Anti-cyclic citrullinated peptide antibody titer predicts time to rheumatoid arthritis onset in patients with undifferentiated arthritis: results from a 2-year prospective study

Introduction

The diagnostic, predictive and prognostic role of anti-cyclic citrullinated peptide (CCP) antibodies in rheumatoid arthritis (RA) patients is widely accepted. Moreover, detection of these antibodies in subjects presenting with undifferentiated arthritis (UA) is associated with a significant risk to develop the disease. On the other hand, clinical and prognostic significance of evaluating anti-CCP levels in subjects with inflammatory arthritis at disease onset has not been fully clarified. The goal of this prospective study is to analyze the value and prognostic significance of anti-CCP titer quantification in UA subjects.

Methods

Serial anti-CCP assays were measured in 192 consecutive patients presenting with UA lasting less than 12 weeks. Clinical and serological data and arthritis outcome were evaluated every 6 months until two years of follow-up.

Results

Anti-CCP positivity, at both low and high titer, and arthritis of hand joints significantly predicted RA at two years, risk increasing in subjects with high anti-CCP titers at baseline. Moreover, time to RA diagnosis was shorter in patients with high anti-CCP2 titers at enrollment with respect to those with low antibody concentration.

Conclusions

Presence of anti-CCP antibodies, at both low and high concentration, is significantly associated with RA development in subjects with recent onset UA. However, time interval from the onset of the first symptoms to the fulfilment of the classification criteria appears to be directly related to the initial anti-CCP level.     

Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to CCl(4)-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), CCl(4)-treated BDL rats, and CCl(4)-treated taurocholate-fed rats. In vitro, we tested whether CCl(4) induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The CCl(4)-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in CCl(4)-treated rats fed taurocholate. CCl(4)-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, CCl(4)-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on CCl(4) effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from CCl(4)-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies.     

Opposing actions of endocannabinoids on cholangiocarcinoma growth: recruitment of Fas and Fas ligand to lipid rafts

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.     

Biogenic amine actions on cholangiocyte function

Biogenic amines, such as serotonin, histamine, dopamine, and the catecholamines epinephrine and norepinephrine, regulate a multitude of cellular responses. A great deal of effort has been invested into understanding the effects of these molecules and their corresponding receptor systems on cholangiocyte secretion, apoptosis, and growth. This review summarizes the results of these efforts and highlights the importance of these regulatory molecules on the physiology and pathophysiology of cholangiocytes.     

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.     

Role of lactoferrin and its receptors on biliary epithelium

Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

Role of stem cells during diabetic liver injury

Diabetes mellitus is one of the most severe endocrine metabolic disorders in the world that has serious medical consequences with substantial impacts on the quality of life. Type 2 diabetes is one of the main causes of diabetic liver diseases with the most common being non‐alcoholic fatty liver disease. Several factors that may explain the mechanisms related to pathological and functional changes of diabetic liver injury include: insulin resistance, oxidative stress and endoplasmic reticulum stress. The realization that these factors are important in hepatocyte damage and lack of donor livers has led to studies concentrating on the role of stem cells (SCs) in the prevention and treatment of liver injury. Possible avenues that the application of SCs may improve liver injury include but are not limited to: the ability to differentiate into pancreatic β‐cells (insulin producing cells), the contribution for hepatocyte regeneration, regulation of lipogenesis, glucogenesis and anti‐inflammatory actions. Once further studies are performed to explore the underlying protective mechanisms of SCs and the advantages and disadvantages of its application, there will be a greater understand of the mechanism and therapeutic potential. In this review, we summarize the findings regarding the role of SCs in diabetic liver diseases.