immunosorbent assay (ELISA) and IL-6 by flow cytometry. The results were presented as median, minimum and maximum; p-value < 0.05 was considered statistically significant. Results: TM levels were significantly higher in the G1 group compared to the others (G1: 8.38; G2: 5.51; G3: 5.88; G4: 6.33 ng/ml, p < 0.0001), and in the R1 group compared to R2 (R1: 6.65; R2: 6.19 ng/ml, p = 0.02). The concentration of IL-6, measured by the mean fluorescence intensity, was higher in C2 group when compared to C1 (C1: 7.9; C2: 13.35, p = 0.03). There was no difference in vWF levels among groups. TM correlated positively with IL-6 and creatinine, and negatively with eGFR. IL-6 also correlated positively with vWF. Conclusion: TM and IL-6 can be identified as potential markers for evaluating renal graft function. TM was more related to the primary cause of CKD compared to vWF and IL-6. RESUMO Introdução: A disfunção endotelial pode contribuir para estados de hipercoagulabilidade e inflamação presentes no transplante renal e na doença renal crônica (DRC) e suas causas, podendo ser avaliada por marcadores como trombomodulina (TM), fator de von Willebrand (FvW) e interleucina 6 (IL-6). Objetivos: Avaliar TM, FvW e IL-6 em receptores do transplante renal (RTR) e associar seus níveis com a causa primária de DRC pré-transplante e função do enxerto. Métodos: Foram alocados 160 RTR em grupos de acordo com a causa primária da DRC (G1: glomerulopatias; G2: nefroesclerose hipertensiva; G3: nefropatia diabética; e G4: outras causas/etiologia desconhecida), os níveis plasmáticos de creatinina (C1 < 1.4 e C2 ≥ 1.4 mg/dl) e o ritmo de filtração glomerular estimado (eRFG) (R1< 60 e R2 ≥ 60 ml/min/1.73 m2). A TM e o FvW foram determinados pelo ensaio de imunoabsorção enzimática (ELISA) e a IL-6, por citometria de fluxo. Os resultados foram apresentados como mediana, mínimo e máximo; p < 0,05 foi considerado significativo. Resultados: Níveis de TM foram significativamente maiores no grupo G1 em comparação com os demais (G1: 8,38; G2: 5,51; G3: 5,88; G4: 6,33 ng/ml, p < 0,0001), e no grupo R1 comparado com o R2 (R1: 6,65; R2: 6,19 ng/ml, p = 0,02). A concentração de IL-6, avaliada pela intensidade média de fluorescência, foi maior no grupo C2 quando comparada com o C1 (C1: 7,9; C2: 13,35, p = 0,03). Não houve diferença entre os grupos para o FvW. TM correlacionou-se positivamente com IL-6 e creatinina e negativamente com eRFG. A IL-6 foi positivamente correlacionada com o FvW. Conclusão: TM e IL-6 podem ser apontadas como potenciais marcadores para avaliar a função do enxerto renal. A TM relacionou-se mais com a causa primária da DRC, se comparada com FvW e IL-6.

Hepatic ischemia-reperfusion injury is seen in a variety of clinical conditions, including hepatic thrombosis, systemic hypotension, and liver transplantation. Calcium (Ca(2+)) signaling mediates several pathophysiological processes in the liver, but it is not known whether and how intracellular Ca(2+) channels are involved in the hepatocellular events secondary to ischemia-reperfusion. Using an animal model of hepatic ischemia-reperfusion injury, we observed a progressive increase in expression of the type 3 isoform of the inositol trisphosphate receptor (ITPR3), an intracellular Ca(2+) channel that is not normally expressed in healthy hepatocytes. ITPR3 expression was upregulated, at least in part, by a combination of demethylation of the ITPR3 promoter region and the increased transcriptional activity of the nuclear factor of activated T-cells (NFAT). Additionally, expression of pro-inflammatory interleukins and necrotic surface area were less pronounced in livers of control animals compared to liver-specific ITPR3 KO mice subjected to hepatic damage. Corroborating these findings, ITPR3 expression and activation of NFAT were observed in hepatocytes of liver biopsies from patients who underwent liver ischemia caused by thrombosis after organ transplant. Together, these results are consistent with the idea that ITPR3 expression in hepatocytes plays a protective role during hepatic injury induced by ischemia-reperfusion.

Background & Aims The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. Methods Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2–3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. Results We observed major immunologic changes in patients with NAS 2–3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. Conclusion The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. Lay summary Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease., Graphical abstract, Highlights • Hepatic immune response is already altered in liver biopsies from patients with mild NAFLD. • We designed a novel mouse model to mimic mild NAFLD, enabling the chronological mapping of liver changes. • This revealed an increased mortality rate upon secondary liver damage and a window of increased susceptibility to infection. • NAFLD diagnosis may be significantly improved by a more profound investigation of changes in hepatic immunology. • These data could guide customized nutritional and therapeutic interventions at different stages of NAFLD.

Background & Aims The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. Methods We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. Results Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. Conclusion In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. Lay summary Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.

Immunosenescence is an age-related reduction of immune system activity that can be associated with frailty. This study aimed to compare cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivations (based on viremias) between young and elderly women who had a chronic CMV and/or EBV infection (i.e., an IgG+ serostatus) without an acute infection (i.e., an IgM- serostatus), and among the elderly group categorized according to frailty status. DNA was extracted from plasma using standard protocols and serostatus was determined by enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction analyses for CMV and EBV were carried out and viral loads were determined. Among elderly women (n = 71), 59% were positive for CMV, in contrast to only 8% of young women (n = 73). Elderly women classified as frail, pre-frail, and non-frail presented 82%, 56%, and 48% positivity for CMV, respectively. Frequency and viral load were significantly higher in the elderly group vs. the young group (p < 0.0001 and p = 0.01, respectively) and in elderly with frailty vs. those without frailty (p = 0.007 and p = 0.03, respectively). The frequency of CMV reactivation presented odds ratios of 11.77 for aging and 6.13 for frailty, and relative risks of 5.39 for aging and 1.93 for frailty. EBV was detected in 30% of the elderly women and 15% of the young women (p = 0.04); however, the viral load did not significantly differ between the two age groups. The frequency of EBV reactivation presented odds ratios of 2.36 for aging and 2.90 for frailty, and relative risks of 1.96 for aging and 2.12 for frailty. However, no difference in EBV viral load among the frailty status subgroups was found. In conclusion, the frequency of CMV reactivation was associated with aging and ongoing frailty, whereas the frequency of EBV reactivation was associated only with aging.

Leprosy is an important cause of disability in the developing world. Early diagnosis is essential to allow for cure and to interrupt transmission of this infection. MicroRNAs (miRNAs) are important factors for host-pathogen interaction and they have been identified as biomarkers for various infectious diseases. The expression profile of 377 microRNAs were analyzed by TaqMan low-density array (TLDA) in skin lesions of tuberculoid and lepromatous leprosy patients as well as skin specimens from healthy controls. In a second step, 16 microRNAs were selected for validation experiments with reverse transcription-quantitative PCR (qRT-PCR) in skin samples from new individuals. Principal-component analysis followed by logistic regression model and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic potential of selected miRNAs. Four patterns of differential expression were identified in the TLDA experiment, suggesting a diagnostic potential of miRNAs in leprosy. After validation experiments, a combination of four miRNAs (miR-101, miR-196b, miR-27b, and miR-29c) was revealed as able to discriminate between healthy control and leprosy patients with 80% sensitivity and 91% specificity. This set of miRNAs was also able to discriminate between lepromatous and tuberculoid patients with a sensitivity of 83% and 80% specificity. In this work, it was possible to identify a set of miRNAs with good diagnostic potential for leprosy.

Yellow fever virus (YFV) is a member of the Flaviviridae family. In Brazil, yellow fever (YF) cases have increased dramatically in sylvatic areas neighboring urban zones in the last few years. Because of the high lethality rates associated with infection and absence of any antiviral treatments, it is essential to identify therapeutic options to respond to YFV outbreaks. Repurposing of clinically approved drugs represents the fastest alternative to discover antivirals for public health emergencies. Other Flaviviruses, such as Zika (ZIKV) and dengue (DENV) viruses, are susceptible to sofosbuvir, a clinically approved drug against hepatitis C virus (HCV). Our data showed that sofosbuvir docks onto YFV RNA polymerase using conserved amino acid residues for nucleotide binding. This drug inhibited the replication of both vaccine and wild-type strains of YFV on human hepatoma cells, with EC50 values around 5 μM. Sofosbuvir protected YFV-infected neonatal Swiss mice and adult type I interferon receptor knockout mice (A129-/-) from mortality and weight loss. Because of its safety profile in humans and significant antiviral effects in vitro and in mice, Sofosbuvir may represent a novel therapeutic option for the treatment of YF. Key-words: Yellow fever virus; Yellow fever, antiviral; sofosbuvir, Author summary Yellow fever virus is transmitted by mosquitoes and its infection may be asymptomatic or lead to a wide clinical spectrum ranging from a mild febrile illness to a potentially lethal viral hemorrhagic fever characterized by liver damage. Although a yellow fever vaccine is available, low coverage allows 80,000–200,000 cases and 30,000–60,000 deaths annually worldwide. There are no specific therapy and treatment relies on supportive care, reinforcing an urgent need for antiviral repourposing. Here, we showed that sofosbuvir, clinically approved against hepatitis C, inhibits yellow fever virus replication in liver cell lines and animal models. In vitro, sofosbuvir inhibits viral RNA replication, decreases the number of infected cells and the production of infectious virus particles. These data is particularly relevante since the liver is the main target of yellow fever infection. Sofosbuvir also protected infected animals from mortality, weight loss and liver injury, especially prophylatically. Our pre-clinical results supports a second use of sofosbuvir against yellow fever.

RACIONAL: Insuficiência hepática pós-operatória devido à remanescente hepático pequeno tem sido complicação temida em pacientes que são submetidos à ressecção hepática extensa. A ligadura da veia porta associada à bipartição do fígado para hepatectomia em dois estágios (ALPPS) foi desenvolvida recentemente com a finalidade de induzir rápida e significante regeneração do fígado para pacientes em que o tumor é previamente considerado irressecável. OBJETIVO: Apresentar a experiência brasileira com o ALPPS. MÉTODO: Foram analisados 39 pacientes submetidos ao procedimento ALPPS em nove hospitais. Ele foi realizado em duas etapas. A primeira operação consistiu em ligadura do ramo direito da veia porta e bipartição hepática. Na segunda, os ramos direito da artéria hepática, via biliar e veia hepática foram ligados e o lobo hepático direito estendido foi removido. Foram 22 pacientes do sexo masculino (56,4%) e 17 do feminino (43,6%). A média de idade foi 57,3 anos (variando de 20 a 83 anos). RESULTADOS: A indicação mais comum foi metástase hepática em 32 pacientes (82,0%), seguida por colangiocarcinoma em três pacientes (7,7%). Dois morreram neste intervalo e não foram submetidos à segunda operação. O intervalo médio da primeira para a segunda operação foi de 14,1 dias (variando de 5-30 dias). O volume do segmento lateral esquerdo apresentou aumento de 83% (variando de 47-211,9%). Morbidade significante foi observada em 23 pacientes (59,0%). A mortalidade foi de 12,8% (cinco pacientes). CONCLUSÃO: O procedimento ALPPS permite ressecção hepática em pacientes com lesões consideradas previamente irressecáveis por induzir rápida hipertrofia do fígado evitando a insuficiência hepática na maioria dos pacientes. Porém ainda apresenta elevada morbidade e mortalidade. BACKGROUND: Postoperative liver failure consequent to insufficiency of remnant liver is a feared complication in patients who underwent extensive liver resections. To induce rapid and significant hepatic hypertrophy, associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been recently developed for patients which tumor is previously considered unresectable. AIM: To present the Brazilian experience with ALPPS approach. METHOD: Were analyzed 39 patients who underwent hepatic resection using ALPPS in nine hospitals. The procedure was performed in two steps. The first operation was portal vein ligation and in situ splitting. In the second operation the right hepatic artery, right bile duct and the right hepatic vein were isolated and ligated. The extended right lobe was removed. There were 22 male (56.4%) and 17 female (43.6%). At the time of the first operation, the median age was 57.3 years (range: 20-83 years). RESULTS: The most common indication was liver metastasis in 32 patients (82.0%), followed by cholangiocarcinoma in three (7.7%). Two patients died (5.2%) during this period and did not undergo the second operation. The mean interval between the first and the second operation was 14.1 days (range: 5-30 days). The volume of the left lateral segment of the liver increased 83% (range 47-211.9%). Significant morbidity after ALPPS was seen in 23 patients (59.0%). The mortality rate was 12.8% (five patients). CONCLUSION: The ALPPS approach can enable resection in patients with lesions previously considered unresectable. It induces rapid liver hypertrophy avoiding liver failure in most patients. However still has high morbidity and mortality.

Sepsis can lead to long-term cognitive changes, including memory and learning deficits, which are known as septic encephalopathy (SE). SE also includes behavioral changes. The underlying mechanism of SE is unknown, and several mechanisms have been proposed. This study investigated late anxiety-like behavior, serum cytokine levels and brain cytokine production in C57BL/6 mice subjected to polymicrobial sepsis induced by sublethal cecum ligature and puncture (CLP). Anxiety-like behavior and locomotor activity were assessed in mice 10 days after sham operation or CLP procedure using the elevated plus maze, contextual fear conditioning, and open field test. Brain and serum concentrations of the cytokines TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were determined by ELISA. We found that mice subjected to polymicrobial sepsis presented anxiety-like behavior, which was accompanied by increased serum TNF-α and brain TNF-α, IFN-γ, IL-1β, and IL-6 levels, 10 days after the surgical procedure. These findings suggest an involvement of central nervous system inflammatory mediators in the anxiety-like symptoms found in SE.

BACKGROUND: The most of the hepatitis C‐infected patients remain undiagnosed until they develop severe liver damage or submitted for serological screening. OBJECTIVE: To evaluate a recombinant multiepitope protein for detection of IgG anti‐hepatitis C virus. Method: A synthetic gene was cloned, expressed in Escherichia coli, and the recombinant protein was purified. Human serum panel consisted of 88 positives (20 HCV genotyped) and 376 negatives for hepatitis C, 6 positives for human acquired immunodeficiency virus, 6 syphilis positives, 6 hepatitis B positives were tested by IgG antihepatitis C virus using the protein by enzyme‐linked immunosorbent assay. In addition, 20 positive (all genotyped samples) and 20 negative samples were also tested by immunoblot and dot blot assays. RESULTS: Positive hepatitis C sera were strongly reactive against the protein by immunoblot assay. In the dot blot assay, positive sera were reactive until 1:1000 dilution and there were no false positive results in the hepatitis C negative sera. In the enzyme‐linked immunosorbent assay, positive and negative sera had significant discrimination. No cross‐reaction was observed in samples positive for syphilis; human acquired immunodeficiency virus and hepatitis B. All 20 genotyped samples were positive by the three methods. CONCLUSION: The multiepitope protein used here has a lower cost compared to production of each antigen separately and could be an alternative for the serological diagnosis of hepatitis C.

Introduction Toll-like receptors (TLRs) play an important role in the recognition of microbial products and in host defense against infection. However, the massive release of inflammatory mediators into the bloodstream following TLR activation following sepsis is thought to contribute to disease pathogenesis. Methods Here, we evaluated the effects of preventive or therapeutic administration of monoclonal antibodies (mAbs) targeting either TLR2 or TLR4 in a model of severe polymicrobial sepsis induced by cecal ligation and puncture in mice. Results Pre-treatment with anti-TLR2 or anti-TLR4 mAb alone showed significant protection from sepsis-associated death. Protective effects were observed even when the administration of either anti-TLR2 or anti-TLR4 alone was delayed (i.e., 3 h after sepsis induction). Delayed administration of either mAb in combination with antibiotics resulted in additive protection. Conclusion Although attempts to translate preclinical findings to clinical sepsis have failed so far, our preclinical experiments strongly suggest that there is a sufficient therapeutic window within which patients with ongoing sepsis could benefit from combined antibiotic plus anti-TLR2 or anti-TLR4 mAb treatment.

Background Succinate is an intermediate of the citric acid cycle as well as an extracellular circulating molecule, whose receptor, G protein-coupled receptor-91 (GPR91), was recently identified and characterized in several tissues, including heart. Because some pathological conditions such as ischemia increase succinate blood levels, we investigated the role of this metabolite during a heart ischemic event, using human and rodent models. Results We found that succinate causes cardiac hypertrophy in a GPR91 dependent manner. GPR91 activation triggers the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), the expression of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and the translocation of histone deacetylase 5 (HDAC5) into the cytoplasm, which are hypertrophic-signaling events. Furthermore, we found that serum levels of succinate are increased in patients with cardiac hypertrophy associated with acute and chronic ischemic diseases. Conclusions These results show for the first time that succinate plays an important role in cardiomyocyte hypertrophy through GPR91 activation, and extend our understanding of how ischemia can induce hypertrophic cardiomyopathy. Electronic supplementary material The online version of this article (doi:10.1186/s12964-014-0078-2) contains supplementary material, which is available to authorized users.

Angiotensin-(1-7) [Ang-(1-7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-{beta}. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas(+/+)) and Mas knockout (Mas (-/-)) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas(+/+) , but not in Mas (-/-) mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1-7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.

Background Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death. Results APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure. Conclusion We suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis.

Postoperative liver failure consequent to insufficiency of remnant liver is a feared complication in patients who underwent extensive liver resections. To induce rapid and significant hepatic hypertrophy, associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) has been recently developed for patients which tumor is previously considered unresectable.To present the Brazilian experience with ALPPS approach.Were analyzed 39 patients who underwent hepatic resection using ALPPS in nine hospitals. The procedure was performed in two steps. The first operation was portal vein ligation and in situ splitting. In the second operation the right hepatic artery, right bile duct and the right hepatic vein were isolated and ligated. The extended right lobe was removed. There were 22 male (56.4%) and 17 female (43.6%). At the time of the first operation, the median age was 57.3 years (range: 20-83 years).The most common indication was liver metastasis in 32 patients (82.0%), followed by cholangiocarcinoma in three (7.7%). Two patients died (5.2%) during this period and did not undergo the second operation. The mean interval between the first and the second operation was 14.1 days (range: 5-30 days). The volume of the left lateral segment of the liver increased 83% (range 47-211.9%). Significant morbidity after ALPPS was seen in 23 patients (59.0%). The mortality rate was 12.8% (five patients).The ALPPS approach can enable resection in patients with lesions previously considered unresectable. It induces rapid liver hypertrophy avoiding liver failure in most patients. However still has high morbidity and mortality.

Laparoscopic liver resections are gaining adherents among surgeons, as they show rapid recovery, shorter hospital stay and better cosmetic results. The use of a laparoscopic radiofrequency device was first carried out successfully in Brazil for resection of hepatocellular carcinoma of the segment VI in two cirrhotic patients. Although intraoperative bleeding remains a major challenge for the surgeon during laparoscopic liver resections, in both cases the hepatic vascular exclusion was expendable and there was no need for blood transfusion. Patients were discharged on the fourth postoperative day.

Acetaminophen (APAP) is a safe analgesic and antipyretic drug. However, APAP overdose leads to massive hepatocyte death. Cell death during APAP toxicity occurs by oncotic necrosis, in which the release of intracellular contents can elicit a reactive inflammatory response. We have previously demonstrated that an intravascular gradient of chemokines and mitochondria-derived formyl peptides collaborate to guide neutrophils to sites of liver necrosis by CXC chemokine receptor 2 (CXCR2) and formyl peptide receptor 1 (FPR1), respectively. Here, we investigated the role of CXCR2 chemokines and mitochondrial products during APAP-induced liver injury and in liver neutrophil influx and hepatotoxicity. During APAP overdose, neutrophils accumulated into the liver, and blockage of neutrophil infiltration by anti–granulocyte receptor 1 depletion or combined CXCR2-FPR1 antagonism significantly prevented hepatotoxicity. In agreement with our in vivo data, isolated human neutrophils were cytotoxic to HepG2 cells when cocultured, and the mechanism of neutrophil killing was dependent on direct contact with HepG2 cells and the CXCR2-FPR1–signaling pathway. Also, in mice and humans, serum levels of both mitochondrial DNA (mitDNA) and CXCR2 chemokines were higher during acute liver injury, suggesting that necrosis products may reach remote organs through the circulation, leading to a systemic inflammatory response. Accordingly, APAP-treated mice exhibited marked systemic inflammation and lung injury, which was prevented by CXCR2-FPR1 blockage and Toll-like receptor 9 (TLR9) absence (TLR9−/− mice). Conclusion: Chemokines and mitochondrial products (e.g., formyl peptides and mitDNA) collaborate in neutrophil-mediated injury and systemic inflammation during acute liver failure. Hepatocyte death is amplified by liver neutrophil infiltration, and the release of necrotic products into the circulation may trigger a systemic inflammatory response and remote lung injury. (HEPATOLOGY 2012;56:1971–1982)

Renal ischemia and reperfusion (I/R) is the major cause of acute kidney injury in hospitalized patients. Mechanisms underlying reperfusion-associated injury include recruitment and activation of leukocytes and release of inflammatory mediators. In this study, we investigated the renal effects of acute administration of AVE0991, an agonist of Mas, the angiotensin-(1–7) receptor, the angiotensin-(1–7) receptor, in a murine model of renal I/R. Male C57BL/6 wild-type or Mas−/−mice were subjected to 30 min of bilateral ischemia and 24 h of reperfusion. Administration of AVE0991 promoted renoprotective effects, as seen by improvement of function, decreased tissue injury, prevention of local and remote leucocyte infiltration, and release of the chemokine, CXCL1. I/R injury was similar in WT and Mas−/−mice, suggesting that endogenous activation of this receptor does not control renal damage under baseline conditions. In conclusion, pharmacological interventions using Mas receptor agonists may represent a therapeutic opportunity for the treatment of renal I/R injury.

PURPOSE: To evaluate the effects of hyperbaric oxygen (HO) therapy in the protection against liver ischemia/reperfusion injury. METHODS: Thirty-two male Wistar rats were divided into four groups of eight animals each: group A - laparotomy and liver manipulation, group B - liver ischemia and reperfusion, group C - HO pretreatment for 60 min followed by liver ischemia and reperfusion, and group D - pretreatment with ambient air at 2.5 absolute atmospheres for 60 min followed by liver ischemia and reperfusion. Plasma was assayed for aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH). Intra-arterial blood pressure was monitored continuously. Myeloperoxidase activity in the liver and lung was assessed 30 min after reperfusion. RESULTS: Plasma AST, ALT and LDH increased after reperfusion in all animals. Plasma ALT values and myeloperoxidase activity in the liver parenchyma were higher in HO-pretreated animals than in groups A, B and D. HO had a negative hemodynamic effect during liver reperfusion. CONCLUSION: Liver preconditioning with hyperbaric oxygen therapy aggravated liver ischemia/reperfusion injury in rats as demonstrated by plasma ALT and liver myeloperoxidase activity. OBJETIVO: Avaliar os efeitos da oxigenoterapia hiperbárica (OH) como método preventivo da lesão de isquemia e reperfusão (LIR) do fígado. MÉTODOS: Trinta e dois ratos machos Wistar foram distribuídos em quatro grupos de oito animais cada: A - laparotomia e manipulação hepática, B - isquemia e reperfusão hepática, C - pré-tratamento com OH por 60 minutos seguido de isquemia e reperfusão hepática e D - pré-tratamento com ar ambiente a 2,5 atmosferas absolutas por 60 minuto e isquemia e reperfusão hepática. Dosagens seriadas de AST, ALT e DHL foram realizadas. A pressão intra arterial foi monitorizada continuamente. O grau de infiltração leucocitária no fígado e pulmões foi inferido pela dosagem de mieloperoxidade tecidual. RESULTADOS: O nível sérico de AST, ALT e DHL aumentou em todos animais. Os animais expostos a OH apresentaram níveis de ALT e infiltração leucocitária hepática maior que os demais. A OH apresentou efeitos hemodinâmicos negativos durante a reperfusão hepática. CONCLUSÃO: O pré-condicionamento hepático por oxigenoteraia hiperbárica agrava a lesão de isquemia e reperfusão hepática em ratos.

A lesão de isquemia e reperfusão (LIR) hepática constitui resposta celular, bioquímica e imunológica a reperfusão do fígado submetido à oclusão temporária de fluxo sanguíneo. São descritos vários métodos cirúrgicos e farmacológicos para atenuar sua intensidade, e diminuirsuas conseqüências deletérias. A oxigenoterapia hiperbárica (OH) é método eficaz no tratamento de diversas doenças relacionadas à má oxigenação tecidual. Com o objetivo de estudar os efeitos do pré-condicionamento por OH na LIR, em ratos submetidos a isquemia hepática segmentar normotérmica foram utilizados 32 ratos Wistar machos, distribuídos aleatoriamente em quatro grupos de oito animais de acordo com o procedimento realizado: grupo A - laparotomia e manipulação do pedículo hepático; grupo B oclusão do pedículo vascular dos lobos mediano e lateral esquerdo do fígado por trintaminutos e reperfusão por trinta minutos; grupo C OH a 2,5 atm por 90 minutos seguida de oclusão do pedículo vascular dos lobos mediano e lateral esquerdo do fígado por trinta minutos e reperfusão por trinta minutos; grupo D exposição ao ar ambiente a 2,5 atm por 90 minutos seguida de oclusão do pedículo vascular dos lobos mediano e lateral esquerdo do fígado por trinta minutos e reperfusão por trinta minutos. O nível sérico de aspartatoaminotransferase, alanina-aminotransferase e desidrogenase láctica foram dosadas antes da isquemia, aos 30 minutos de isquemia e após 30 minutos de reperfusão. A pressão arterial durante o procedimento foi aferida por canulação da artéria carótida esquerda. Foi dosada a concentração tecidual de mieloperoxidase do fígado e do pulmão ao término da reperfusão.O pré-condicionamento por OH acentuou o aumento da concentração sérica de alanina-aminotransferase, causou maior infiltração tecidual de leucócitos no fígado e pulmão e não atenuou a queda de pressão intra-arterial ocorrida na reperfusão do fígado, comparativamente aos grupos não tratados. Em conclusão, o pré-condicionamento por OH agravou a lesão hepatocelular e aumentou a infiltração leucocitária no parênquima hepático e pulmonar em ratos submetidos a isquemia e reperfusão do fígado. Liver ischemia/reperfusion injury (IR injury) is a cellular and immunological insult. It occurs in various clinical situations in which blood flow to the liver is interrupted. Hyperbaric oxygenation (HO) has been used as a treatment option in several ischemic diseases. Howeverthe effectiveness of this method as protective against IR injuries is controversial. To determine the effect of HO in IR liver injury, a model of partial warm liver ischemia was used. Hilar area of the left lateral and medial hepatic lobes were clamped for 30 minutes followed by 30 reperfusion. For the study was conducted on 32 male Wistar rats which divided into four groups of eight animals each: group A animals submitted to laparotomy and liver manipulation, group B animals submitted to IR, group C animals pre-treated with 90 minutes of HO before IR and group C animals pre-treated with room air at 2,5 atm absolute before IR. Before ischemia episode and after 30 minutes of reperfusion, plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehidrogenase (LDH) analyses wereperformed. Intra-arterial blood pressure was monitored continuously. Myeloperoxidase (MPO) activity in liver and lung were assessed after 30 minutes of reperfusion. AST, ALT and LDH increased after reperfusion in all the animals. ALT values and MPO activity in the liver parenchyma of HO pre-treated animals were higher than in groups A,B and D. Lung parenchyma MPO activity was higher in HO treated animal groups C and D. HO had negative hemodynamic effects during liver IR. We conclude that liver preconditioning with HO aggravated liver IR injury in rats.

As ressecções hepáticas laparoscópicas vêm ganhando adeptos entre cirurgiões pois evidenciam recuperação rápida, menor permanência hospitalar e melhor resultado estético. A utilização do dispositivo laparoscópico de radiofreqüência foi pela primeira vez utilizada, com sucesso, no Brasil, para a ressecção de carcinoma hepatocelular no segmento VI, em dois pacientes cirróticos. Apesar do sangramento intraoperatório continuar sendo um grande desafio para o cirurgião durante ressecções hepáticas laparoscópicas, em ambos os casos, a exclusão vascular hepática foi dispensável e não houve necessidade de hemotransfusão. Os pacientes receberam alta hospitalar no quarto dia pós-operatório.

INTRODUÇÃO: O transplante hepático é o tratamento de escolha da doença hepática terminal. Apesar dos avanços técnicos, o transplante ainda enfrenta complicações que impactam na sobrevida e qualidade de vida dos pacientes. Entre as complicações pós-transplante, as estenoses biliares e complicações infecciosas são as mais frequentes. As estenoses biliares são classificadas como anastomóticas e não-anastomóticas. Diversos fatores estão relacionados ao surgimento de estenoses biliares, como infecção por CMV, tempo de isquemia fria prolongado, idade do doador, MELD, entre outros. OBJETIVOS: Avaliar a associação da infecção por citomegalovírus no desenvolvimento de estenose biliar em pacientes submetidos ao transplante hepático. MÉTODOS: Estudo de coorte retrospectivo de 175 pacientes submetidos a transplante hepático no Hospital Felício Rocho durante o período de janeiro 2011 a setembro de 2017. RESULTADOS: 201 pacientes foram transplantados no Hospital Felício Rocho no período de Janeiro de 2011 a Setembro de 2017. Dentre estes, 175 foram incluídos neste trabalho. A idade média dos receptores foi de 54 anos, 129/175 (73,7%) eram do sexo masculino. 21/175 (12%) desenvolveram complicações biliares sendo que 9/175 (5,1%) apresentaram mais de uma complicação. A principal complicação biliar foi estenose (9,1%), seguida de colangite , fístula (2,8%) e cálculo (2,2%). Dentre as complicações biliares 16/21 (76%) pacientes desenvolveram estenose sendo 14/16 (87,5%) do tipo anastomóticas e 02/16 (12,5%) do tipo não-anastomóticas. A infecção por CMV foi confirmada em 40/175 (22,9%) pacientes. Entre os pacientes CMV positivos, 2/40 pacientes (5%) desenvolveram complicações biliares após o diagnóstico de CMV. Na análise univariada, a presença de CMV prévio apresentou associação com desenvolvimento de complicações biliares (p=0,014), bem como estenose em geral (p=0,008). No entanto na avaliação multivariada não houve significância, bem como o tipo de estenose. A média MELD no grupo dos pacientes que desenvolveram estenose biliar foi 21, enquanto a média MELD no grupo dos pacientes que não desenvolveram foi 17 (p=0,033). Dentre os demais fatores avaliados (Idade do doador, Idade do receptor, Na doador, TIF, trombose de artéria e MELD), apenas o MELD > 21 se apresentou como fator de risco independente para o desenvolvimento de complicações biliares em geral (p=0,013) e estenose biliar (p=0,019) na análise multivariada. CONCLUSÃO: A infecção por CMV não apresentou associação para o desenvolvimento de estenose biliar não-anastomótica pós-transplante. Entretanto a presença de CMV, assim como MELD > 21 foram fatores de risco para o desenvolvimento de complicações biliares em geral e estenose biliar na análise univariada. Apenas MELD > 21 apresentou significância para o surgimento de complicações biliares e estenose na análise multivariada. INTRODUCTION: Liver transplantation is the treatment of choice for end-liver stage disease. Despite technical advances, transplantation still faces complications that impact patients' survival and quality of life. Among post-transplant complications, biliary strictures and infectious complications are the most frequent. Biliary strictures are classified as anastomotic and non-anastomotic. Several factors are related to the appearance of biliary strictures, such as CMV infection, prolonged cold ischemia time, donor age, MELD, among others. OBJECTIVES: To evaluate the association of cytomegalovirus infection, as well as its prevalence in the development of biliary stenosis in patients undergoing liver transplantation. METHODS: Retrospective cohort study of 175 patients undergoing liver transplantation at the Felicio Rocho Hospital from January 2011 to September 2017. RESULTS: 201 patients were transplanted at the Felicio Rocho Hospital from January 2011 to September 2017. Among these, 175 were included in this study. The average age of the recipients was 54 years, 129/175 (73.7%) were male. 21/175 (12%) developed biliary complications and 9/175 (5.1%) had more than one complication. The main biliary complication was stenosis (9.1%), followed by cholangitis, fistula (2.8%) and calculus (2.2%). Among the biliary complications 16/21 (76%) patients developed stenosis, 14/16 (87.5%) of the anastomotic type and 02/16 (12.5%) of the non-anastomotic type. CMV infection was confirmed in 40/175 (22.9%) patients. Among CMV positive patients, 2/40 patients (5%) developed biliary complications after the diagnosis of CMV. In the univariate analysis, the presence of previous CMV was associated with the development of biliary complications (p = 0.014), as well as stenosis (p = 0.008). However, in the multivariate assessment there was no significance, as well as the type of stenosis. The average MELD in the group of patients who developed biliary stenosis was 21, while the average MELD in the group of patients who did not develop was 17 (p = 0.033). Among the other factors evaluated (Age of the donor, Age of the recipient, sodium donor, CIT, thrombosis of the artery and MELD), only MELD> 21 presented itself as an independent risk factor for the development of biliary complications (p = 0.013) and biliary stenosis (p = 0.019) in the multivariate analysis. CONCLUSION: CMV infection was not associated with the development of post-transplant non-anastomotic biliary stenosis. However, the presence of CMV, as well as MELD> 21, were a risk factor for the development of biliary complications and biliary stenosis in univariate analysis. Only MELD> 21 showed significance for the appearance of biliary complications and stenosis in the multivariate analysis.

O objetivo deste estudo foi investigar o efeito das características demográficas, clínicas e laboratoriais do binômio doador-receptor e de variáveis relativas ao procedimento cirúrgico na sobrevida do enxerto hepático em 30 dias, tanto individualmente como agrupadas em índices de risco previamente descritos (MELD, D-MELD, DRI, SOFT, BAR e TRI). Adicionalmente, um novo índice de risco foi calculado com as variáveis que foram associadas à sobrevida do enxerto hepático na população estudada e comparado aos demais índices utilizados neste estudo. Trata-se de um estudo retrospectivo que utilizou informações de 471 transplantes hepáticos realizados no Hospital das Clínicas da Universidade Federal de Minas Gerais entre 2008 e 2018. A variável desfecho foi tempo de vida útil do enxerto hepático (período entre a data do transplante hepático e a data do óbito/re-transplante). As variáveis explicativas foram: características demográficas, clínicas, laboratoriais tanto do receptor como do doador e variáveis relativas ao procedimento. Curvas de sobrevida de Kaplan-Meier e modelos de regressão de Cox foram utilizados. Todas as variáveis que foram associadas à falha do enxerto na análise multivariada foram combinadas para calcular novo índice de risco (Belo Horizonte Risk Index - BHRI). A habilidade de predição de falha do enxerto (óbito ou re-transplante) em 30 dias do BHRI e de cada índice de risco calculado foi realizada comparando a área sob a curva ROC. Os resultados da análise multivariada evidenciaram que o IMC do doador (HR: 2,10, IC95%: 1,19-3,74) e do receptor (HR: 2,40, IC95%: 1,45-4,00), hospitalização do receptor imediatamente antes do transplante (HR: 3,70, IC95%: 2,25-6,07) e trombose de veia porta no receptor antes do transplante hepático (HR:2,14, IC95%: 1,20-3,81) foram independentemente associados a falha do enxerto hepático. Encontramos baixo poder de predição na falha do enxerto hepático dos índices de risco previamente descritos na literatura, com a área sob a curva ROC para esses índices variando de 0,47 para o DRI a 0,62 para o BAR. Uma performance discretamente melhor foi observada no índice desenvolvido neste estudo (BHRI) que obteve uma área sob a curva ROC de 0,66. Nossos resultados apontam que os fatores relacionados ao binômio doador-receptor devem ser levados em consideração pela equipe responsável pelo transplante dada a possibilidade de pior prognóstico em relação a sobrevida do enxerto, sem, entretanto, serem utilizados para contra-indicar o procedimento. O baixo poder de predição da falha do enxerto hepático dos índices de risco previamente descritos na presente população provavelmente é decorrente das diferenças entre as populações utilizadas para a elaboração destes. Estudos adicionais que promovam adaptações desses índices para as características de cada população podem trazer um melhor pareamento doador-receptor, permitindo a utilização de doadores com critérios expandidos e diminuindo a lista de espera para o enxerto hepático. The aim of this study was to investigate the effect of the demographic, clinical, and laboratory characteristics of the donor-recipient binomial and of variables related to the procedure on liver graft survival in 30 days, either individually or grouped in previously described risk indices (MELD, D-MELD, DRI, SOFT, BAR and TRI). Additionally, a new risk index was calculated with the variables that were associated with liver graft survival in the population studied and compared to the other indices used in this study. We carried out a retrospective study that used information from 471 liver transplants performed at the Hospital das Clínicas of Universidade Federal de Minas Gerais from 2008 through 2018. The outcome was the survival time of the liver graft (period between the date of the liver transplant and the date of death/re-transplant). The explicative variables were: demographic, clinical, and laboratory characteristics of both recipient and donor, and variables related to the procedure. Kaplan-Meier survival curves and Cox regression models were used. All variables that were associated with graft failure in the multivariate analysis were used to calculate a new risk index (Belo Horizonte Risk Index - BHRI). The ability to predict graft failure within 30 days of the BHRI and each calculated risk index was performed comparing the area under the ROC curve. The multivariate analysis showed that the BMI of the donor (HR: 2.10, 95%CI: 1.19-3.74) and of the recipient (HR: 2.40, 95%CI: 1.45-4.00), hospitalization of the recipient immediately before transplantation (HR: 3.70, 95%CI: 2.25-6.07) and portal vein thrombosis in the recipient before liver transplantation (HR: 2.14, 95%CI: 1.20-3.81) were independently associated with liver graft failure. The risk indices previously described in the literature demonstrated a low predictive power of the hepatic graft failure and the area under the ROC curve for these indices ranged from 0.47 for the DRI to 0.62 for the BAR. A slightly better performance was observed in the index developed in this study (BHRI), which obtained an area under the ROC curve of 0.66. Our results suggest that the characteristics related to the donor-recipient binomial should be taken into account by the surgical team at the transplant, given the possibility of a worse prognosis in relation to graft survival, however this information should not be used to contraindicate the procedure. The low predictive power for liver graft failure obtained using the risk indices previously described in the literature that we found in the present study is probably due to the differences between the populations used in the current analysis from other populations used for the elaboration of the previous indices. Additional studies to promote adaptations of these indices to the characteristics of each population can bring a better donor-recipient matching, allowing the use of expanded criteria donors and reducing the waiting list for the liver graft.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior O Citomegalovírus (HCMV) é o maior vírus pertencente à classe dos herpesvírus humanos, da família Herpesviridae. Em pacientes imunocompetentes, a infecção primária por HCMV é geralmente oligoassintomática e sua reativação muito infrequente. Entretanto, em pacientes imunussuprimidos, a doença primária é acompanhada de alta morbimortalidade e os episódios de reativação muito recorrentes e sintomáticos. Em pacientes transplantados de órgãos ou tecidos trata-se de um problema praticamente universal. O HLA é um importante sistema que participa da resposta imune adaptativa a agentes infecciosos, sendo o principal responsável pela apresentação de antígenos derivados de patógenos a linfócitos T. O HLA é uma molécula extremamente polimórfica e cada tipo específico apresenta maior ou menor afinidade com os antígenos apresentados. O objetivo deste estudo foi determinar o grau de interação do HLA com os peptídeos das 10 principais proteínas antigênicas do HCMV e sua relação com o polimorfismo HLA. A sequência peptídica das proteínas foi obtida da base de dados UniProt e os peptídeos imunogênicos foram testados in silico através da predição de ligação de epítopos às moléculas do MHC. Redes neurais artificiais foram exploradas na ferramenta do website Immune Epitope Database para o cálculo do grau de afinidade (Ic50) dos 27 tipos de HLAs do tipo I quando testados com as 10 proteínas. As afinidades dos peptídeos foram classificadas por Ic50 em alta ( 500 nM). A maioria dos peptídeos previstos (98,30%, variando de 82,3% a 99,7% para as 10 proteínas) foram de baixa afinidade, com pouca chance de interagir com os HLAs. Em média, 0,41 peptídeos mostram alta afinidade de Ic50 com alguns HLAs. Quando classificado pelo número de peptídeos imunogênicos (Ic50 abaixo de 500 nM), a UL48 (maior) foi a mais imunogênica seguido por UL55, UL29, UL32, UL28, Pp65 (mais estudada), UL122, UL123, US3, US32. Essas análises revelaram que alguns HLA são ótimos para apresentar epítopos de HCMV, como A*02:03, A*02:06, A*31:01, A*68:01; enquanto outros são maus apresentadores como A*01:01, A*26:01, B*51:01, B*44:02. O HLA A *02:03 foi o melhor HLA para responder contra os peptídeos do HCMV que exibem níveis elevados de interação com todos os peptídeos da proteína, exceto o US32. Em contraste, os HLAs A*26:01, A*01:01, B*51:01 foram os piores HLAs a apresentar peptídeos de HCMV, exibindo nenhuma ou pouca interação com peptídeos previstos de todas as proteínas. No contexto clínico, conhecer a interação entre os HLAs e as proteínas do HCMV, pode ajudar a inferir o risco de infecção viral no pós-transplante. Cytomegalovirus (HCMV) is a common human herpesvirus mostly asymptomatic in immunocompetent population. In immunosuppression, as transplantation, HCMV can reactive and affect transplant outcome, causing changes in clinical status. HLA is an important system that participates in antigen presentation and T cell activation, which imposes important barrier to transplantation. Matches between HLA guides donor-receptor compatibility. We asked if different HLA would be associated with resistance to HCMV. We chose 10 most immunogenic HCMV proteins related to CD8+ response. The proteins sequence was taken from UniProt Database and immunogenic peptides for MHC-I were predicted by T Cell Epitopes MHC binding Prediction using Artificial Neural Networks to calculate predicted Ic50 at Immune Epitope Database website against 27 types of I HLAs. The peptides affinities were classified by Ic50 into high (500 nM). The majority of predicted peptides (98.30%, varying 82.3% to 99.7% for the 10 proteins) were low affinity with little chance to interact with HLAs. On average, 0.41 peptides show high affinity Ic50 in the HLA. When classified by the number of immunogenic peptides (Ic50 below 500 nM), UL48 (biggest) were found to be the most immunogenic followed by UL55, UL29, UL32, UL28, Pp65 (most studied), UL122, UL123, US3, US32. These analyses revealed that some HLA are optimal to present HCMV epitopes, as A*02:03, A*02:06, A*31:01, A*68:01; while others are poor presenters as A*01:01, A*26:01, B*51:01, B*44:02. HLA A*02:03 was the best HLA to respond against HCMV peptides displaying high levels of interaction with all protein peptides but US32. In contrast HLA A*26:01, A*01:01, B*51:01 were the worst HLAs to presents HCMV peptides displaying none or just few interactions with predicted peptides from all proteins. Clinical context, knowing the interaction between the HLAs and HCMV proteins might help infer the risk of virus infection on post transplantation.

O adenocarcinoma ductal do pâncreas representa 2% de todos os tipos de câncer e 4% das mortes por neoplasia no Brasil. A ressecção cirúrgica representa a única opção terapêutica com chance de cura. O objetivo deste estudo foi identificar fatores prognósticos em pacientes com adenocarcinoma ductal da cabeça de pâncreas (ADCP), submetidos a tratamento cirúrgico com intenção curativa. No período de 2005 a 2018, dados clinicodemográficos, laboratoriais, peroperatórios e histopatológicos foram prospectivamente coletados. A análise de sobrevida foi feita pelo método de Kaplan-Meier e a comparação entre as curvas pelo teste de log-rank. Para a análise multivariada utilizou-se o modelo de regressão de Cox. A idade dos pacientes variou de 34 a 89 anos (média de 60,1 anos, desvio padrão de 12 anos e mediana de 59,5 anos). Os fatores com impacto na sobrevida na análise univariada foram a idade 70 anos (p=0,012), história de câncer prévio (p=0,026) e complicações pós-operatórias III e IV de Clavien-Dindo (0,015). Na análise multivariada, pacientes com CA 19.9 sérico pré-operatório entre de 38 a 554 U/ml apresentaram 3,15 vezes maior chance de óbito do que aqueles com a dosagem normal (HR 3,15; IC 95% 1,01 - 9,82; p=0,047), enquanto que os pacientes com o marcador acima de 554 U/ml evoluíram com chance 3,96 vezes maior de óbito (HR 3,96; IC 95% 1,19 - 13,10; p=0,024). Pacientes com comorbidades prévias evoluíram com chance 2,90 vezes maior de óbito que doentes sem outras condições associadas (HR 2,90; IC 95% 1,10 - 7,67; p=0,032). Em conclusão, o adenocarcinoma ductal da cabeça do pâncreas mostrou ser doença agressiva, para a qual os fatores de pior prognóstico foram a idade avançada, presença de comorbidades, história prévia de câncer, níveis de CA 19.9 elevados e complicações maiores no pós-operatório. Pancreatic ductal adenocarcinoma represents 2% of all types of cancer and 4% of deaths due to neoplasia in Brazil. Surgical resection is the only curative therapeutic option. The aim of this study was to identify prognostic factors in patients with ductal adenocarcinoma of the head of the pancreas, who underwent surgical treatment with curative intent. From 2005 to 2018, clinico-demographic, laboratory, perioperative and histopathological data were prospectively collected. Survival analysis was performed by the Kaplan-Meier method and the comparison between the curves by the log-rank test. For the multivariate analysis, the Cox regression model was used. The patients' ages ranged from 34 to 89 years (mean 60.1 years, standard deviation 12 years, median 59.5 years). The factors with impact on survival in the univariate analysis were age 70 years (p=0.012), previous cancer history (p=0.026) and Clavien-Dindo postoperative complications III and IV (p=0.015). In the multivariate analysis, patients with preoperative serum CA 19.9 level between 38 and 554 U/ml had a 3.15 times higher chance of death than those with normal level (HR 3.15; 95% CI 1.01 - 9,82; p=0.047), whereas patients with the marker above 554 U/ml had 3.96 times greater chance of death (HR 3.96; 95% CI 1.19 - 13.10; p=0.024). Patients with comorbidities had 2.90 times greater chance of death than patients without other associated conditions (HR 2.90; 95% CI 1.10 - 7.67; p=0.032). In conclusion, ductal adenocarcinoma of the pancreas head is an aggressive disease, for which the worst prognostic factors were advanced age, presence of comorbidities, previous history of cancer, high serum CA 19.9 levels and major postoperative complications.

O fígado é indispensável para manter a homeostase do corpo por ser responsável pelos principais processos metabólicos do organismo. Além disso, é um órgão que desenvolveu como característica importante a capacidade de regenerar-se. Apesar de muitos estudos sobre a regeneração do fígado, as doenças a ele relacionadas ainda são causas de óbito pela resposta insuficiente do órgão à regeneração, dependendo do grau de lesão. Portanto, sistemas bioartificiais tem sido propostos como substitutos na insuficiência hepática em estágio final. Aqui, relatamos uma plataforma biológica polimérica que induziu a proliferação sequencial do parênquima hepático dentro da plataforma em camundongos. Os discos porosos de poliéterpoliuretano foram colocados cirurgicamente no fígado e removidos nos dias 4, 8, 12 e 25 após a implantação. Não foram adicionados fatores de crescimento exógenos ou componentes da matriz extracelular na plataforma. Este dispositivo bio-híbrido proporcionou um preenchimento gradual com parênquima hepático e células não parenquimatosas, como detectado por análise histológica. Nos dias 12 e 25, uma estrutura semelhante ao fígado preenchia 2/3 da plataforma, sua organização se assemelhava ao do fígado controle, inclusive componentes vasculares, hepatócitos e outras células foram positivas para o marcador de proliferação celular por imunohistoquímica. Por coloração com Picrosirius, demonstrou-se que a deposição de colágeno no parênquima hepático no implante era semelhante ao fígado controle. A atividade de MPO foi maior no primeiro tempo avaliado (8 dias pós-implantação) e a atividade de NAG foi maior no dia 25 pós-implantação. Os níveis de TNF- foram elevados nos dias 8 e 12, e no dia 25 retornou ao mesmo nível inicial, os níveis de VEGF atingiram o pico no dia 8 e os níveis de TGF-1 foram semelhantes em todos os tempos avaliados. A concentração sérica de proteínas totais, albumina e AST não apresentou alterações durante o período experimental, apenas ALT aumentou inicialmente após a implantação, mas retornou gradualmente aos níveis de controle. A plataforma porosa sintética (sem adição de fatores exógenos) colocada adjacente ao fígado dos animais exibe potencial de ser explorado para investigar mecanismos fisiológicos de regeneração hepática. The liver is indispensable for maintaining body homeostasis by acting in the metabolic processes. It is an organ that has developed an important characteristic of regenerating itself. Despite of many studies about on liver regeneration, liver diseases are still causes of death by subsequent failure depending on the lesion degree. Therefore, bioartificial liver systems have been proposed as surrogates for end-stage liver failure. Here, we report a polymeric biological platform that induced sequential proliferation of liver parenchyma inside the scaffold in mice. Porous discs of polyetherpolyurethane were surgically placed on the liver and removed at days 4, 8, 12 and 25 after implantation. No exogenous growth factors or extracellular matrix components were added to the scaffold. This biohybrid device provided a scaffold that was gradually filled with parenchymal and non-parenchymal liver tissue as detected by histological analysis. By days 12 and 25 a liver-like structure filled 2/3 of the scaffold, its organization resembled that of a mature liver, inclusive vascular components. Therefore, hepatocytes and other cells were stained to immunohistochemistry for cell proliferation marker. By Picrosirius staining it was shown that collagen deposition in hepatic tissue into implant was similar to the control liver. MPO activity was higher in the first time evaluated (8 days post-implantation) and NAG activity was higher at day 25 post-implantation. TNF- levels were elevated on days 8 and 12, returning to baseline at 25 days, VEGF levels peaked at day 8 and TGF-1 levels were similar at all-time points evaluated. The serum concentration of total proteins, albumin and AST did not change during the experimental period, only ALT increased initially after implantation, but gradually returned to control levels. The synthetic porous scaffold (without addition of exogenous factors) placed adjacent to the native animal liver exhibits the potential to be exploited to investigate physiological mechanisms of liver regeneration.

Pacientes com doenças graves de fígado que demandam transplante hepático (TH) comumente evoluem com alterações do estado nutricional e do balanço energético pré e pós-operação. Essas alterações podem impactar negativamente a morbimortalidade desses enfermos. O objetivo deste estudo foi avaliar diferentes parâmetros de avaliação nutricional para predizer desfechos clínicos no momento pré-transplante e avaliar a evolução nutricional e do balanço energético após o TH. Ainda, avaliar se essas alterações impactam no tempo de internação (no centro de terapia intensiva CTI; e hospitalar) e na morbidade e mortalidade desses pacientes. No pré-operatório e pós-transplante precoce foram avaliados estado nutricional por meio da Avaliação Global Subjetiva (AGS), antropometria (circunferência do braço CB; dobra cutânea tricipital DCT e área muscular do braço AMB), dinamometria manual (DM), Teste de 6 minutos de Caminhada (T6MC) e ângulo de fase padronizado (AFP). Gasto energético de repouso (GER) foi mensurado por calorimetria indireta, ingestão alimentar foi estimada por registro alimentar e, foi realizado cálculo do balanço energético (BE). Desfechos clínicos no pré-transplante compreenderam complicações de cirrose e mortalidade e, no pós-transplante o tempo de internação no CTI e hospitalar, a presença de complicações pós-operatórias e a mortalidade. Essas variáveis foram coletadas a partir de prontuário médico do paciente. Análise uni e multivariada e curva ROC foram realizadas, sendo p