Bioenergetic adaptations of the human liver in the ALPPS procedure - how liver regeneration correlates with mitochondrial energy status.

BACKGROUND: The Associating Liver Partition and Portal Ligation for Staged Hepatectomy (ALPPS) depends on a significant inter-stages kinetic growth rate (KGR). Liver regeneration is highly energy-dependent. The metabolic adaptations in ALPPS are unknown. AIMS: i) Assess bioenergetics in both stages of ALPPS (T1 and T2) and compare them with control patients undergoing minor (miHp) and major hepatectomy (MaHp), respectively; ii) Correlate findings in ALPPS with volumetric data; iii) Investigate expression of genes involved in liver regeneration and energy metabolism. METHODS: Five patients undergoing ALPPS, five controls undergoing miHp and five undergoing MaHp. Assessment of remnant liver bioenergetics in T1, T2 and controls. Analysis of gene expression and protein content in ALPPS. RESULTS: Mitochondrial function was worsened in T1 versus miHp; and in T2 versus MaHp (p < 0.05); but improved from T1 to T2 (p < 0.05). Liver bioenergetics in T1 strongly correlated with KGR (p < 0.01). An increased expression of genes associated with liver regeneration (STAT3, ALR) and energy metabolism (PGC-1α, COX, Nampt) was found in T2 (p < 0.05). CONCLUSION: Metabolic capacity in ALPPS is worse than in controls, improves between stages and correlates with volumetric growth. Bioenergetic adaptations in ALPPS could serve as surrogate markers of liver reserve and as target for energetic conditioning.

Functional hepatocellular regeneration in elderly patients undergoing hepatectomy.

BACKGROUND & AIMS: More than 50% of liver tumours occur in patients aged 65 years or more. Assessment of functional liver regeneration capacity is crucial to minimize postoperative liver failure. We aimed to study functional hepatocellular regeneration, through scintigraphic quantification of Mebrofenin hepatic extraction fraction (HEF), after partial hepatectomy, comparing elderly patients with younger ones. METHODS: One hundred and two patients undergoing partial hepatectomy for primary or secondary hepatic lesions were prospectively included and divided in two groups: Group A - 58 patients aged <65 years (33 men, 53.9 ± 8.7 years), Group B - 44 patients aged ≥65 years (32 men, 71 ± 5 years). Groups were comparable in several aspects except for the presence of cirrhosis (more common in Group B, all patients Child-Pugh score A) and the initial diagnosis (Group B - primary lesions, Group A - metastases). The scintigraphic evaluation of Mebrofenin-HEF was performed before surgery, on the 5th and 30th day post-hepatectomy. RESULTS: Mortality and morbidity were 3.4 and 12.1%, respectively, in Group A and 2.3 and 11.4% in Group B (n.s.). HEF values (%), T1/2 (min) and Tmax (min) showed no significant differences between the two groups: Group A (preoperative: HEF = 99.2 ± 1.5%, T1/2 = 36.7 ± 21.3, Tmax = 15 ± 6. Day 5: HEF = 96.3 ± 10.8%, T1/2 = 76.4 ± 75.9; Tmax = 13.3 ± 4.9. Day 30: HEF = 98.4 ± 5.5%, T1/2 = 38.6 ± 7.7, Tmax = 12.8 ± 3.6) and Group B (preoperative: HEF = 95.3 ± 13%, T1/2 = 38.1 ± 24.1; Tmax = 15.9 ± 9.4. Day 5: HEF = 98.4 ± 2.6%, T1/2 = 106.6 ± 131.7; Tmax = 15.1 ± 6.2. Day 30: HEF = 99 ± 2.1%, T1/2 = 40.5 ± 27; Tmax = 15.5 ± 6.7). CONCLUSION: Our results suggest that functional hepatocellular regeneration is early, fast and similar between elderly and younger patients. Thus, age alone, does not appear to represent an absolute contraindication to hepatectomy.

CD8+ Treg cells play a role in the obesity-associated insulin resistance.

Obesity-related chronic low-grade inflammation may trigger insulin resistance and type 2 diabetes (T2D) development. Cells with regulatory phenotype have been shown to be reduced during obesity, especially CD4+ Treg cells. However, little is known about the CD8+ Treg cells. Therefore, we aim to characterize the CD8+ Treg cells in human peripheral blood and adipose tissue, specifically, to address the effect of obesity and insulin resistance in this regulatory immune cell population. A group of 42 participants with obesity (OB group) were recruited. Fourteen of them were evaluated pre- and post-bariatric surgery. A group of age- and sex-matched healthy volunteers (n = 12) was also recruited (nOB group). CD8+ Treg cell quantification and phenotype were evaluated by flow cytometry, in peripheral blood (PB), subcutaneous (SAT), and visceral adipose tissues (VAT). The OB group displayed a higher percentage of CD8+ Treg cells in PB, compared to the nOB. In addition, they were preferentially polarized into Tc1- and Tc1/17-like CD8+ Treg cells, compared to nOB. Moreover, SAT displayed the highest content of CD8+ Tregs infiltrated, compared to PB or VAT, while CD8+ Tregs infiltrating VAT displayed a higher percentage of cells with Tc1-like phenotype. Participants with pre-diabetes displayed a reduced percentage of TIM-3+CD8+ Tregs in circulation, and PD-1+CD8+ Tregs infiltrated in the VAT. An increase in the percentage of circulating Tc1-like CD8+ Treg cells expressing PD-1 was observed post-surgery. In conclusion, obesity induces significant alterations in CD8+ Treg cells, affecting their percentage and phenotype, as well as the expression of important immune regulatory molecules.

Identification and characterization of circulating and adipose tissue infiltrated CD20+T cells from subjects with obesity that undergo bariatric surgery.

T cells play critical roles in adipose tissue (AT) inflammation. The role of CD20+T cell in AT dysfunction and their contributing to insulin resistance (IR) and type 2 diabetes progression, is not known. The aim was to characterize CD20+T cells in omental (OAT), subcutaneous (SAT) and peripheral blood (PB) from subjects with obesity (OB, n = 42), by flow cytometry. Eight subjects were evaluated before (T1) and 12 months post (T2) bariatric/metabolic surgery (BMS). PB from subjects without obesity (nOB, n = 12) was also collected. Higher percentage of CD20+T cells was observed in OAT, compared to PB or SAT, in OB-T1. CD20 expression by PB CD4+T cells was inversely correlated with adiposity markers, while follicular-like CD20+T cells were positively correlated with impaired glucose tolerance (increased HbA1c). Notably, among OB-T1, IR establishment was marked by a lower percentage and absolute number of PB CD20+T cells, compared nOB. Obesity was associated with higher percentage of activated CD20+T cells; however, OAT-infiltrated CD20+T cells from OB-T1 with diabetes displayed the lowest activation. CD20+T cells infiltrating OAT from OB-T1 displayed a phenotype towards IFN-γ-producing Th1 and Tc1 cells. After BMS, the percentage of PB CD4+CD20+T cells increased, with reduced Th1 and increased Th17 phenotype. Whereas in OAT the percentage of CD20+T cells with Th1/17 and Tc1/17 phenotypes increased. Interestingly, OAT from OB pre/post BMS maintained higher frequency of effector memory CD20+T cells. In conclusion, CD20+T cells may play a prominent role in obesity-related AT inflammation.

Bariatric Surgery Induces Alterations in the Immune Profile of Peripheral Blood T Cells.

Low-grade inflammation is closely linked to obesity and obesity-related comorbidities; therefore, immune cells have become an important topic in obesity research. Here, we performed a deep phenotypic characterization of circulating T cells in people with obesity, using flow cytometry. Forty-one individuals with obesity (OB) and clinical criteria for bariatric surgery were enrolled in this study. We identified and quantified 44 different circulating T cell subsets and assessed their activation status and the expression of immune-checkpoint molecules, immediately before (T1) and 7-18 months after (T2) the bariatric surgery. Twelve age- and sex-matched healthy individuals (nOB) were also recruited. The OB participants showed higher leukocyte counts and a higher percentage of neutrophils. The percentage of circulating Th1 cells were negatively correlated to HbA1c and insulin levels. OB Th1 cells displayed a higher activation status and lower PD-1 expression. The percentage of Th17 and Th1/17 cells were increased in OB, whereas the CD4+ Tregs' percentage was decreased. Interestingly, a higher proportion of OB CD4+ Tregs were polarized toward Th1- and Th1/17-like cells and expressed higher levels of CCR5. Bariatric surgery induced the recovery of CD4+ Treg cell levels and the expansion and activation of Tfh and B cells. Our results show alterations in the distribution and phenotype of circulating T cells from OB people, including activation markers and immune-checkpoint proteins, demonstrating that different metabolic profiles are associated to distinct immune profiles, and both are modulated by bariatric surgery.

Hepatic extraction fraction: interest of its quantification in patients with hepatic tumours.

BACKGROUND/AIMS: This prospective study aimed to estimate the usefulness of the hepatic extraction fraction (HEF) in the context of the pre- and postoperative evaluation of patients with hepatic tumours. METHODOLOGY: Seventy patients with colorectal metastases (n=25), hepatocellular carcinoma (n=25), cholangiocellular carcinoma (n=6), gastric cancer metastases (n=5), hemangioma (n=5) and others (n=4) were included. Thirty patients underwent hepatectomy. Child-Pugh score, prothrombin, albumin, ALT, AST, AF, LDH, total, direct and indirect bilirubin, platelet number as well as the HEF were evaluated in the preoperative period and one month after hepatectomy. RESULTS: Preoperative evaluation of HEF values between Child-Pugh A (93.6±17.3%) and Child-Pugh B/C (n=13; 58.1±28.6%) demonstrated significant differences (p=0.001). We found a high negative correlation between the preoperative HEF and ALT (p<0.001), AST (p<0.001), AF (p<0.001), TB (p<0.001), IB (p<0.001) and DB (p<0.001), and also a high positive correlation between the preoperative HEF and albumin (p<0.001) or prothrombin (p<0.01). All operated patients had a normal HEF and a positive correlation between the postoperative HEF and albumin (p<0.05) at one month after surgery. CONCLUSIONS: The HEF allows a dynamic evaluation of hepatocellular function, which is not possible with other clinical, biological and radiological methods.

Cholangiocarcinoma: from molecular biology to treatment.

Cholangiocarcinoma is a rare tumor originating in the bile ducts, which, according to their anatomical location, is classified as intrahepatic, extrahepatic and hilar. Nevertheless, incidence rates have increased markedly in recent decades. With respect to tumor biology, several genetic alterations correlated with resistance to chemotherapy and radiotherapy have been identified. Here, we highlight changes in KRAS and TP53 genes that are normally associated with a more aggressive phenotype. Also IL-6 and some proteins of the BCL-2 family appear to be involved in the resistance that the cholangiocarcinoma presents toward conventional therapies. With regard to diagnosis, tumor markers most commonly used are CEA and CA 19-9, and although its use isolated appears controversial, their combined value has been increasingly advocated. In imaging terms, various methods are needed, such as abdominal ultrasound, computed tomography and cholangiopancreatography. Regarding therapy, surgical modalities are the only ones that offer chance of cure; however, due to late diagnosis, most patients cannot take advantage of them. Thus, the majority of patients are directed to other therapeutic modalities like chemotherapy, which, in this context, assumes a purely palliative role. Thus, it becomes urgent to investigate new therapeutic options for this highly aggressive type of tumor.

Fluorine-18 Fluorodeoxyglucose Uptake in Hepatocellular Carcinoma: Correlation with Glucose Transporters and p53 Expression.

INTRODUCTION: Hepatocellular Carcinoma (HCC) is one of most lethal cancers worldwide. The prognosis is very poor and therapeutic options are limited. The aim of this study was to determine the correlation of the [(18)F]FDG uptake profile of three HCC cell lines with p53 and glucose transporters (GLUTs) 1, 2, 3, 5 and 12 expression and with the glucose level present in the cell culture medium. METHODS: Cell lines used are HepG2 (wp53), HuH7 (overexpress p53) and Hep3B2.1-7 (p53null). An immunocytochemical analysis was performed to evaluate p53 expression. Through uptake studies were analyzed the [(18)F]FDG uptake profiles of all cell lines under study. The expression of GLUTs were quantified by flow cytometry. The [(18)F]FDG uptake studies GLUTs expression analysis were performed on cells that grew in a high and low glucose medium in order to determine the effect of glucose concentration on GLUTs expression and on [(18)F]FDG uptake. RESULTS: Immunocytochemical analysis confirmed the p53 expression profiles of all cell lines. It was found out that for all cell lines, [(18)F]FDG uptake is higher when cells grow in low glucose medium, however, the glucose level doesn't affect mostly the GLUTs expression. The Hep3B2.1-7 (p53null) is always the one that have higher [(18)F]FDG uptake. It was found that not always GLUT1 and GLUT3 are the most expressed by these cell lines. CONCLUSIONS: Our results shown that the p53 expression influences [(18)F]FDG uptake. This suggests that [(18)F]FDG may be used in HCC diagnosis, and may even provide some information about the genetic profile of the tumor.

Influence of P53 on the radiotherapy response of hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. METHODS: Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. RESULTS: The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. CONCLUSIONS: These results suggest that P53 plays a key role in the radiotherapy response of HCC.

Positron emission tomography diagnostic imaging in multidrug-resistant hepatocellular carcinoma: focus on 2-deoxy-2-(18F)Fluoro-D-Glucose.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. Surgical resection and liver transplantation are still the best options for treatment. Nevertheless, as the number of patients who may benefit from these therapies is limited, alternative therapies have been developed, including chemotherapy. However, partly due to the expression of multidrug resistance (MDR) proteins, it has been found that HCC is a highly chemoresistant tumor. The major family of MDR proteins is the ATP-binding cassette (ABC) transporter superfamily, which includes P-glycoprotein (Pgp) and MDR-associated protein 1 (MRP1). Positron emission tomography using the radiolabeled analog of glucose, 2-deoxy-2-((18)F)fluoro-D-glucose ([(18)F]FDG), has been used in diagnostic imaging of various types of tumors. Clinical studies are inconsistent but experimental studies have shown that [(18)F]FDG uptake is associated with tumor grade and is inversely proportional to Pgp expression in HCC. These studies unveil that [(18)F]FDG can be a substrate of Pgp, although that relationship remains unclear. This review sums up the relationship between MDR expression in HCC, and [(18)F]FDG uptake by tumor cells, showing that this radiopharmaceutical may provide a useful tool for the study of chemoresistance in HCC, and that the use of this marker may contribute to the therapeutic choice on this highly aggressive tumor.

Hepatectomy and liver regeneration: from experimental research to clinical application.

BACKGROUND: The mechanisms and kinetics of hepatic growth have continuously been investigated. This study concerns liver regeneration in animal and patients who underwent partial hepatectomy evaluated by the hepatic extraction fraction (HEF) calculated through radioisotopic methods. METHODS: Thirty normal Wistar rats were submitted to an 85% hepatectomy, and 95 patients with primary and secondary liver tumours were included. In animal study, the liver regeneration kinetics was assessed by HEF using 99mTc-mebrofenin, the ratio liver/bodyweight and by using bromodeoxyuridine deoxyribonucleic acid incorporation. In patient study, the liver regeneration was evaluated by calculation of HEF before surgery, 5 and 30 days after hepatectomy. RESULTS: In animal, we verified a positive correlation between HEF kinetics and liver/bodyweight ratio or hepatocyte proliferation evaluated by bromodeoxyuridine deoxyribonucleic acid staining after 85% hepatectomy. In the clinical arm, no statistical differences of the HEF before hepatectomy, 5 and 30 days after hepatectomy, were observed. CONCLUSIONS: Our results support the view that human liver regeneration commences early, is fast, non-anatomical and functionally complete 5 days after hepatectomy. The fast functional liver regeneration may have a high clinical impact particularly concerning the post-operative oncological therapeutic approaches.