Increased Expression of S100B and RAGE in a Mouse Model of Bile Duct Ligation-induced Liver Fibrosis.

BACKGROUND: Liver fibrosis is defined as the accumulation of the extracellular matrix and scar formation. The receptor for advanced glycation end products (RAGE) has been demonstrated to participate in fibrogenesis. S100B is a ligand of RAGE and exerts extracellular functions by inducing a series of signal transduction cascades. However, the involvement of S100B and RAGE in cholestasis-induced liver fibrosis remains unclear. In this study, we investigated S100B and RAGE expression during liver fibrosis in mice that underwent common bile duct ligation (BDL). METHODS: BDL was performed in 10-week-old male C57BL/6J mice with sham control (n = 26) and BDL (n = 26) groups. Expression levels of S100B, RAGE and fibrotic markers in the livers from both groups at week 1 and 3 after BDL were examined by western blot and quantitative real-time reverse transcription polymerase chain reaction analysis. Liver fibrotic changes were examined by histological and ultrastructural analysis. RESULTS: Histological staining with Sirius Red and the evaluation of the messenger RNA expression of fibrotic markers showed noticeable periportal fibrosis and bile duct proliferation. S100B was mainly present in bile duct epithelial cells, and its expression was upregulated in proportion to the ductular reaction during fibrogenesis by BDL. RAGE expression was also increased, and interestingly, triple immunofluorescence staining and transmission electron microscopy showed that both S100B and RAGE were expressed in proliferating bile duct epithelial cells and activated hepatic stellate cells (HSCs) of the BDL livers. In addition, in rat HSCs (HSC-T6), treatment with recombinant S100B protein significantly increased fibrotic markers in a dose-dependent manner, and RAGE small interfering RNA (siRNA) suppressed S100B-stimulated upregulation of fibrotic markers compared with cells treated with scramble siRNA and S100B. CONCLUSION: These findings suggest that the increased expression of S100B and RAGE and the interaction between S100B and RAGE may play an important role in ductular reaction and liver fibrosis induced by BDL.

Accumulation of citrullinated glial fibrillary acidic protein in a mouse model of bile duct ligation-induced hepatic fibrosis.

Hepatic stellate cells (HSCs) play pivotal roles in hepatic fibrosis as they synthesize glial fibrillary acidic protein (GFAP), which is increased in activated HSCs. GFAP-expressing HSCs and myofibroblasts accumulate in and around hepatic fibrosis lesions. Peptidylarginine deiminase 2 (PAD2) is responsible for the citrullination of GFAP (cit-GFAP). However, the involvement of PAD2 and cit-GFAP in hepatic fibrosis remains unclear. To determine the expression of PAD2 and cit-GFAP in hepatic fibrosis, C57BL/6 mice underwent bile duct ligation (BDL) or a sham operation. In BDL livers, the expression of PAD2 and its enzyme activity were significantly increased compared with controls. In addition, PAD2-postitive cells were rarely observed in only the portal vein and the small bile duct in sham-operated livers, whereas an increased number of PAD2-positive cells were detected in the bile duct and Glisson's sheath in BDL livers. Interestingly, PAD2 was colocalized with α-SMA-positive cells and CK19-positive cells in BDL livers, indicating upregulated PAD2 in activated HSCs and portal fibroblasts of the livers of BDL mice. We also found that citrullinated proteins were highly accumulated in the livers of BDL mice compared with controls. Moreover, the expression level of GFAP and the amount of cit-GFAP were higher in BDL livers than in control livers. In correlation with PAD2 localization, cit-GFAP was observed in α-SMA-positive and CK19-positive cells in the livers of BDL mice. These results suggest that the increased expression and activation of PAD2 along with increased citrullinated proteins, specifically cit-GFAP, may play important roles in the pathogenesis of hepatic fibrosis.

Myelin Basic Protein Citrullination, a Hallmark of Central Nervous System Demyelination, Assessed by Novel Monoclonal Antibodies in Prion Diseases.

Myelin basic protein (MBP) citrullination by peptidylarginine deiminase (PAD) enzymes leads to incomplete protein-lipid bilayer interactions and vulnerability to proteolytic enzymes, resulting in disorganization of the myelin sheath in the central nervous system. Therefore, citrullinated MBP (citMBP) has been suggested as a hallmark of demyelination, but how citMBP is implicated in prion diseases remains unknown. For the first time, we developed mouse monoclonal anti-citMBP IgG1 (clones 1B8, 1H1, and 3C6) and IgM (clone 3G5) antibodies that recognize human citMBP at its R25, R122, and R130 residues and at its C-terminal region (or the corresponding sites in mouse MBP), respectively. Using a biochemical, immunohistochemical, and immunogold-silver staining for electron microscopy techniques, we found that MBP residue R23 (corresponding to human R25) was specifically citrullinated, was stained as intense punctae in the corpus callosum, the striatum, and the cerebellar white matter, and was predominantly localized in disorganized myelin in the brains of scrapie-infected mice. In the brains of Creutzfeldt-Jakob disease (CJD) patients, MBP residues R25, R122, and R130 were markedly citrullinated and were stained as fibrils and punctae. In particular, white matter regions, such as the midbrain and the medulla, exhibited high levels of citMBP compared to other regions. However, the high levels of citMBP were not correlated with PAD2 expression. The clone 3G5 recognized significantly increased expression of the 18.5 kDa and/or 21.5 kDa variants of MBP in prion disease. Our findings suggest that significantly increased levels of citMBP may reflect demyelinating neuropathology, and that these newly developed antibodies may be useful for identifying demyelination.

Upregulation of Connexin 43 Expression Via C-Jun N-Terminal Kinase Signaling in Prion Disease.

Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

Mass spectrometric identification of citrullination sites and immunohistochemical detection of citrullinated glial fibrillary acidic protein in Alzheimer's disease brains.

Peptidylarginine deiminases (PADs) are posttranslational modification enzymes that convert protein arginine to citrulline residues in a calcium ion-dependent manner. Previously, we reported the abnormal accumulation of citrullinated proteins and the increase in the amount of PAD2 in hippocampi from Alzheimer's disease (AD) patients. Moreover, glial fibrillary acidic protein (GFAP), an astrocyte-specific marker protein, and vimentin were identified as citrullinated proteins by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. To clarify the substrate specificity of PADs against GFAP, we prepared recombinant human (rh)PAD1, rhPAD2, rhPAD3, rhPAD4, and rhGFAP. After incubation of rhGFAP with rhPAD1, rhPAD2, rhPAD3, and rhPAD4, citrullinated (cit-)rhGFAP was detected by Western blotting. The citrullination of rhGFAP by rhPAD2 was unique, specific, and time dependent; additionally, rhPAD1 slightly citrullinated rhGFAP. We then generated eight anti-cit-rhGFAP monoclonal antibodies, CTGF-125, -128, -129, -1212, -1213, -1221, -122R, and -1224R, which reacted specifically with cit-rhGFAP. Two of those eight monoclonal antibodies, CTGF-122R and -1224R, reacted with both cit-rhGFAP and rhGFAP in Western blots. By using the CTGF-1221 antibody and a tandem mass spectrometer, we identified the two independent citrullination sites (R270Cit and R416Cit) of cit-rhGFAP. Immunohistochemical analysis with CTGF-1221 antibody revealed cit-GFAP staining in the hippocampus of AD brain, and the cit-GFAP-positive cells appeared to be astrocyte-like cells. These collective results strongly suggest that PAD2 is responsible for the citrullination of GFAP in the progression of AD and that the monoclonal antibody CTGF-1221, reacting with cit-GFAP at R270Cit and R416Cit, is useful for immunohistochemical investigation of AD brains.

[A case of spontaneous bacterial peritonitis caused by Listeria monocytogenes].

Spontaneous bacterial peritonitis (SBP) is a life-threatening complication in patients with ascites caused by advanced liver disease. While gram negative bacteria, such as Escherichia coli and Klebsiella pneumonia are the common pathogens, Listeria monocytogenes has been recognized as a very rare pathogen. Empirical treatment with third generation cephalosporins does not provide adequate antibiotics coverage against L. monocytogenes. Diagnosis is often delayed as it requires confirmation from ascitic fluid culture. Herein, we describe the first case of SBP caused by L. monocytogenes in a patient with advanced alcoholic liver cirrhosis in Korea. Clinicians should be aware of the atypical pathogens, especially in patients with inadequate response to empirical antibiotics.

Pathological characterization of TgElk mice injected with brain homogenate from elk with chronic wasting disease.

Chronic wasting disease (CWD) is classified as a transmissible spongiform encephalopathy or prion disease that affects cervids. CWD has been reported in 15 US states, two Canadian provinces, and in imported elk on several farms in Korea. This study was conducted to examine the molecular biological and pathogenic characteristics of a CWD-associated prion isolated in Korea. The epidemiological origin of this pathogen was also determined. Homozygous TgElk mice were infected with a CWD-affected elk brain pool prepared from the brain of an imported Canadian elk. We measured the incubation time of the pathogen, neuropathological changes by immunohistochemical staining, the pattern(s) of scrapie prion protein (PrPSc) deposition, and PrPSc protein profiles by Western blotting. We found that TgElk mice infected with brain homogenate from the elk suffering from CWD showed incubation times, vacuolar degeneration, and PrPSc accumulation similar to those previously reported in the literature. Our results suggest that homozygous TgElk mice efficiently transmit CWD with short incubation times and that this animal can serve a valuable research model and reliable in vivo diagnostic tool.

Familial Creutzfeldt-Jakob disease with a mutation at codon 180 presenting with an atypical phenotype.

The clinical features of familial Creutzfeldt-Jakob disease (fCJD) with a mutation at codon 180 (V180I) are less typical than those of patients with sporadic CJD. We describe a patient with pathologically confirmed CJD carrying the V180I mutation who had atypical cerebrospinal fluid and electroencephalography findings. Similar to other prion protein mutations, this report suggests that the V180I mutation is not the exclusive determinant of the phenotype.

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases.

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ϵ-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.

Dura mater graft-associated Creutzfeldt-Jakob disease: the first case in Korea.

Since 1987, dura mater graft-associated iatrogenic Creutzfeldt-Jakob disease (dCJD) has been reported in many countries. We report the first case of dCJD in Korea. A 54-yr-old woman, who underwent resection of the meningioma in the left frontal region and received a dura mater graft 23 yr ago presented with dysesthesia followed by psychiatric symptoms and ataxia. Her neurological symptoms rapidly progressed to such an extent that she exhibited myoclonus, dementia, and pyramidal and extrapyramidal signs within 8 weeks. The 14-3-3 protein was detected in her cerebrospinal fluid; however, an electroencephalogram did not reveal characteristic positive sharp wave complexes. Diffusion-weighted magnetic resonance images, obtained serially over 64 days, revealed the rapid progression of areas of high signal intensity in the caudate nucleus and cingulate gyrus to widespread areas of high signal intensity in the cortex and basal ganglia. Pathological examination of brain biopsy specimens confirmed the presence of spongiform changes and deposition of prion protein in the neurons and neuropils.

Association of endothelial nitric oxide synthase and mitochondrial dysfunction in the hippocampus of scrapie-infected mice.

The elevation of nitric oxide (NO) within the central nervous system (CNS) is known to be associated with the pathogenesis of neurodegenerative diseases such as HIV-associated dementia (HAD), brain ischemia, Parkinson's disease, and Alzheimer's disease. NO is enzymatically formed by the enzyme nitric oxide synthase (NOS). There are two forms of NOS, the constitutive and the inducible form. The constitutive form is present in endothelial cells (eNOS) and neurons (nNOS). The inducible form (iNOS) is expressed in various cell types including astroglia and microglia of the CNS. Using an animal model, we investigated the involvement of eNOS in the pathology of prion disease. We showed dramatic upregulation of eNOS immunoreactivity in reactive astroglial cells in the hippocampus in the prion disease animal model, scrapie in mice. Expression of eNOS was upregulated in cytosolic and mitochondrial fractions of whole brain. In the hippocampal region, eNOS was widely overexpressed in various components of the cell. We found that eNOS dramatically accumulated in hippocampal mitochondria and was particularly prevalent in structurally dysfunctional mitochondria. In association with the accumulation of eNOS in mitochondria, we showed that mitochondrial superoxide dismutase (Mn-SOD or SOD2), cytochrome c, and ATP activity were downregulated both in whole brain and in the hippocampal region. These results indicate that eNOS plays a role in the development of dysfunctional mitochondria and this, in turn, could induce some of the histopathological changes seen in prion diseases.

A Drosophila model of GSS syndrome suggests defects in active zones are responsible for pathogenesis of GSS syndrome.

We have established a Drosophila model of Gerstmann-Sträussler-Scheinker (GSS) syndrome by expressing mouse prion protein (PrP) having leucine substitution at residue 101 (MoPrP(P101L)). Flies expressing MoPrP(P101L), but not wild-type MoPrP (MoPrP(3F4)), showed severe defects in climbing ability and early death. Expressed MoPrP(P101L) in Drosophila was differentially glycosylated, localized at the synaptic terminals and mainly present as deposits in adult brains. We found that behavioral defects and early death of MoPrP(P101L) flies were not due to Caspase 3-dependent programmed cell death signaling. In addition, we found that Type 1 glutamatergic synaptic boutons in larval neuromuscular junctions of MoPrP(P101L) flies showed significantly increased numbers of satellite synaptic boutons. Furthermore, the amount of Bruchpilot and Discs large in MoPrP(P101L) flies was significantly reduced. Brains from scrapie-infected mice showed significantly decreased ELKS, an active zone matrix marker compared with those of age-matched control mice. Thus, altered active zone structures at the molecular level may be involved in the pathogenesis of GSS syndrome in Drosophila and scrapie-infected mice.

Involvement of peptidylarginine deiminase-mediated post-translational citrullination in pathogenesis of sporadic Creutzfeldt-Jakob disease.

Peptidylarginine deiminases (PADs)-mediated post-translational citrullination processes play key roles in protein functions and structural stability through the conversion of arginine to citrulline in the presence of excessive calcium concentrations. In brain, PAD2 is abundantly expressed and can be involved in citrullination in disease. Recently, we have reported pathological characterization of PAD2 and citrullinated proteins in scrapie-infected mice, but the implication of protein citrullination in the pathophysiology in human prion disease is not clear. In the present study, we explored the molecular and biological involvement of PAD2 and the pathogenesis of citrullinated proteins in frontal cortex of patients with sporadic Creutzfeldt-Jakob disease (sCJD). We found increased expression of PAD2 in reactive astrocytes that also contained increased levels of citrullinated proteins. In addition, PAD activity was significantly elevated in patients with sCJD compared to controls. From two-dimensional gel electrophoresis and MALDI-TOF mass analysis, we found various citrullinated candidates, including cytoskeletal and energy metabolism-associated proteins such as vimentin, glial fibrillary acidic protein, enolase, and phosphoglycerate kinase. Based on these findings, our investigations suggest that PAD2 activation and aberrant citrullinated proteins could play a role in pathogenesis and have value as a marker for the postmortem classification of neurodegenerative diseases.

Spontaneous immortalization of oligodendroglial cells derived from an SV40 T antigen-positive human glioblastoma multiforme.

The polyoma group of viruses, including SV40, is known to be oncogenic in certain species. Here we report for the first time naturally occurring, immortalized tumor cells from a patient with glioblastoma multiforme (GBM); the cells were shown to be oligodendroglia; cells had developed remarkable chromosomal changes and were positive for SV40 T antigen. Therefore, we postulated that the main cause of immortalization of these cells was the expression of SV40 T antigen gene and protein. Since the cells are naturally generated, they will provide a useful model to study the function of oligodendroglial cells and the development of GBM.

3D polycaprolactone scaffolds with controlled pore structure using a rapid prototyping system.

Designing a three-dimensional (3-D) ideal scaffold has been one of the main goals in biomaterials and tissue engineering, and various mechanical techniques have been applied to fabricate biomedical scaffolds used for soft and hard tissue regeneration. Scaffolds should be biodegradable and biocompatible, provide temporary support for cell growth to allow cell adhesion, and consist of a defined structure that can be formed into customized shapes by a computer-aided design system. This versatility in preparing scaffolds gives us the opportunity to use rapid prototyping devices to fabricate polymeric scaffolds. In this study, we fabricated polycaprolactone scaffolds with interconnecting pores using a 3-D melt plotting system and compared the plotted scaffolds to those made by salt leaching. Scanning electron microscopy, a laser scanning microscope, micro-computed tomography, and dynamic mechanical analysis were used to characterize the geometry and mechanical properties of the resulting scaffolds and morphology of attached cells. The plotted scaffolds had the obvious advantage that their mechanical properties could be easily manipulated by adjusting the scaffold geometry. In addition, the plotted scaffolds provided more opportunity for cells to expand between the strands of the scaffold compared to the salt-leached scaffold.

[A case of appendicular tuberculosis presenting as acute appendicitis].

Tuberculosis may affect primarily all organs and tissues of the body, although some of these show high immunity against the infection. The most common forms of non-pulmonary tuberculosis are tuberculosis of bones and joints (30%), urinary system (24%), lymph nodes (13%), sexual organs (8%), cerebrospinal meninges (4%), and alimentary system (3%). Especially, the commonest presentation of abdominal tuberculosis is ileocecal disease, but isolated appendicular involvement is also rarely seen, occurring in only 1.5% to 3% of cases in the absence of pulmonary or other abdominal involvement. The appendix may either be involved secondary to ileocecal tuberculosis, or to tuberculosis at another site within the abdomen, or may occur in the even, rarer "isolated" form, without the evidence of disease elsewhere. We report a case of acute appendicitis underwent appendectomy and histopathologic examination of appendix revealed appendicular tuberculosis.

[Pulmonary toxicity by pegylated interferon alpha-2a in a patient with chronic hepatitis C].

The combination therapy with pegylated interferon alpha and ribavirin has increasingly prescribed for chronic hepatitis C. Although many side effects of interferon such as flu-like symptoms, gastrointestinal and neuropsychiatric symptoms are well known, only several cases of interferon-induced pulmonary toxicity have been reported. Interferon-induced pulmonary toxicity usually develops from 2 weeks to 12 weeks after treatment for HCV infection. Diagnosis is commonly based on clinical findings such as a dry cough, dyspnea, hypoxemia, and a restrictive pattern in pulmonary function testing, bilateral diffuse parenchymal infiltrations, histopathological findings of interstitial pneumonitis, and exclusion of any other causative agents. Prompt withdrawal of the drug is the cornerstone of treatment. We report a case of PEG-IFN alpha-2a induced pulmonary toxicity in a 50-year-old male patient with hepatitis C. To our knowledge, this is the first case of pegylated interferon alpha-2a induced pulmonary toxicity in Korea.

[Comparison of model for end-stage liver disease score with discriminant function and child-Turcotte-Pugh scores for predicting short-term mortality in Korean patients with alcoholic hepatitis].

BACKGROUND/AIMS: Alcoholic hepatitis is an acute or acute-on-chronic inflammatory syndrome associated with significant morbidity and mortality. Traditionally, Maddrey discriminant function (DF) score and Child-Turcott-Pugh (CTP) score have been used for stratifying the prognosis of alcoholic hepatitis. Recently, the model for end-stage liver disease (MELD) score has been applied to alcoholic hepatitis and some investigators consider MELD score as a better prognostic indicator for severe alcoholic hepatitis. Therefore, this analysis was aimed to compare MELD score with DF and CTP scores for predicting the short-term mortality in Korean patients with alcoholic hepatitis. METHODS: The medical records of patients hospitalized with alcoholic hepatitis between January 1, 1999 and December 31, 2004 at Hanyang University Guri-Hospital were analyzed retrospectively. RESULTS: Of the 138 medical records reviewed, 74 cases fulfilled the inclusion criteria (61 males and 13 females; mean age 47.1 years). Twelve patients (16.2%) died within 90 days after admission. Univariate analysis demonstrated that variables such as ascites, hepatic encephalopathy, splenomegaly, international normalized ratio, CTP, and DF scores were significantly correlated with increased 90-day mortality while MELD score was not. According to the multivariate analysis, only CTP score was statistically significant (p=0.012) while DF and MELD scores were not significant for predicting 90-day mortality. The survival analysis with Cox regression test showed higher DF and CTP scores, but not MELD score, significantly increased the risk of in-hospital mortality. CONCLUSIONS: This study demonstrates that DF and CTP scores are independent predictors of short-term mortality in patients with alcoholic hepatitis.

[A case of primary omental torsion presenting as an acute abdominal pain].

Torsion of greater omentum is a rare cause of acute abdomen. However, it should be included in the differential diagnoses in addition to acute cholecystitis, acute appendicitis, cecal diverticulitis, and other variable causes of acute abdomen. Diagnosis is usually made at laparotomy for suspected appendicitis. In some cases, computed tomography demonstrates a successful preoperative detection of omental torsion. We report a case of surgically and pathologically proven torsion with subsequent infarction of greater omentum presented as an acute abdominal pain.