Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

Inhibition of mapk signalling promotes cell cycle arrest and sensitises intrahepatic cholangiocarcinoma cells to chemotherapy

Introduction: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary hepatic malignancy, accounting for approximately 15% of cases of primary liver cancer. Although new treatments have increased survival for many other cancers, including the more common primary hepatocellular carcinoma, treatment strategies and survival for patients with ICC have seen little improvement. Our previous studies suggest that the mitogen-activated protein kinase (MAPK) signalling plays a central role in the regulation of cell proliferation in human ICC. However the molecular mechanisms are poorly understood. In this study, we aim to explore whether inhibition of the MAPK pathway and its downstream effectors enhances the sensitisation of ICC cells to the chemotherapeutic agent cisplatinum. Method: We used a combinatorial approach of immunohistochemical and gene expression analyses to investigate the expression of MAPK-related genes in ICC tumours. Furthermore, by using in-vitroand in-vivoanalyses we have characterised the function of a novel MAPK downstream effector in ICC cells. Results: The expression of MAPK signalling was determined by immunohistochemical staining in tumour samples from a cohort of 14 ICC patients. High expression of phospho-activated MAPK was observed in 71.4% (10/14) of ICC cases as compared with surrounding nontumour tissue. Likewise, expression of JDP, a downstream effector of the MAPK signalling, was scored as high intensity in 64.3% (9/14). Strikingly, elevated expression of JDP transcripts was also observed in two independent cohorts of human ICC (n = 149 and n = 109 per group, respectively) compared to surrounding normal liver tissue. Consistent with the in-vivo analyses of human samples, immunoblotting analyses showed constitutive activation of MAPK and expression of JDP in ICC-derived cells (i.e. SG231, CCLP-1 and HuCCT1). Using loss-of-function analyses, we demonstrates that knockdown of JDP in ICC-derived cells resulted in cell cycle arrest and reduced expression of cell cycle regulators (i.e. cyclins), and had minimal effect on apoptosis. Chemical inhibition of JDP significantly sensitises ICC-derived cells to cisplatinum (P < 0.001). Conclusion: These results demonstrate that enhanced activation of MAPK signalling is important for ICC cell proliferation and suggest that targeting its downstream effectors is a potential therapeutic strategy for ICC

Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1–2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin

Nuclear localization of phosphorylated c-Myc protein in human tumor cells.

Using immunocytochemical techniques at light and electron microscopy, we analysed the distribution of phosphorylated c-Myc in actively proliferating human HeLa cells. The distribution pattern of c-Myc was also compared with those of other ribonucleoprotein (RNP)-containing components (PANA, hnRNP-core proteins, fibrillarin) or RNP-associated nuclear proteins (SC-35 splicing factor). Our results provide the first evidence that phosphorylated c-Myc accumulates in the nucleus of tumor cells, where it colocalizes with fibrillarin, both in the nucleolus and in extranucleolar structures

Conditional Genetic Elimination of Hepatocyte Growth Factor in Mice Compromises Liver Regeneration after Partial Hepatectomy

Hepatocyte growth factor (HGF) has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ERT transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70-80%) of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH), which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4)-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth. © 2013 Nejak-Bowen et al

Chronic Cerebrospinal Venous Insufficiency (CCSVI) IN Meniere Disease. Case or Cause?

Abstract CCSVI is the acronym for Chronic Cerebrospinal Venous Insufficiency, initially described by P.Zamboni, as being strongly associated with multiple sclerosis (MS). It is a syndrome characterized by stenosis of the internal jugular veins (IJVs) and/or azygous vein (AZ) with opening of collaterals and insufficient drainage. Bavera PM carried out 823 Duplex exams on a control group of 60 patients without MS. As expected CCSVI was found only in few subjects of the control group, three, two females and one male, but all affected with Sudden Sensorineural Hearing Loss (SSHL). Successively, we reported a case of bilateral SSHL with vertigo, showing evidence of the CCSVI pattern at Duplex examination (not associated with MS). To the best of the authors&apos; knowledge, this kind of association has never been reported. We studied 52 patients affected with cochleo-vestibular disturbances subdivided into two groups of out-patients:Definite unilateral Meniere (Men): 12 subjects (8 males and 4.females, mean age 41,6.yy) according to international AOO-HNS 1995 diagnostic criteria -No-Meniere (No-Men): 14 subjects (6.males and 8 females, mean age 44,7.yy) affected with unilateral cocleo-vestibular impairment A third group of subjects have been considered, as a &quot;normal&quot; group, 13 patients (8 females and 5 males, mean age 45,5 yy) affected with Benign Paroxismal Positioning Vertigo (BPPV) with cochlear involvement Asymmetrical artherious flow in VA or CA was revealed in 2 Men 9 no-Men and 1 BPPV, respectively 12,5 -60,7 -and 8,6 %. Differences between Men and NoMen and between each of this group with respect to BPPV were highly significant (p&lt;0.001). Asymmetrical venous flow in IJV or VV was detected in 9 patients in MEN group and in 4 in no-MEN and 2 BPPV, respectively 79 -28,5 and 13 %. Differences between Men and No-Men and between each of this group with respect to BBV were highly significant (p&lt;0.001

HIV Protease Inhibitors Sensitize Human Head and Neck Squamous Carcinoma Cells to Radiation by Activating Endoplasmic Reticulum Stress

Background Human head and neck squamous cell carcinoma (HNSCC) is the sixth most malignant cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, there is no significant improvement of mortality rates for this disease in the past decades. Radiotherapy is the core component of the clinical combinational therapies for HNSCC. However, the tumor cells have a tendency to develop radiation resistance, which is a major barrier to effective treatment. HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear. Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress. The aim of this study was to examine the role of ER stress in HIV PI-induced radiosensitivity in human HNSCC. Methodology and Principal Findings HNSCC cell lines, SQ20B and FaDu, and the most commonly used HIV PIs, lopinavir and ritonavir (L/R), were used in this study. Clonogenic assay was used to assess the radiosensitivity. Cell viability, apoptosis and cell cycle were analyzed using Cellometer Vision CBA. The mRNA and protein levels of ER stress-related genes (eIF2α, CHOP, ATF-4, and XBP-1), as well as cell cycle related protein, cyclin D1, were detected by real time RT-PCR and Western blot analysis, respectively. The results demonstrated that L/R dose-dependently sensitized HNSCC cells to irradiation and inhibited cell growth. L/R-induced activation of ER stress was correlated to down-regulation of cyclin D1 expression and cell cycle arrest under G0/G1 phase. Conclusion and Significance HIV PIs sensitize HNSCC cells to radiotherapy by activation of ER stress and induction of cell cycle arrest. Our results provided evidence that HIV PIs can be potentially used in combination with radiation in the treatment of HNSCC

Autoantibody profile in rheumatoid arthritis during long-term infliximab treatment

The aim of the present study was to investigate the effect of long-term infliximab treatment on various autoantibodies in patients with rheumatoid arthritis. Serum samples from 30 consecutive patients, who were prospectively followed during infliximab and methotrexate therapy for refractory rheumatoid arthritis, were tested at baseline and after 30, 54 and 78 weeks. At these points, median values of the Disease Activity Score were 6.38 (interquartile range 5.30-6.75), 3.69 (2.67-4.62), 2.9 (2.39-4.65) and 3.71 (2.62-5.06), respectively. Various autoantibodies were assessed by standard indirect immunofluorescence and/or ELISA. Initially, 50% of patients were positive for antinuclear antibodies, and this figure increased to 80% after 78 weeks (P=0.029). A less marked, similar increase was found for IgG and IgM anticardiolipin antibody titre, whereas the frequency of anti-double-stranded DNA antibodies (by ELISA) exhibited a transient rise (up to 16.7%) at 54 weeks and dropped to 0% at 78 weeks. Antibodies to proteinase-3 and myeloperoxidase were not detected. The proportion of patients who were positive for rheumatoid factor (RF) was similar at baseline and at 78 weeks (87% and 80%, respectively). However, the median RF titre exhibited a progressive reduction from 128 IU/ml (interquartile range 47-290 IU/ml) to 53 IU/ml (18-106 IU/ml). Anti-cyclic citrullinated peptide (CCP) antibodies were found in 83% of patients before therapy; anti-CCP antibody titre significantly decreased at 30 weeks but returned to baseline thereafter. In conclusion, the presence of anti-double-stranded DNA antibodies is a transient phenomenon, despite a stable increase in antinuclear and anticardiolipin antibodies. Also, the evolution of RF titres and that of anti-CCP antibody titres differed during long-term infliximab therapy

Maternal Diet Intervention Before Pregnancy Primes Offspring Lipid Metabolism in Liver

Nonalcoholic fatty liver disease (NAFLD) has a developmental origin and is influenced in utero. We aimed to evaluate if maternal diet intervention before pregnancy would be beneficial to reduce the risk of offspring NAFLD. In our study, female mice were either on a normal-fat diet (NF group), or a high-fat diet for 12 weeks and continued on this diet throughout pregnancy and lactation (HF group), or switched from HF-to-NF diet 1 week (H1N group), or 9 weeks (H9N group) before pregnancy. Compared with the NF offspring, the H1N and HF, but not the H9N offspring, displayed more severe hepatic steatosis and glucose intolerance. More specifically, an abnormal blood lipid panel was seen in the H1N offspring and abnormal hepatic free fatty acid composition was present in both the HF and H1N offspring, while the H9N offspring displayed both at normal levels. These physiological changes were associated with desensitized hepatic insulin/AKT signaling, increased expression of genes and proteins for de novo lipogenesis and cholesterol synthesis, decreased expression of genes and proteins for fatty acid oxidation, increased Pcsk9 expression, and hypoactivation of 5' AMP-activated protein kinase (AMPK) signaling in the HF and H1N offspring. However, these effects were completely or partially rescued in the H9N offspring. In summary, we found that early maternal diet intervention is effective in reducing the risk of offspring NAFLD caused by maternal HF diet. These findings provide significant support to develop effective diet intervention strategies and policies for prevention of obesity and NAFLD to promote optimal health outcomes for mothers and children