COMMD1-deficient dogs accumulate copper in hepatocytes and provide a good model for chronic hepatitis and fibrosis

New therapeutic concepts developed in rodent models should ideally be evaluated in large animal models prior to human clinical application. COMMD1-deficiency in dogs leads to hepatic copper accumulation and chronic hepatitis representing a Wilson's disease like phenotype. Detailed understanding of the pathogenesis and time course of this animal model is required to test its feasibility as a large animal model for chronic hepatitis. In addition to mouse models, true longitudinal studies are possible due to the size of these dogs permitting detailed analysis of the sequence of events from initial insult to final cirrhosis. Therefore, liver biopsies were taken each half year from five new born COMMD1-deficient dogs over a period of 42 months. Biopsies were used for H&E, reticulin, and rubeanic acid (copper) staining. Immunohistochemistry was performed on hepatic stellate cell (HSC) activation marker (alpha-smooth muscle actin, α-SMA), proliferation (Ki67), apoptosis (caspase-3), and bile duct and liver progenitor cell (LPC) markers keratin (K) 19 and 7. Quantitative RT-PCR and Western Blots were performed on gene products involved in the regenerative and fibrotic pathways. Maximum copper accumulation was reached at 12 months of age, which coincided with the first signs of hepatitis. HSCs were activated (α-SMA) from 18 months onwards, with increasing reticulin deposition and hepatocytic proliferation in later stages. Hepatitis and caspase-3 activity (first noticed at 18 months) increased over time. Both HGF and TGF-β1 gene expression peaked at 24 months, and thereafter decreased gradually. Both STAT3 and c-MET showed an increased time-dependent activation. Smad2/3 phosphorylation, indicative for fibrogenesis, was present at all time-points. COMMD1-deficient dogs develop chronic liver disease and cirrhosis comparable to human chronic hepatitis, although at much higher pace. Therefore they represent a genetically-defined large animal model to test clinical applicability of new therapeutics developed in rodent models

Fibrosis.

<p>Fibrosis scoring: α-SMA (hepatic stellate cell activation) and reticulin grading (collagen type III) in five COMMD1-deficient dogs in a time-dependent copper-induced hepatitis. Data are presented as median (range).</p><p>α-SMA grading; 0: no staining of HSCs; 1: a few positive HSCs; 2: diffuse staining of HSCs; 3: mild increased staining of HSCs; 4: marked increased staining of HSC and compared with age matches normal controls.</p><p>Reticulin grading; grade 0: normal, grade 1: local mild centrilobular increase, grade 2: multifocal mild to moderate centrilobular increase, grade 3: moderate increase with centro-central bridging or nodular transformation. The uncorrected p-values for comparison of each time point with 6 months of age are reported in the table. No significant differences are present when correcting these p-values for the number of tests.</p

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understand-ing the way this nutrient affects plantigrowth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (ay) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measure-ments revealed that vacuolar aK declined linearly with de-creases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data hav

DYRK1A Is a Regulator of S-Phase Entry in Hepatic Progenitor Cells

Plant cells contain two major pools of K+, one in the vacuole and one in the cytosol. The behavior of K+ concentrations in these pools is fundamental to understand-ing the way this nutrient affects plantigrowth. Triple-barreled microelectrodes have been used to obtain the first fully quantitative measurements of the changes in K+ activity (ay) in the vacuole and cytosol of barley (Hordeum vulgare L.) root cells grown in different K+ concentrations. The electrodes incorporate a pH-selective barrel allowing each measurement to be assigned to either the cytosol or vacuole. The measure-ments revealed that vacuolar aK declined linearly with de-creases in tissue K+ concentration, whereas cytosolic aK initially remained constant in both epidermal and cortical cells but then declined at different rates in each cell type. An unexpected finding was that cytoplasmic pH declined in parallel with cytosolic aK, but acidification of the cytosol with butyrate did not reveal any short-term link between these two parameters. These measurements show the very different responses of the vacuolar and cytosolic K+ pools to changes in K+ availability and also show that cytosolic K+ homeostasis differs quantitatively in different cell types. The data hav

Histological description.

<p>COMMD1-deficient dog livers were stained with H&E and RA to assess inflammation and copper accumulation, respectively, and stained for α-SMA and reticulin (collagen type III) to assess fibrosis. Representative pictures of a COMMD1-deficient dog over a period of 42 months are shown. Numbers indicate age in months.</p

HGF pathway.

<p>(<b>A</b>) Gene-expression profiling. Interaction plots of Q-PCR data of important mediators of regeneration in a time-dependent copper-induced hepatitis in five COMMD1-deficient dogs. Q-PCR results were normalized against the expression of six control dogs (one to three years of age). Linear mixed-effect modeling was used; * significant difference corrected for multiple testing. (B) Western blot analysis on the activation HGF-signalling (HGF, phosphorylated c-Met, (phosphorylated) STAT3) of five COMMD1-deficient dogs at 12, 30, and 42 months. β-actin (ACTB) served as loading control.</p

Blood examinations.

<p>ALT: alanine aminotransferase; AP: alkaline phosphatase.</p><p>Blood examinations during aging in five COMMD1-deficient dogs. Values are expressed as median (range).</p>*<p>significantly increased serum concentration compared with 12 months of age.</p

TGF-β1 pathway.

<p>(<b>A</b>) Gene-expression profiling. Interaction plots of Q-PCR data of important mediators of fibrogenesis in a time-dependent copper-induced hepatitis in five COMMD1-deficient dogs. Q-PCR results were normalized against the expression of six control dogs (one to three years of age). Linear mixed-effect modeling was used; * significant difference corrected for multiple testing. (<b>B</b>) Western blot analysis on the activation of TGF-beta signalling (total Smad2 and phosphorylated Smad2 (Ser727)) of five COMMD1-deficient dogs at 12, 30, and 42 months. β-actin (ACTB) served as loading control.</p

The Kaposi's Sarcoma-associated Herpesvirus-encoded vIRF-3 Inhibits Cellular IRF-5*S⃞

Kaposi's sarcoma-associated herpesvirus encodes four genes with homology to the family of interferon regulatory factors (IRFs). At least one of these viral IRFs, vIRF-3, is expressed in latently Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma (PEL) cells and is essential for the survival of PEL cells. We now report that vIRF-3 interacts with cellular IRF-5, thereby inhibiting binding of IRF-5 to interferon-responsive promoter elements. Consequently, vIRF-3 blocked IRF-5-mediated promoter activation. A central double helix motif present in vIRF-3 was sufficient to abrogate both DNA binding and transcriptional transactivation by IRF-5. Upon DNA damage or activation of the interferon or Toll-like receptor pathways, cytoplasmic IRF-5 has been reported to be translocated to the nucleus, which results in induction of both p53-independent apoptosis and p21-mediated cell cycle arrest. We report here that IRF-5 is present in the nuclei of PEL cells without interferon stimulation. Silencing of vIRF-3 expression in PEL cells was accompanied by increased sensitivity to interferon-mediated apoptosis and up-regulation of IRF-5 target genes. In addition, vIRF-3 antagonized IRF-5-mediated activation of the p21 promoter. The data presented here indicate that vIRF-3 contributes to immune evasion and sustained proliferation of PEL cells by releasing IRF-5 from transcription complexes