Liver Stiffness by Transient Elastography Predicts Liver-Related Complications and Mortality in Patients with Chronic Liver Disease

Background

Liver stiffness measurement (LSM) by transient elastography (TE, FibroScan) is a validated method for noninvasively staging liver fibrosis. Most hepatic complications occur in patients with advanced fibrosis. Our objective was to determine the ability of LSM by TE to predict hepatic complications and mortality in a large cohort of patients with chronic liver disease.

Methods

In consecutive adults who underwent LSM by TE between July 2008 and June 2011, we used Cox regression to determine the independent association between liver stiffness and death or hepatic complications (decompensation, hepatocellular carcinoma, and liver transplantation). The performance of LSM to predict complications was determined using the c-statistic.

Results

Among 2,052 patients (median age 51 years, 65% with hepatitis B or C), 87 patients (4.2%) died or developed a hepatic complication during a median follow-up period of 15.6 months (interquartile range, 11.0–23.5 months). Patients with complications had higher median liver stiffness than those without complications (13.5 vs. 6.0 kPa; P<0.00005). The 2-year incidence rates of death or hepatic complications were 2.6%, 9%, 19%, and 34% in patients with liver stiffness <10, 10–19.9, 20–39.9, and ≥40 kPa, respectively (P<0.00005). After adjustment for potential confounders, liver stiffness by TE was an independent predictor of complications (hazard ratio [HR] 1.05 per kPa; 95% confidence interval [CI] 1.03–1.06). The c-statistic of liver-stiffness for predicting complications was 0.80 (95% CI 0.75–0.85). A liver stiffness below 20 kPa effectively excluded complications (specificity 93%, negative predictive value 97%); however, the positive predictive value of higher results was sub-optimal (20%).

Conclusions

Liver stiffness by TE accurately predicts the risk of death or hepatic complications in patients with chronic liver disease. TE may facilitate the estimation of prognosis and guide management of these patients.     

Genetical genomic determinants of alcohol consumption in rats and humans

Background

We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

Results

In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.

Conclusion

Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.     

Clusterin in cerebrospinal fluid: analysis of carbohydrates and quantification of native and glycosylated forms

Clusterin is suggested to be involved in the pathogenesis of Alzheimer's disease. Clusterin expression is increased in brain tissue in affected regions of Alzheimer patients, and intense clusterin staining is found in both senile plaques and in neuronal and glia cells. In contrast, the cerebrospinal fluid level of clusterin in Alzheimer patients has, thus far, been found unchanged. Clusterin is a glycosylated protein, and an alteration of its glycosylation in Alzheimer's disease might influence accurate quantification in cerebrospinal fluid through interference of antibody binding to the protein. Using enzymatic deglycosylation of clusterin isolated from cerebrospinal fluid, we found that the carbohydrates attached to clusterin were of the N-linked type and sialic acids. Based on this finding, cerebrospinal fluid samples from Alzheimer patients (n = 99) and controls (n = 39) were analysed. The samples were treated with peptide: N-glycanase F, cleaving off N-linked carbohydrates, and clusterin was quantified before and after deglycosylation using a new sandwich enzyme-linked immunosorbent assay. Clusterin was significantly increased in Alzheimer patients, in both native (7.17 ± 2.43 AU versus 5.73 ± 2.09 AU; p = 0.002), and deglycosylated samples (12.19 ± 5.00 AU versus 9.68 ± 4.38 AU; p = 0.004). Deglycosylation led to increased measured levels of clusterin by 70% (p < 0.001) in Alzheimer patients and 67% (p < 0.001) in controls. These findings indicate that glycosylation of proteins may interfere with their quantification. The results show that clusterin is significantly increased in cerebrospinal fluid from Alzheimer patients as a group, supporting that clusterin might be involved in the pathogenesis of Alzheimer's disease. However, the individual clusterin levels overlap between the two groups, and thus cerebrospinal fluid clusterin measurement is not suitable as a biochemical marker in the diagnosis of Alzheimer's disease.     

UDP-N-acetylglucosamine:alpha-6-D-mannoside beta1,6 N-acetylglucosaminyltransferase V (Mgat5) deficient mice

Targeted gene mutations in mice that cause deficiencies in protein glycosylation have revealed functions for specific glycans structures in embryogenesis, immune cell regulation, fertility and cancer progression. UDP-N-acetylglucosamine:alpha-6-D-mannoside beta1,6 N-acetylglucosaminyltransferase V (GlcNAc-TV or Mgat5) produces N-glycan intermediates that are elongated with poly N-acetyllactosamine to create ligands for the galectin family of mammalian lectins. We generated Mgat5-deficient mice by gene targeting methods in embryonic stem cells, and observed a complex phenotype in adult mice including susceptibility to autoimmune disease, reduced cancer progression and a behavioral defect. We found that Mgat5-modified N-glycans on the T cell receptor (TCR) complex bind to galectin-3, sequestering TCR within a multivalent galectin-glycoprotein lattice that impedes antigen-dependent receptor clustering and signal transduction. Integrin receptor clustering and cell motility are also sensitive to changes in Mgat5-dependent N-glycosylation. These studies demonstrate that low affinity but high avidity interactions between N-glycans and galectins can regulate the distribution of cell surface receptors and their responsiveness to agonists.     

TRPM channels, calcium and redox sensors during innate immune responses

Melastatin-related TRPM ion channels have emerged as novel therapeutic targets due to their potential ability to modulate the function and fate of immune cells during inflammation, innate, and adaptive immunity. Four family members, TRPM1, TRPM2, TRPM4 and TRPM7 have a strong presence in the immune system. TRPM channels regulate ion-homeostasis by sensing cellular redox status and cytoplasmic calcium levels. TRPM2 for example, is highly expressed in phagocytes. This channel is activated by intracellular ADP-ribose upon exposure to oxidative stress and induces cell death. Here we will review the functional links between TRPM-mediated ion conductance, chemotaxis, apoptosis, and innate immunity.     

Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth

Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Multiple mechanisms contribute to Schizosaccharomyces pombe origin recognition complex-DNA interactions

Eukaryotic DNA replication requires the assembly of multiprotein pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication. Here we describe the interactions of highly purified Schizosaccharomyces pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and with ars1 origin DNA. We show that SpORC binds DNA in at least two steps. The first step likely involves electrostatic interactions between the AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the formation of a salt-sensitive complex. In the second step, the salt-sensitive complex is slowly converted to a salt-stable complex that involves additional interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is facilitated by negative supercoiling and is accompanied by changes in DNA topology, suggesting that SpORC-DNA complexes contain underwound or negatively writhed DNA. Purified human origin recognition complex (ORC) induces similar topological changes in origin DNA, indicating that this property of ORC is conserved in eukaryotic evolution and plays an important role in ORC function. We also show that SpCdc18 and SpCdt1 form a binary complex that has greater affinity for DNA than either protein alone. In addition, both proteins contribute significantly to the stability of the initial SpORC-DNA complex and enhance the SpORC-dependent topology changes in origin DNA. Thus, the formation of stable protein-DNA complexes at S. pombe origins of replication involves binary interactions among all three proteins, as well as interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings demonstrate that SpORC is not the sole determinant of origin recognition.     

Buildup from birth onward of short telomeres in human hematopoietic cells

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere-Shortest-Length-Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age-dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth-89 years) from the general population, and 18 patients with dyskeratosis congenita-telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL-mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans.     

Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells

Humans are genetically incapable of producing the mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to an inactivating mutation in the enzyme synthesizing it. Despite this, human cells and tissues appear capable of metabolically incorporating Neu5Gc from exogenous sources, including dietary red meat and dairy products. All normal humans studied are now shown to have circulating Abs against Neu5Gc, with marked differences in isotype levels. The question arises whether such Abs can adversely affect Neu5Gc-expressing human cells or tissues. In this study, we show that although normal human PBMC do not incorporate Neu5Gc during in vitro incubation, activated T cells do. Primary human leukemia cells and human leukemic cell lines are even more efficient at incorporation. Human sera containing naturally high levels of anti-Neu5Gc IgG Abs (hereafter abbreviated GcIg) deposited complement on Neu5Gc-expressing leukemic cells and activated T cells, but not on normal cells. The binding of GcIg resulted in complement-mediated cytotoxicity, which was inhibited by heat inactivation. Low anti-Neu5Gc IgG-containing human sera did not mediate any of these effects. Mixed killing assays confirmed the 15-fold selective killing of leukemic cells over PBMC by GcIg following Neu5Gc feeding. This approach could potentially serve as novel way to target malignant cells for death in vivo using either natural Abs or anti-Neu5Gc Abs prepared for this purpose. Further studies are needed to determine whether deposition of natural GcIg and complement can also target healthy proliferating immune cells for death in vivo following incorporation of dietary Neu5Gc.