Serum ficolin-2 correlates worse than fecal calprotectin and CRP with endoscopic Crohn's disease activity.

BACKGROUND AND AIMS Ficolin-2 is an acute phase reactant produced by the liver and targeted to recognize N-acetyl-glucosamine which is present in bacterial and fungal cell walls. We recently showed that ficolin-2 serum levels were significantly higher in CD patients compared to healthy controls. We aimed to evaluate serum ficolin-2 concentrations in CD patients regarding their correlation with endoscopic severity and to compare them with clinical activity, fecal calprotectin, and CRP. METHODS Patients provided fecal and blood samples before undergoing ileo-colonoscopy. Disease activity was scored clinically according to the Harvey-Bradshaw Index (HBI) and endoscopically according to the simplified endoscopic score for CD (SES-CD). Ficolin-2 serum levels and fecal calprotectin levels were measured by ELISA. RESULTS A total of 136 CD patients were prospectively included (mean age at inclusion 41.5±15.4 years, 37.5% females). Median HBI was 3 [2-6] points, median SES-CD was 5 [2-8], median fecal calprotectin was 301 [120-703] μg/g, and median serum ficolin-2 was 2.69 [2.02-3.83] μg/mL. SES-CD correlated significantly with calprotectin (R=0.676, P<0.001), CRP (R=0.458, P<0.001), HBI (R=0.385, P<0.001), and serum ficolin-2 levels (R=0.171, P=0.047). Ficolin-2 levels were higher in CD patients with mild endoscopic disease compared to patients in endoscopic remission (P=0.015) but no difference was found between patients with mild, moderate, and severe endoscopic disease. CONCLUSIONS Ficolin-2 serum levels correlate worse with endoscopic CD activity when compared to fecal calprotectin or CRP.

Computed tomography findings in liver fibrosis and cirrhosis

Abstract PRINCIPLES: Computed tomography (CT) is inferior to the fibroscan and laboratory testing in the noninvasive diagnosis of liver fibrosis. On the other hand, CT is a frequently used diagnostic tool in modern medicine. The auxiliary finding of clinically occult liver fibrosis in CT scans could result in an earlier diagnosis. The aim of this study was to analyse quantifiable direct signs of liver remodelling in CT scans to depict liver fibrosis in a precirrhotic stage. METHODS: Retrospective review of 148 abdominal CT scans (80 liver cirrhosis, 35 precirrhotic fibrosis and 33 control patients). Fibrosis and cirrhosis were histologically proven. The diameters of the three main hepatic veins were measured 1-2 cm before their aperture into the inferior caval vein. The width of the caudate and the right hepatic lobe were divided, and measured horizontally at the level of the first bifurcation of the right portal vein in axial planes (caudate-right-lobe ratio). A combination of both (sum of liver vein diameters divided by the caudate-right lobe ratio) was defined as the ld/crl ratio. These metrics were analysed for the detection of liver fibrosis and cirrhosis. RESULTS: An ld/crl-r <24 showed a sensitivity of 83% and a specificity of 76% for precirrhotic liver fibrosis. Liver cirrhosis could be detected with a sensitivity of 88% and a specificity of 82% if ld/crl-r <20. CONCLUSION: An ld/crl-r <24 justifies laboratory testing and a fibroscan. This could bring forward the diagnosis and patients would profit from early treatment in a potentially reversible stage of disease.

Clinical Course of acute-on-chronic liver failure syndrome and effects on prognosis

Acute-on-chronic liver failure (ACLF) is characterized by acute decompensation (AD) of cirrhosis, organ failure(s), and high 28-day mortality. We investigated whether assessments of patients at specific time points predicted their need for liver transplantation (LT) or the potential futility of their care. We assessed clinical courses of 388 patients who had ACLF at enrollment, from February through September 2011, or during early (28-day) follow-up of the prospective multicenter European Chronic Liver Failure (CLIF) ACLF in Cirrhosis study. We assessed ACLF grades at different time points to define disease resolution, improvement, worsening, or steady or fluctuating course. ACLF resolved or improved in 49.2%, had a steady or fluctuating course in 30.4%, and worsened in 20.4%. The 28-day transplant-free mortality was low-to-moderate (6%-18%) in patients with nonsevere early course (final no ACLF or ACLF-1) and high-to-very high (42%-92%) in those with severe early course (final ACLF-2 or -3) independently of initial grades. Independent predictors of course severity were CLIF Consortium ACLF score (CLIF-C ACLFs) and presence of liver failure (total bilirubin ≥12 mg/dL) at ACLF diagnosis. Eighty-one percent had their final ACLF grade at 1 week, resulting in accurate prediction of short- (28-day) and mid-term (90-day) mortality by ACLF grade at 3-7 days. Among patients that underwent early LT, 75% survived for at least 1 year. Among patients with ≥4 organ failures, or CLIF-C ACLFs >64 at days 3-7 days, and did not undergo LT, mortality was 100% by 28 days. CONCLUSIONS Assessment of ACLF patients at 3-7 days of the syndrome provides a tool to define the emergency of LT and a rational basis for intensive care discontinuation owing to futility.

Subjective well -being as predictor of mortality, heart disease, and obesity: Prospective evidence from the Alameda County Study

Based on the World Health Organization's (1965) definition of health, understanding of health requires understanding of positive psychological states. Subjective Well-being (SWB) is a major indicator of positive psychological states. Up to date, most studies of SWB have been focused on its distributions and determinants. However, study of its consequences, especially health consequences, is lacking. This dissertation research examined Subjective Well-being, as operationally defined by constructs drawn from the framework of Positive Psychology, and its sub-scores (Positive Feelings and Negative Feelings) as predictors of three major health outcomes—mortality, heart disease, and obesity. The research used prospective data from the Alameda County Study over 29 years (1965–1994), based on a stratified, randomized, representative sample of the general public in Alameda County, California (Baseline N = 6928). ^ Multivariate analyses (Survival analyses using sequential Cox Proportional Hazard models in the cases of mortality and heart disease, and sequential Logistic Regression analyses in the case of obesity) were performed as the main methods to evaluate the associations of the predictors and the health outcomes. The results revealed that SWB reduced risks of all-cause mortality, natural-cause mortality, and cardiovascular mortality. Positive feelings not only had an even stronger protective effect against all-cause, natural-cause and cardiovascular mortality, but also predicted decreased unnatural-cause mortality which includes deaths from suicide, homicide, accidents, mental disorders, drug dependency, as well as alcohol-related liver diseases. These effects were significant even after adjusted for age, gender, education, and various physical health measures, and, in the case of cardiovascular mortality, obesity and health practices (alcohol consumption, smoking, and physical activities). However, these two positive psychological indicators, SWB and positive feelings, did not predict obesity. And negative feelings had no significant effect on any of the health outcomes evaluated, i.e., all-cause mortality, natural- and unnatural-cause mortality, cardiovascular mortality, or obesity, after covariates were controlled. These findings were discussed (1) in comparison with relevant existing studies, (2) in terms of their implications in health research and promotion, (3) in terms of the independence of positive and negative feelings, and (4) from a Positive Psychology perspective and its significance in Public Health research and practice. ^

Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model. STAT5b gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in STAT5b−/− males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient Little mice, STAT5b−/− mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in STAT5b−/− mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.

Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified α-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered α-gal A, (ii) to assess the pharmacokinetics of i.v.-administered α-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. α-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified α-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of α-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of α-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 × 106 infectious units of recombinant adeno-associated virus encoding the human β-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.

Aging accentuates and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice

Fabry disease is an X-linked metabolic disorder caused by a deficiency of α-galactosidase A (α-Gal A). The enzyme defect leads to the systemic accumulation of glycosphingolipids with α-galactosyl moieties consisting predominantly of globotriaosylceramide (Gb3). In patients with this disorder, glycolipid deposition in endothelial cells leads to renal failure and cardiac and cerebrovascular disease. Recently, we generated α-Gal A gene knockout mouse lines and described the phenotype of 10-week-old mice. In the present study, we characterize the progression of the disease with aging and explore the effects of bone marrow transplantation (BMT) on the phenotype. Histopathological analysis of α-Gal A −/0 mice revealed subclinical lesions in the Kupffer cells in the liver and macrophages in the skin with no gross lesions in the endothelial cells. Gb3 accumulation and pathological lesions in the affected organs increased with age. Treatment with BMT from the wild-type mice resulted in the clearance of accumulated Gb3 in the liver, spleen, and heart with concomitant elevation of α-Gal A activity. These findings suggest that BMT may have a potential role in the management of patients with Fabry disease.

Localization of the Wilson’s disease protein product to mitochondria

Wilson’s disease (WND) is an inherited disorder of copper homeostasis characterized by abnormal accumulation of copper in several tissues, particularly in the liver, brain, and kidney. The disease-associated gene encodes a copper-transporting P-type ATPase, the WND protein, the subcellular location of which could be regulated by copper. We demonstrate that the WND protein is present in cells in two forms, the 160-kDa and the 140-kDa products. The 160-kDa product was earlier shown to be targeted to trans-Golgi network. The 140-kDa product identified herein is located in mitochondria as evidenced by the immunofluorescent staining of HepG2 cells with specific mitochondria markers and polyclonal antibody directed against the C terminus of the WND molecule. The mitochondrial location for the 140-kDa WND product was confirmed by membrane fractionation and by analysis of purified human mitochondria. The antibody raised against a repetitive sequence in the N-terminal portion of the WND molecule detects an additional 16-kDa protein, suggesting that the 140-kDa product was formed after proteolytic cleavage of the full-length WND protein at the N terminus. Thus, the WND protein is a P-type ATPase with an unusual subcellular localization. The mitochondria targeting of the WND protein suggests its important role for copper-dependent processes taking place in this organelle.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids.

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as "eat me" signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

Imaging liver-stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.

The influence of viral genotypes and rejection episodes on the recurrence of hepatitis C after liver transplantation

Purpose. The aim of this study was to report the influence of hepatitis C virus (HCV) genotype and rejection episodes on the outcome of orthotopic liver transplantation (OLT), hepatitis recurrence, and progression to graft cirrhosis after OLT. Methods. Fifty-three patients who all had undergone OLT for end-stage liver cirrhosis were selected for this study. Hepatitis C genotype was determined. Recurrent hepatitis and rejection were diagnosed based on elevated liver function tests and a liver biopsy. Results. The patients were followed up for a mean of 51.9 +/- 34.3 months. The cumulative survival rate was no different in OLT for hepatitis C and OLT for all other liver diseases. After OLT, serum HCV RNA was detected in 93%. Histological recurrence occurred in 85% of all patients. The 1-, 3-, and 5-year recurrence rates were 48%, 77%, and 85%, respectively. Of the 41 patients with recurrent hepatitis C, 4 (10%) had cirrhosis, 18 (44%) had hepatitis with fibrosis, and 91 (46%) had hepatitis without fibrosis at the end of follow-up. A total of 32% of the patients were infected by HCV genotype 1b and 68% by other HCV genotypes. The recurrence rates were significantly higher in patients infected with genotype 1b than in those with other genotypes (p = 0.04). Twenty of 48 patients (42%) experienced acute rejection. There was a strong association between the number of rejection episodes and the incidence of HCV-related cirrhosis (p < 0.01). Conclusion. Our findings showed the genotype 1b to result in a higher recurrence rate after OLT. On the other hand, rejection episodes were associated with a more rapid progression to graft cirrhosis.