Results of laboratory tests in four patients with HBV-associated acute liver failure.

<p>To convert the values for creatinine to µmol/L, multiply by 88.4; to convert the values for total and conjugated bilirubin to µmol/L, multiply by 17.1.</p>†<p>Normal range, 0.80 to 1.20 international normalized ratio (INR).</p>‡<p>Normal range, <10.0 ng/mL. NA denotes not available.</p

Histopathologic features of HBV-associated acute liver failure (ALF) showing the severity of hepatic necrosis in 4 patients.

<p>At the time of liver transplantation there was massive hepatic necrosis (MHN) with no viable hepatocytes in Patient 241, MHN (nearly 100%) with few scattered hepatocytes in Patient 31, and submassive hepatic necrosis (SHN) in Patients 219 (70 to 80% necrosis) and 32 (60% necrosis) (hematoxylin and eosin, x400).</p

Hierarchical clustering.

<p>Hierarchical cluster analysis of 17 liver specimens from 4 patients with acute liver failure and 10 specimens from individual liver donors using all 11,597 transcripts that passed the filtering criteria. All specimens were grouped into 3 main clusters, which correspond to the 3 liver conditions (ALF with MHN, ALF with SHN and liver donors). The unsupervised nature of the analysis and the 100% correct classification of samples suggest a high specificity and sensitivity of gene expression differences between ALF and normal livers.</p

Multidimensional scaling.

<p>Three-dimensional (3D) projection of 17 liver specimens from 4 patients with ALF and 10 specimens from individual liver donors by multidimensional scaling using all 11,597 transcripts that passed the filtering criteria. In the 3D projection, each point represents an individual liver specimen, and the distance between points is proportional to the overall dissimilarity of gene expression profiling. The plot illustrates how the gene expression profiles differentiate between ALF and liver donors, as well as among ALF patients. ALF patients with massive hepatic necrosis (MHN) (Patients 241 and 31) and those with submassive hepatic necrosis (SHN) (Patients 219 and 32) form two distinct clusters whose distance from liver donors reflects the extent of liver injury.</p

Liver Regeneration Signature in Hepatitis B Virus (HBV)-Associated Acute Liver Failure Identified by Gene Expression Profiling

<div><h3>Introduction</h3><p>The liver has inherent regenerative capacity via mitotic division of mature hepatocytes or, when the hepatic loss is massive or hepatocyte proliferation is impaired, through activation of hepatic stem/progenitor cells (HSPC). The dramatic clinical course of acute liver failure (ALF) has posed major limitations to investigating the molecular mechanisms of liver regeneration and the role of HSPC in this setting. We investigated the molecular mechanisms of liver regeneration in 4 patients who underwent liver transplantation for hepatitis B virus (HBV)-associated ALF.</p> <h3>Methods and Findings</h3><p>Gene expression profiling of 17 liver specimens from the 4 ALF cases and individual specimens from 10 liver donors documented a distinct gene signature for ALF. However, unsupervised multidimensional scaling and hierarchical clustering identified two clusters of ALF that segregated according to histopathological severity massive hepatic necrosis (MHN; 2 patients) and submassive hepatic necrosis (SHN; 2 patients). We found that ALF is characterized by a strong HSPC gene signature, along with ductular reaction, both of which are more prominent in MHN. Interestingly, no evidence of further lineage differentiation was seen in MHN, whereas in SHN we detected cells with hepatocyte-like morphology. Strikingly, ALF was associated with a strong tumorigenesis gene signature. MHN had the greatest upregulation of stem cell genes (EpCAM, CK19, CK7), whereas the most up-regulated genes in SHN were related to cellular growth and proliferation. The extent of liver necrosis correlated with an overriding fibrogenesis gene signature, reflecting the wound-healing process.</p> <h3>Conclusion</h3><p>Our data provide evidence for a distinct gene signature in HBV-associated ALF whose intensity is directly correlated with the histopathological severity. HSPC activation and fibrogenesis positively correlated with the extent of liver necrosis. Moreover, we detected a tumorigenesis gene signature in ALF, emphasizing the close relationship between liver regeneration and liver cancer.</p> </div

Liver fibrosis in HBV-associated ALF.

<p>Delicate fibrosis is evident in the zones of collapsed necrotic parenchyma. The mature collagen of portal areas stains dark blue, while the newer collagen stains light blue (Masson’s trichrome, x200).</p

Differential gene expression between HBV-associated ALF and liver donors showing the up-regulated transcripts related to liver regeneration.

<p>Data of the 10 liver donors represent individual samples, whereas data of the 4 ALF patients represent the average of multiple samples analyzed (up to 5 liver specimens for each patient). Each cell represents the expression of a particular transcript (rows) of a particular liver specimen (columns). The color in each cell reflects the level of expression of the corresponding gene in the corresponding sample, relative to its mean level of expression in the entire set of 14 samples. Ratios were log2-transformed and row-wise standardized. According to the color scale, up-regulated genes are shown in shades of red, down-regulated genes in shades of green. MHN denotes massive hepatic necrosis. SHN denotes submassive hepatic necrosis. LD denotes liver donors.</p

Pedigrees showing Telomerase Mutations and Disease Phenotypes.

<p><b>(A)</b> The <i>TERT</i> K570N mutation tracked with hematological disorders and severe liver disease (lower pedigree) in Family A. In the extended family (upper pedigree), several disorders are found, including autoimmune diseases, anemia, thyroid diseases, liver diseases, and multiple miscarriages; however, the mutation was only associated with liver disease and multiple miscarriages. Two consanguineous relationships are not show: Subject A-IV-17 is a grand-daughter of Subjects A-II-7 and A-II-8, and Subject A-IV-7 is a grandson of Subjects A-III-14 and A-III-15. The genetic status for the immediate family (lower pedigree) and its association with bone marrow failure have been previously reported by us <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007926#pone.0007926-Xin1" target="_blank">[15]</a>. In smaller pedigrees, <b>(B)</b><i>TERC</i> nucleotide 341-360 deletion tracked to liver disease in family B, <b>(C)</b> liver disease occurred in a family with a <i>TERC</i> nucleotide 28–34 deletion, and <b>(D)</b> in a family with <i>TERC</i> nucleotide 109–123 deletion. The following are denoted by their abbreviations: common variable immunodeficiency (CVID), aplastic anemia (AA), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), idiopathic thrombocytic purpura (ITP), and non-alcoholic steatohepatitis (NASH).</p

Functional analysis of telomerase mutations.

<p><b>(A)</b> Telomere length in peripheral-blood total white blood cells (ordinate) from patients and their relatives with or without telomerase gene mutations as a function of age (abscissa) compared to healthy controls. Telomere lengths were measured by flow fluorescence <i>in situ</i> hybridization (flow-FISH). Small gray circles represent the telomere lengths for 400 healthy volunteers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007926#pone.0007926-Yamaguchi1" target="_blank">[10]</a>, and the curve marks the 50^th percentile for healthy controls as a function of age. <b>(B)</b> Telomerase activity - measured by telomeric-repeat amplification assay - of lysates of telomerase-negative WI38-VA13 cells cotransfected with mutated <i>TERC</i> and wild-type <i>TERT</i> expression vectors (2 µg per vector per transfection reaction). Enzymatic activity was normalized to <i>TERC</i> expression as measured by Real Time RT-PCR and to the telomerase activity of wild-type <i>TERC</i>, which was set at 100%. Quadruplicate measurements were performed using one microgram of cell lysate protein per reaction. “Empty vector” refers to protein from VA13 cells transfected with an empty pcDNA3-Flag vector in lieu of <i>TERC</i>.</p

Hematoxylin and Eosin Bone Marrow and Liver Sections from Probands and Relatives with Aplastic Anemia, Acute Myeloid Leukemia, and Severe Liver Disease.

<p><b>(A)</b> Family A proband's bone marrow was hypocellular with isolated regions of normal cellularity (hematoxylin and eosin [H&E] staining; low power magnification). <b>(B)</b> Proband's father's bone marrow smear (Subject A-IV-29), illustrating dysplastic changes and increased number of blasts (H&E, high power magnification). <b>(C)</b> Subject A-IV-23's liver biopsy revealing islands of liver surrounded by zones of necrosis and parenchymal collapse (H&E, low magnification). The necrosis was far enough in the past that hepatocytes have mostly disappeared. In the inset, in some of the areas where hepatocytes were preserved there was still necrosis near the central veins. Little evidence of inflammation exists. <b>(D)</b> Subject's A-IV-25's liver biopsy showing small portal areas and poorly formed veins (H&E, low power magnification). <b>(E)</b> Same liver biopsy exhibiting widened hepatocyte plates on the reticulin stain (high power magnification), but clear changes of nodular regenerative hyperplasia were not seen. <b>(F)</b> The CD34 stain by was positive in sinusoidal endothelial cells consistent with an abnormal proportion of arterial blood flow to the sinuses (immunohistochemistry, low power magnification). <b>(G)</b> Liver biopsy of Subject A-III-11 in which the hepatic architecture is distorted by bridging fibrosis (low power magnification); the inset gives a close-up of the fibrosis. The biopsy revealed moderate inflammation but not elevated levels of plasma cells relative to other inflammatory cells. Other changes included interface hepatitis and cholatestasis. <b>(H)</b> Subject B-II-3's liver biopsy demonstrating portal inflammation with interface hepatitis (H&E, low power magnification). In the inset, Masson staining shows sclerosis around central vein with perisinusoidal fibrosis. <b>(I)</b> Subject B-III-7's liver biopsy with mild, macrovesicular steatosis in a zone 3 distribution. The inset indicates that there is mild lymphocytic portal inflammation with focal interface hepatitis (H&E). <b>(J)</b> Subject C-III-3's liver biopsy shows mild hepatocellular iron accumulation in a pericanalicular pattern; the sinusoidal-lining cells show mild to moderate iron accumulation. The inset illustrates mild variation in hepatocyte nuclear size. <b>(K)</b> Subject C-III-3's reticulin staining exemplifying several zones in which the hepatocyte plates were abnormally widened, consistent with regeneration. <b>(L)</b> Subject E-II-1's liver biopsy revealing some portal areas with mild inflammation and all with poorly formed, slit-like veins (H&E). <b>(M)</b> The reticulin stain showed evidence of nodular regenerative hyperplasia, with zones of plate widening alternating with areas of compression. <b>(N)</b> CD34 stain was abnormally positive in the sinusoidal endothelial cells by immunohistochemistry, indicating abnormal proportion of arterial blood flow to the sinuses.</p