bluuebunny/crossref_metadata_2025 huggingface dataset:
Definition and management of anemia in patients infected with hepatitis C virus
Authors: McHutchison John G. , Manns Michael P. , Longo Dan L.
Published: April 2006

Abstract:Chronic infection with hepatitis C virus (HCV) can progress to cirrhosis, hepatocellular carcinoma, and end‐stage liver disease. The current best treatment for HCV infection is combination therapy with pegylated interferon and ribavirin. Although this regimen produces sustained virologic responses (SVRs) in approximately 50% of patients, it can be associated with a potentially dose‐limiting hemolytic anemia. Hemoglobin concentrations decrease mainly as a result of ribavirin‐induced hemolysis, and this anemia can be problematic in patients with HCV infection, especially those who have comorbid renal or cardiovascular disorders. In general, anemia can increase the risk of morbidity and mortality, and may have negative effects on cerebral function and quality of life. Although ribavirin‐associated anemia can be reversed by dose reduction or discontinuation, this approach compromises outcomes by significantly decreasing SVR rates. Recombinant human erythropoietin has been used to manage ribavirin‐associated anemia but has other potential disadvantages. Viramidine, a liver‐targeting prodrug of ribavirin, has the potential to maintain the virologic efficacy of ribavirin while decreasing the risk of hemolytic anemia in patients with chronic hepatitis C.
Novel markers of cell kinetics to evaluate progression from cirrhosis to hepatocellular carcinoma
Authors: Quaglia Alberto , Mcstay Mary , Stoeber Kai , Loddo Marco , Caplin Martyn , Fanshawe Thomas , Williams Gareth , Dhillon Amar
Published: April 2006

Abstract: Background: We investigated cell cycle kinetics of nodular lesions in cirrhosis to differentiate hepatocellular carcinoma (HCC) from its precursor lesions.

Methods: Twelve small HCC, 10 regenerative (RN), six large regenerative (LRN), and five dysplastic nodules (DN), identified in explant cirrhotic livers of five consecutive patients transplanted at Royal Free Hospital in 2002. Immunoperoxidase for MCM2, geminin and Ki67 was performed and the percentage of positive cells counted.

Results: The proportion of cells expressing MCM2 was more than those expressing Ki67, which in turn was more than those expressing geminin (overall median=16%, 2% and 0.5%, respectively, P<0.001). There was a statistically significant trend of increasing Ki67 expression (P=0.006), from RN to HCC; this trend was not statistically significant for geminin (P=0.18) or MCM2 (P=0.51). The median percentage of cells expressing Ki67 was 1% in RN, 0.5% in LRN, 2.2% in DN and 5.4% in HCC. The combination of these markers identified four different cell kinetics patterns: ‘resting’ (G0 cells: MCM2 −ve, Ki67 −ve, geminin −ve); ‘licensed’ (MCM2 +ve, Ki67 −ve, geminin −ve); ‘slowly growing’ (G1 phase arrest, MCM2 +ve, Ki67 +ve, low (0.4%) geminin) and expanding (MCM2 +ve, Ki67 +ve, geminin +ve) nodules.

Conclusions: The combination of MCM2, geminin and Ki67 could represent a valuable tool in the understanding of HCC progression in cirrhosis.
Expression pattern of angiogenic factors and prognosis after hepatic resection in hepatocellular carcinoma: importance of angiopoietin‐2 and hypoxia‐induced factor‐1a
Authors: Wada Hiroshi , Nagano Hiroaki , Yamamoto Hirofumi , Yang Yubo , Kondo Motoi , Ota Hideo , Nakamura Masato , Yoshioka Shinichi , Kato Hitoshi , Damdinsuren Bazarragchaa , Tang Di , Marubashi Sigeru , Miyamoto Atsushi , Takeda Yutaka , Umeshita Koji , Nakamori Shoji , Sakon Masato , Dono Keizo , Wakasa Kenichi , Monden Morito
Published: April 2006

Abstract:
Background: Hepatocellular carcinoma (HCC) is a hypervascular tumor and angiogenesis plays an important role in its progression. Angiogenesis is regulated by a balance between pro and antiangiogenic molecules. The aim of this study was to investigate the expressions of angiogenic factors and elucidate their roles in angiogenesis in HCC.

Methods: We investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang‐1 and Ang‐2), hypoxia‐induced factor‐1α (HIF‐1α) and thrombospondin‐1 (TSP‐1) in 60 specimens of surgically resected HCC. We investigated the relationship between their expressions and clinicopathological factors or prognosis.

Results: Ang‐2 staining had a significant correlation with the grade of differentiation of HCC cells (P=0.0082). VEGF and Ang‐2 expression correlated positively with microvessel density (MVD) (P=0.0061 and 0.0374, respectively). MVD of well‐differentiated HCC were significantly lower than those of moderately and poorly differentiated HCC. The disease‐free survival time of patients with high Ang‐2 and/or HIF‐1α expression was significantly shorter than that of the low expression group (P=0.0278 and 0.0374, respectively).

Conclusion: Our study showed that the expression of VEGF and Ang‐2 correlated with MVD. Strong Ang‐2 expression and/or high nuclear expression of HIF‐1α is a significant predictive factor for recurrence after curative resection in HCC patients.
Management of spontaneous haemorrhage and rupture of hepatocellular adenomas. A single centre experience
Authors: Erdogan Deha , Busch Olivier R.C. , Van Delden Otto M. , Ten Kate Fiebo J.W. , Gouma Dirk J. , Van Gulik Thomas M.
Published: April 2006

Abstract: Background: Hepatocellular adenomas (HCA) may present with spontaneous haemorrhage and rupture. The aim of this study was to assess management in 22 patients treated for haemorrhage and/or rupture of HCA.

Patients and methods: Between May 1990 and July 2005, 22 female patients were diagnosed with acute haemorrhage and/or rupture of lesions highly suspicious of HCA. Preoperative imaging diagnostics and pathologic specimens were reviewed.

Results: Twelve haemodynamically stable and four unstable patients could be treated conservatively. One patient underwent acute partial liver resection, whereas four patients underwent laparotomy with initial packing of the liver. In one patient, selective embolisation of the left hepatic artery was performed. Fifteen patients eventually underwent resection after a mean time of 8 months after initial treatment. Six patients did not undergo resection and showed no complications or rebleeding after a mean follow‐up of 24.6 months. Only in seven patients, histopathological examination showed HCA, and in one patient, HCA with focal nodular hyperplasia.

Conclusion: HCA with haemorrhage and/or rupture does not necessarily require immediate liver resection. Conservative treatment is justified in stable patients. In case of an instable patient with or without hemoperitoneum, laparotomy with packing or selective embolisation can stop the bleeding.
Relationship between oxidative stress levels and activation state on a hepatic stellate cell line
Authors: Guimarães E. L. M. , Franceschi M. F. S. , Grivicich I. , Dal‐pizzol F. , Moreira J. C. F. , Guaragna R. M. , Borojevic R. , Margis R. , Guma F. C. R.
Published: April 2006

Abstract: Background/Aims: Oxidative stress plays an important role in liver fibrosis. Under pathological conditions, hepatic stellate cells (HSC) undergo an activation process, developing a myofibroblast‐like phenotype from the lipocyte phenotype. In this study, we determined the levels of oxidative stress and proliferation in different activation states of an experimental model of mouse HSC, the GRX cell line. These cells can be induced in vitro to display a more activated state or a quiescent phenotype.

Methods/Results: We observed increased oxidative damage and higher levels of reactive oxygen species, measured by thiobarbituric acid reactive species and 2′,7′‐dichlorofluorescein diacetate, respectively, and diminished catalase activity in activated cells. Activation decreased proliferation and increased the number of cells in G2/M. Antioxidants N‐acetylcysteine and Trolox varied in their capacity to correct the oxidative stress and proliferation status.

Conclusions: The differences in physiological functions of stellate cell phenotypes suggest a relationship between oxidative stress levels and activation state.
Successful liver transplantation and delivery in a woman with fulminant hepatic failure occurring during the second trimester of pregnancy
Authors: Jarufe Nicolás , Soza Alejandro , Pérez‐Ayuso Rosa María , Poblete José Andrés , González Robinson , Guajardo Matías , Hernandez Verónica , Riquelme Arnoldo , Arrese Marco , Martínez Jorge
Published: April 2006

Abstract: Background: Severe liver dysfunction occurring during pregnancy is an unusual but dramatic event that poses special technical and ethical issues because it involves two lives.

Methods and Results: We report the case of a 35‐year‐old woman with cryptogenic fulminant hepatic failure who underwent successful orthotopic liver transplantation at 22 weeks of pregnancy. After a relatively uneventful post‐operative course she delivered a normal offspring at the 27th week of gestation. There were no obstetrical complications and neonatal outcome was excellent. After a year of follow‐up, the patient is doing well,and the newborn has exhibited normal psychomotor and weight/height development.

Conclusion: This case illustrates the challenge of treating fulminant hepatic failure during pregnancy and demonstrates that liver transplantation is a feasible therapeutic option for treatment of patients with this condition, allowing successful completion of pregnancy.
Salmonellosis with liver abscess mimicking hepatocellular carcinoma in a diabetic and cirrhotic patient: a case report and review of the literature
Authors: Chou Yeh‐Pin , Changchien Chi‐Sin , Chiu King‐Wah , Kuo Chung‐Mou , Kuo Fang‐Ying , Kuo Chung‐Huang
Published: April 2006

Abstract: Salmonellosis with liver abscess in a cirrhotic liver is extremely rare. We report the first case of Salmonellosis with septic shock and liver abscess in a diabetic and cirrhotic patient. The image studies of liver initially favored hepatocellular carcinoma. But no definite focus of sepsis was found. After close follow‐up of the liver space‐occupied lesion, ultrasound examination revealed the features of liver abscesses at space‐occupied lesion later. Ultrasound‐guided liver aspiration proved abscess. The clinical and radiological responses were good after antibiotics treatment.
Liver protection by hypothermic perfusion at different temperatures during total vascular exclusion
Authors: Dinant Sander , Van Veen Suzanne Q. , Roseboom Hendrik J. , Van Vliet Arlène K. , Van Gulik Thomas M.
Published: April 2006

Abstract: Introduction: In
situ hypothermic perfusion (HP) can be applied to attenuate ischemia and reperfusion (I/R) injury during liver resection under total vascular exclusion (TVE). This study examines the protective effect of cooling by HP at 20 and 28°C as compared with no HP during TVE in a porcine liver I/R model.

Methods: Twenty‐one pigs underwent 60 min TVE of the liver followed by 24 h reperfusion. HP was performed via the portal vein using ringerlactate solution of 4°C. Pigs were assigned to three groups: TVE without HP (no‐HP, n=9), TVE with HP at 28°C (HP‐28, n=6) and TVE with HP at 20°C (HP‐20, n=6).

Results: Perfusion volumes during TVE were 5.1±0.5 and 17.3±1.7 l in HP‐28 and HP‐20, respectively (P<0.05). Aspartate aminotransferase (AST) after 24 h reperfusion was 1172±440 U/l in no‐HP as compared with 223±69 and 180±22 U/l in HP‐28 and HP‐20, respectively (P<0.05). No differences in liver function or histopathology were found between the HP‐28 and HP‐20 groups.

Conclusions: HP at 20°C is equally effective in preserving liver function and preventing hepatocellular injury under TVE as compared with HP at 28°C. HP at 28°C is advised, because of the lesser perfusion volume necessary for cooling of the liver.
Hyaluronic acid and aspartate aminotransferase levels normalized by liver function can reflect sinusoidal impairment in chronic liver disease
Authors: Testa Emanuela , Malfatti Federica , Milazzo Sara , Cordiviola Cinzia , Cotellessa Tiziana , Marabotto Elisa , Giannini Edoardo , Ceppa Paola , Mamone Mario , Risso Domenico , Testa Roberto
Published: April 2006

Abstract: Background/Aim: To evaluate the relationship between hyaluronic acid/aminopyrine breath test (HA/ABT) ratio and fibrosis score in chronic hepatitis, and between HA/ABT and clinical staging (child‐turcotte‐pugh'score, CTP; and model for end stage liver disease, MELD) in cirrhosis, as well as to evaluate the aspartate aminotransferase (AST)/ABT in relation to the HA/ABT.

Methods: We studied 48 patients with histologically proven chronic hepatitis C (CHC) and 35 patients with compensated cirrhosis (CIR).

Results: HA/ABT and AST/ABT showed a more significant correlation with the fibrosis score than HA or ABT or AST alone in the 48 CHC patients: r=0.568 (P<0.0001), r=0.610 (P<0.0001), r=0.450 (P=0.0021), r=−0.449 (P=0.0021), and r=0.472(P=0.0012), respectively. Progressive liver damage (fibrosis 1–2 vs fibrosis 3–6 vs cirrhosis) was significantly (P<0.05) reflected by both HA/ABT (mean±SEM: 4.0±0.9 vs 18.1±4.2 vs 149.9±33.1) and AST/ABT (6.3±1.8 vs 12.7±1.6 vs 42.1±14.6). A strong relationship was found between HA/ABT and AST/ABT (r=0.755 P<0.0001). In cirrhotic patients, the most significant relationship was observed between HA/ABT and CTP r=0.483 and P=0.0049, and MELD r=0.523 and P=0.0023.

Conclusion: Considering that HA levels in chronic hepatitis depend on the progressive impairment of sinusoidal endothelial cells (SEC), related to progressive fibrosis, HA/ABT ratio would seem to be the most specific reflection of progressive impairment of the SEC. AST/ABT could be used as a possible surrogate of HA in identifying SEC impairment in chronic hepatitis.
Tumor necrosis factor‐a antisense transfer remarkably improves hepatic graft viability
Authors: Yoshizumi Tomoharu , Yonemitsu Yoshikazu , Ikeda Yasuhiro , Kaneda Yasufumi , Yanaga Katsuhiko , Sugimachi Keizo , Sueishi Katsuo
Published: April 2006

Abstract: Background: Cold ischemia/reperfusion injury of the hepatic graft, an unsolved problem in liver transplantations, is attributed to the release of inflammatory cytokines, especially the tumor necrosis factor‐ (TNF) α, from activated Kupffer cells (KC). Therefore, the specific inhibition of TNF‐α could improve the viability of the hepatic graft upon reperfusion.

Methods: We assessed the efficacy of TNF‐α antisense (TNF‐AS) oligodeoxynucleotides (ODNs) delivery to KC in a rodent liver transplantation model.

Results: Seventy‐one percent of the animals that received 6 hours preserved grafts in baths of lactated Ringer's solution (4°C) and were treated with TNF‐AS survived for over 14 days. Eighty percent of the animals treated with vehicle, sense ODNs, or balanced salt saline (BSS) died. Four hours after reperfusion of the liver, a significant reduction was noted in livers treated with TNF‐AS in the release of cytosolic enzymes from the hepatocytes and the serum TNF‐α (P<0.05). The expressions of TNF‐α on KC and of intercellular adhesion molecule‐1 on sinusoidal endothelial cells were completely suppressed in TNF‐AS‐treated livers.

Conclusions: TNF‐AS delivery improves the viability of the hepatic graft, and this technique may solve hepatic graft nonfunction in a clinical setting.
PDC‐E3BP is not a dominant T‐cell autoantigen in primary biliary cirrhosis
Authors: McHugh Anna , Robe Amanda J. , Palmer Jeremy M. , Jones David E. J.
Published: April 2006

Abstract: Background: Autoantibody responses reactive with the E2 and E3BP components of pyruvate dehydrogenase complex (PDC), which characterise primary biliary cirrhosis (PBC) crossreact, precluding the identification, from serological studies, of the antigen to which the principal breakdown of tolerance occurs. Although autoreactive T‐cell responses to PDC‐E2 have been well characterised it is, at present, unclear whether T‐cell tolerance breakdown also occurs to PDC‐E3BP. The aims of this study were to characterise autoreactive T‐cell responses to PDC‐E3BP in PBC and potential factors regulating their expression.

Methods: Peripheral blood T‐cell proliferative responses to purified recombinant human PDC‐E2 and PDC‐E3BP at a range of concentrations were characterised in PBC patients and control subjects.

Results: T‐cell proliferative responses to both E2 and E3BP were absent from control subjects (median peak stimulation index (SI) to PDC‐E2 1.2 [range 0.3–1.9], 0/10 positive (SI>2.32), median peak SI to PDC‐E3BP 1.1 [0.7–2.1]], 0/10 positive). Significant responses to PDC‐E2 were seen in the majority of patients (median peak SI 11.4 [0.4–24.4], 17/20 (85%) positive) but to PDC‐E3BP in only a minority (median peak SI 1–9 [0.6–9.95], 8/20 (40%) positive). Where responses to PDC‐E3BP were seen they were universally secondary to responses to PDC‐E2.

Conclusions: Despite the presence of antibodies reactive with PDC‐E3BP in the majority of PBC patients this self‐protein is not a dominant T‐cell autoantigen in PBC.
Human intrahepatic biliary epithelial cells function in innate immunity by producing IL‐6 and IL‐8 via the TLR4‐NF‐κB and ‐MAPK signaling pathways
Authors: Yokoyama Terufumi , Komori Atsumasa , Nakamura Minoru , Takii Yasushi , Kamihira Takashi , Shimoda Shinji , Mori Tsuyoshi , Fujiwara Shinsuke , Koyabu Makiko , Taniguchi Ken , Fujioka Hikaru , Migita Kiyoshi , Yatsuhashi Hiroshi , Ishibashi Hiromi
Published: April 2006

Abstract: Background: Human intrahepatic biliary epithelial cells (HIBECs) may play active roles in both the innate and adaptive immune responses. Little is known, however, about the role of toll‐like receptors (TLRs) on HIBECs in inflammatory cholangiopathies.

Methods: The expression of TLR1–9 and the biological responses to their ligands, lipopolysaccharide (LPS) or lipoteichoic acid (LTA), were studied in cultured HIBECs by reverse transcription‐polymerase chain reaction, immunoblotting, and enzyme‐linked immunosorbent assay.

Results: HIBECs constitutively expressed transcripts encoding TLR1–6 and 9, as well as myeloid differentiation factor 88 (MyD88), MD2, and CD14. Stimulation of HIBECs with LPS resulted in translocation of NF‐κB subunits from the cytoplasmic to the nuclear fraction, followed by increased secretion of a variety of chemokines/cytokines, including interleukin‐8 (IL‐8), monocyte chemotactic protein‐1 (MCP‐1), and IL‐6. Treatment with BAY11‐7082 efficiently inhibited the LPS‐induced transcription and secretion of these chemokines/cytokines. In HIBECs, the mitogen‐activated protein kinases (MAPKs) were also activated by LPS stimulation. These results indicated that LPS activates HIBECs via a TLR4‐MyD88‐dependent pathway. Stimulation of HIBECs with LTA induced the secretion of a similar profile of cytokines/chemokines via a TLR2‐MyD88‐dependent pathway.

Conclusions: In HIBECs, at least TLR2 and 4 are capable of mediating innate immune system function in vitro. This result, in conjunction with our recent finding that TLR4 expression is increased in biliary epithelial cells in primary biliary cirrhosis, suggests the involvement of TLRs in the development of chronic inflammatory cholangiopathies.
Peg‐interferon improves liver histology in patients with HBeAg‐positive chronic hepatitis B: no additional benefit of combination with lamivudine
Authors: Van Zonneveld Monika , Zondervan Pieter E , Cakaloglu Yilmaz , Simon Christopher , Akarca Ulus S , So Thomas MK , Flink Hajo J , De Man Robert A , Schalm Solko W , Janssen Harry LA ,   for the HBV 99‐01 Study Group
Published: April 2006

Abstract: Background: The effect of pegylated interferon or its combination with lamivudine on liver histology of patients with chronic hepatitis B (CHB) is unknown. In a double‐blinded, randomized, multi‐center study we assessed histological changes in 110 hepatitis B e‐antigen (HBeAg)‐positive CHB patients treated for 52 weeks with Pegylated interferon α‐2b (PEG‐IFN) in combination with either lamivudine or placebo. Liver biopsies were taken before and at the end of treatment. All biopsies were blinded and scored according to the Ishak system.

Results: Necroinflammatory score improved (defined as a decrease of at least two points) in 25 patients (48%) of the PEG‐IFN/lamivudine combination therapy group and in 31 patients (53%) of the PEG‐IFN monotherapy group. The fibrosis score improved (decrease of at least 1 point) in 17 patients (33%) of the combination therapy group vs. 13 patients (22%) of the PEG‐IFN monotherapy group (P=0.23). Responders (n=42), defined as serum HBeAg negative at the end of therapy, showed a larger decline in necroinflammatory score than non‐responders (mean decline 2.3 and 1.2 points, respectively, P=0.02). Among patients receiving PEG‐IFN monotherapy necroinflammation improved more frequently in responders (78% of responders vs. 43% of non‐responders, P=0.01) and in patients who showed normalization of ALT (76% of patients with normal ALT vs. 40% of patients with abnormal ALT, P=0.01). Fibrosis score in the PEG‐IFN monotherapy group improved more often in responders (39%) than in non‐responders (15%, P=0.04). In the PEG‐IFN/lamivudine combination therapy group, we found no significant association between virological and biochemical endpoints and histological improvement.

Conclusions: Treatment with PEG‐IFN therapy improves liver necroinflammation in HBeAg‐positive CHB patients, particularly in responders to therapy. PEG‐IFN also improves fibrosis in responders. Addition of lamivudine to PEG‐IFN did not further improve the histological outcome.
Back‐Channel Negotiation: International Bargaining in the Shadows
Authors: Wanis‐St. John Anthony
Published: April 2006

Back‐channel negotiations (BCNs) are officially sanctioned negotiations conducted in secret between the parties to a dispute. These extraordinary negotiations operate in parallel with, or replace, acknowledged front channels of negotiation. Back channels are like the black markets of negotiation; they are separate tables where bargaining takes place in the shadows. When front‐channel negotiations fail, they are sometimes eclipsed by successful BCNs even though the same principals, conflicts, and sociopolitical contexts are involved. This article asks: Why do decision makers deploy back channels? What is the impact of BCN on international peace processes? The Palestinian–Israeli peace process, in which both back and front channels have been used consistently, provides the basis for comparing channels and offering initial answers to these questions. The author concludes that while BCN can facilitate breakthrough agreements, it can also damage a peace process by helping to reinforce some of the uncertainties that gave rise to the use of back channels in the first place.
Integrative and Distributive Bargaining Situations in the European Union: What Difference Does It Make?
Authors: Da Conceição‐Heldt Eugénia
Published: April 2006

Despite the distinction made between integrative and distributive bargaining situations in European studies literature, few studies have focused specifically on how these two situations differ. This article attempts to close this gap by examining two key bargaining situations involving fisheries that led to the Europeanization of this policy field. The integrative bargaining situation dealt with the negotiations about the structural policy and common organization of the market for fish products. In contrast, the distributive bargaining case focused on the protracted negotiations over resource conservation and management policies. The article focuses primarily on how the unanimity decision rule may affect negotiation patterns in the European Union and on how the specific type of bargaining situation affects policy outcomes. The thesis is that different bargaining tools are used to reach an agreement.
Domestic and International Politics during an EU Intergovernmental Conference: Bridging the Gap between Negotiation Theory and Practice
Authors: Thurner Paul W. , Pappi Franz Urban
Published: April 2006

This article describes selective results of a large‐scale study of within‐governmental coordination processes and international negotiations. We examined a European Union intergovernmental conference (IGC), the so‐called IGC 1996, which led to the Amsterdam Treaty, from a quantitative negotiation analysis perspective. Our focus of attention has been the embeddedness of international negotiations within formal governmental organizations and within informal communication networks. We have identified the relative impact on negotiation dynamics and on negotiation performance of each of the parties by using various statistical techniques. We argue that such insights can be used for a research‐based consulting in which social scientists respond to practitioners’“what‐if” questions with evidence‐based simulations and scenarios.
Is Teaching Negotiation Too Easy, Too Hard, or Both?
Authors: Wheeler Michael
Published: April 2006

Teaching negotiation is easy because teachers and students find the topic fun, interesting, and relevant, which makes most negotiation courses well received. At the same time, teachers may underestimate the challenges in getting their students to think and behave differently in negotiation, which can make it difficult to teach it well. The author examines three teaching challenges in particular: dealing with ethical issues, addressing power imbalances (including those implicated by gender and racial differences), and putting theory into practice in the form of real‐world behavior change. This piece is an adaptation of the keynote address that the author delivered on November 14, 2005 at the PON‐IRENE conference, New Trends in Negotiation Teaching: Toward a Transatlantic Network, in Cergy, France.
Like Talking to a Brick Wall: Implications of Emotion Metaphors for Mediation Practice
Authors: Jameson Jessica Katz , Bodtker Andrea M. , Jones Tricia S.
Published: April 2006

In this article, the authors discuss the role of emotions in mediation with the goal of providing practical insights that can improve the mediation process. Their assumption is that emotion is ever‐present, particularly in conflict, and that acknowledging and addressing underlying emotions facilitates conflict transformation during the mediation process.
Investigating the Origin and Spread of Hepatitis C Virus Genotype 5a
Authors: Verbeeck Jannick , Maes Piet , Lemey Philippe , Pybus Oliver G. , Wollants Elke , Song Ernie , Nevens Frederik , Fevery Johan , Delport Wayne , Van der Merwe Schalk , Van Ranst Marc
Published: April 2006

Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.
An Alternative Splice Product of IκB Kinase (IKKγ), IKKγ-Δ, DifferentiallyMediates Cytokine and Human T-Cell Leukemia Virus Type 1 Tax-Induced NF-κB Activation
Authors: Hai Tao , Yeung Man-Lung , Wood Thomas G. , Wei Yuanfen , Yamaoka Shoji , Gatalica Zoran , Jeang Kuan-Teh , Brasier Allan R.
Published: April 2006

NF-κB
is an inducible transcription factor mediating innate immune responses
whose activity is controlled by the multiprotein IκB kinase
(IKK) “signalsome”. The core IKK consists of two
catalytic serine kinases, IKKα and IKKβ, and a
noncatalytic subunit, IKKγ. IKKγ is required for IKK
activity by mediating kinase oligomerization and serving to couple the
core catalytic subunits to upstream mitogen-activated protein 3-kinase
cascades. We have discovered an alternatively spliced IKKγ mRNA
isoform, encoding an in-frame deletion of exon 5, termed
IKKγ-Δ. Using a specific reverse transcription-PCR
assay, we find that IKKγ-Δ is widely expressed in
cultured human cells and normal human tissues. Because
IKKγ-Δ protein is lacking a critical coiled-coil domain
important in protein-protein interactions, we sought to determine its
signaling properties by examining its ability to self associate, couple
to activators of the canonical pathway, and mediate human T-cell
leukemia virus type 1 (HTLV-1) Tax-induced NF-κB activity.
Coimmunoprecipitation and confocal colocalization assays indicate
IKKγ-Δ has strong homo- and heterotypic association
with wild-type (WT) IKKγ and, like IKKγ WT, associates
with the IKKβ kinase. Similarly, IKKγ-Δ
mediates IKK kinase activity and downstream NF-κB-dependent
transcription in response to tumor necrosis factor (TNF) and the
NF-κB-inducing kinase-IKKα signaling pathway.
Surprisingly, however, in contrast to IKKγ WT,
IKKγ-Δ is not able to mediate HTLV-1 Tax-induced
NF-κB-dependent transcription, even though
IKKγ-Δ binds and colocalizes with Tax. These
observations suggest that IKKγ-Δ is a functionally
distinct alternatively spliced mRNA product differentially mediating
TNF-induced, but not Tax-induced, signals converging on the IKK
signalsome. Differing levels of IKKγ-Δ expression,
therefore, may affect signal transduction cascades coupling to
IKK.
Generation of Measles Virus with a Segmented RNA Genome
Authors: Takeda Makoto , Nakatsu Yuichiro , Ohno Shinji , Seki Fumio , Tahara Maino , Hashiguchi Takao , Yanagi Yusuke
Published: April 2006

Viruses classified in the order
Mononegavirales
have a single nonsegmented RNA molecule as the genome and employ similar strategies for genome replication and gene expression. Infectious particles of
Measles virus
(MeV), a member of the family
Paramyxoviridae
in the order
Mononegavirales
, with two or three RNA genome segments (2 seg- or 3 seg-MeV) were generated using a highly efficient reverse genetics system. All RNA segments of the viruses were designed to have authentic 3′ and 5′ self-complementary termini, similar to those of negative-stranded RNA viruses that intrinsically have multiple RNA genome segments. The 2 seg- and 3 seg-MeV were viable and replicated well in cultured cells. 3 seg-MeV could accommodate up to six additional transcriptional units, five of which were shown to be capable of expressing foreign proteins efficiently. These data indicate that the MeV genome can be segmented, providing an experimental insight into the divergence of the negative-stranded RNA viruses with nonsegmented or segmented RNA genomes. They also illustrate a new strategy to develop mononegavirus-derived vectors harboring multiple additional transcriptional units.
The E7 Oncoprotein Is Translated from Spliced E6*I Transcripts in High-Risk Human Papillomavirus Type 16- or Type 18-Positive Cervical Cancer Cell Lines via Translation Reinitiation
Authors: Tang Shuang , Tao Mingfang , McCoy J. Philip , Zheng Zhi-Ming
Published: April 2006

High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105
Rb
and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.
Reconstitution of Herpes Simplex Virus Microtubule-Dependent Trafficking In Vitro
Authors: Lee Grace E. , Murray John W. , Wolkoff Allan W. , Wilson Duncan W.
Published: April 2006

Microtubule-mediated anterograde transport of herpes simplex virus (HSV) from the neuronal cell body to the axon terminal is crucial for the spread and transmission of the virus. It is therefore of central importance to identify the cellular and viral factors responsible for this trafficking event. In previous studies, we isolated HSV-containing cytoplasmic organelles from infected cells and showed that they represent the first and only destination for HSV capsids after they emerge from the nucleus. In the present study, we tested whether these cytoplasmic compartments were capable of microtubule-dependent traffic. Organelles containing green fluorescent protein-labeled HSV capsids were isolated and found to be able to bind rhodamine-labeled microtubules polymerized in vitro. Following the addition of ATP, the HSV-associated organelles trafficked along the microtubules, as visualized by time lapse microscopy in an imaging microchamber. The velocity and processivity of trafficking resembled those seen for neurotropic herpesvirus traffic in living axons. The use of motor-specific inhibitors indicated that traffic was predominantly kinesin mediated, consistent with the reconstitution of anterograde traffic. Immunocytochemical studies revealed that the majority of HSV-containing organelles attached to the microtubules contained the
trans
-Golgi network marker TGN46. This simple, minimal reconstitution of microtubule-mediated anterograde traffic should facilitate and complement molecular analysis of HSV egress in vivo.
Bromodomain Protein 4 Mediates the Papillomavirus E2 Transcriptional Activation Function
Authors: Schweiger Michal-Ruth , You Jianxin , Howley Peter M.
Published: April 2006

The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis.
Role for TLR2 in NK Cell-Mediated Control of Murine Cytomegalovirus In Vivo
Authors: Szomolanyi-Tsuda Eva , Liang Xueya , Welsh Raymond M. , Kurt-Jones Evelyn A. , Finberg Robert W.
Published: April 2006

Natural killer (NK) cells are essential for the early control of murine cytomegalovirus (MCMV) infection. Here, we demonstrate that toll-like receptor 2 (TLR2) plays a role in the NK cell-mediated control of MCMV. TLR2 knockout (KO) mice had elevated levels of MCMV in the spleen and liver on day 4 postinfection compared to C57BL/6 mice. In vivo depletion of NK cells with anti-NK1.1 antibodies, however, eliminated the differences in viral titers between the two groups, suggesting that the effect of TLR2 on MCMV clearance on day 4 was NK cell mediated. The defect in early antiviral control was associated with a decreased NK cell population in the spleen and liver and reduced amounts of interleukin-18 and α/β interferon secreted in the TLR2 KO mice. Our studies suggest that in addition to the reported involvement of TLR9 and TLR3, TLR2 is also involved in innate immune responses to MCMV infection.
Targeting of p300/CREB Binding Protein Coactivators by Simian Virus 40 Is Mediated through p53
Authors: Borger Darrell R. , DeCaprio James A.
Published: April 2006

The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT.
Crystal Structure of the Simian Virus 40 Large T-Antigen Origin-Binding Domain
Authors: Meinke Gretchen , Bullock Peter A. , Bohm Andrew
Published: April 2006

The origins of replication of DNA tumor viruses have a highly conserved feature, namely, multiple binding sites for their respective initiator proteins arranged as inverted repeats. In the 1.45-Å crystal structure of the simian virus 40 large T-antigen (T-ag) origin-binding domain (obd) reported herein, T-ag obd monomers form a left-handed spiral with an inner channel of 30 Å having six monomers per turn. The inner surface of the spiral is positively charged and includes residues known to bind DNA. Residues implicated in hexamerization of full-length T-ag are located at the interface between adjacent T-ag obd monomers. These data provide a high-resolution model of the hexamer of origin-binding domains observed in electron microscopy studies and allow the obd's to be oriented relative to the hexamer of T-ag helicase domains to which they are connected.
Role of the Alpha/Beta Interferon Response in the Acquisition of Susceptibility to Poliovirus by Kidney Cells in Culture
Authors: Yoshikawa Tomoki , Iwasaki Takuya , Ida-Hosonuma Miki , Yoneyama Mitsutoshi , Fujita Takashi , Horie Hitoshi , Miyazawa Miwako , Abe Shinobu , Simizu Bunsiti , Koike Satoshi
Published: April 2006

Replication of poliovirus (PV) is restricted to a few sites, including the brain and spinal cord. However, this neurotropism is not conserved in cultured cells. Monkey kidney cells become susceptible to PV infection after cultivation in vitro, and cell lines of monolayer cultures from almost any tissue of primates are susceptible to PV infection. These observations suggest that cellular changes during cultivation are required for acquisition of susceptibility. The molecular basis for the cellular changes during this process is not known. We investigated the relationship between PV susceptibility and interferon (IFN) response in primary cultured kidney and liver cells derived from transgenic mice expressing human PV receptor and in several primate cell lines. Both kidneys and liver in vivo showed rapid IFN response within 6 h postinfection. However, monkey and mouse kidney cells in culture and primate cell lines, which were susceptible to PV, did not show such rapid response or showed no response at all. On the other hand, primary cultured liver cells, which were partially resistant to infection, showed rapid IFN induction. The loss of IFN inducibility in kidney cells was associated with a decrease in expression of IFN-stimulated genes involved in IFN response. Mouse kidney cells pretreated with a small dose of IFN, in turn, restored IFN inducibility and resistance to PV. These results strongly suggest that the cells in culture acquire PV susceptibility during the process of cultivation by losing rapid IFN response that has been normally maintained in extraneural tissues in vivo.
Enhanced Replication of Simian Immunodeficiency Virus Adjacent to Catecholaminergic Varicosities in Primate Lymph Nodes
Authors: Sloan Erica K. , Tarara Ross P. , Capitanio John P. , Cole Steve W.
Published: April 2006

Clinical and in vitro studies have shown that activity of the autonomic nervous system (ANS) can stimulate lentivirus replication. To define the potential anatomical basis for this effect, we analyzed the spatial relationship between catecholaminergic neural fibers and sites of simian immunodeficiency virus (SIV) replication in lymph nodes from rhesus macaques experimentally infected with SIVmac251. Viral replication was mapped by in situ hybridization for SIVenv,gag, andnefRNA, and catecholaminergic varicosities from the ANS were mapped by sucrose phosphate glyoxylic acid chemofluorescence. Spatial statistical analyses showed that the likelihood of active SIV replication increased by 3.9-fold in the vicinity of catecholaminergic varicosities (P< 0.0001). The densities of both ANS innervation and SIV replication differed across cortical, paracortical, and medullary regions of the lymph node, but analyses of each region separately continued to show increased replication of SIV adjacent to catecholaminergic varicosities. Ancillary analyses ruled out the possibility that SIV-induced alterations in lymph node architecture might create a spurious spatial association. These data support human clinical studies and in vitro molecular analyses showing that catecholamine neurotransmitters from the ANS can increase lentiviral replication by identifying a specific anatomic context for interactions between ANS neural fibers and replication of SIV in lymphoid tissue.
Repair and Polyadenylation of a Naturally Occurring Hepatitis C Virus 3′ Nontranslated Region-Shorter Variant in Selectable Replicon Cell Lines
Authors: van Leeuwen Hans C. , Liefhebber Jolanda M. P. , Spaan Willy J. M.
Published: April 2006

The 3′ nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific
cis
-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3′ end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3′ NTR may also be essential in host defense against exoribonucleases. During 3′-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3′ end of the genome. These shorter 3′ ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3′ ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3′ terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3′ ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3′ end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.
Identification of Functionally Important Negatively Charged Residues in the Carboxy End of Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein
Authors: Verma Sandhya , Bednar Valerie , Blount Andrew , Hogue Brenda G.
Published: April 2006

The coronavirus nucleocapsid (N) protein is a multifunctional viral gene product that encapsidates the RNA genome and also plays some as yet not fully defined role in viral RNA replication and/or transcription. A number of conserved negatively charged amino acids are located within domain III in the carboxy end of all coronavirus N proteins. Previous studies suggested that the negatively charged residues are involved in virus assembly by mediating interaction between the membrane (M) protein carboxy tail and nucleocapsids. To determine the importance of these negatively charged residues, a series of alanine and other charged-residue substitutions were introduced in place of those in the N gene within a mouse hepatitis coronavirus A59 infectious clone. Aspartic acid residues 440 and 441 were identified as functionally important. Viruses could not be isolated when both residues were replaced by positively charged amino acids. When either amino acid was replaced by a positively charged residue or both were changed to alanine, viruses were recovered that contained second-site changes within N, but not in the M or envelope protein. The compensatory role of the new changes was confirmed by the construction of new viruses. A few viruses were recovered that retained the D
441
-to-arginine change and no compensatory changes. These viruses exhibited a small-plaque phenotype and produced significantly less virus. Overall, results from our analysis of a large panel of plaque-purified recovered viruses indicate that the negatively charged residues at positions 440 and 441 are key residues that appear to be involved in virus assembly.