bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperacute rejection of hDAF‐transgenic pig organ xenografts in cynomolgus monkeys: influence of pre‐existing anti‐pig antibodies and prevention by the αGAL glycoconjugate GAS914
Authors: Lam Tuan T. , Hausen Bernard , Boeke‐Purkis Katrin , Paniagua Ricardo , Lau Macy , Hook Laurie , Berry Gerry , Higgins John , Duthaler Rudolf O. , Katopodis Andreas G. , Robbins Robert , Reitz Bruce , Borie Dominique , Schuurman Henk‐Jan , Morris Randall E.
Published: October 2004

Abstract:  Background:  Our introductory pig‐to‐cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay‐accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti‐pig antibodies. We evaluated the efficacy of GAS914, a Galα1–3Gal trisaccharide linked to a poly‐l‐lysine backbone, in inhibition of HAR.

Methods:  hDAF transgenic heterotopic heart (n = 15) or life‐supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti‐thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti‐pig antibody levels were determined in various assays.

Results:  Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre‐treatment, did not show HAR (chi‐square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre‐treatment.

Conclusions:  HAR of hDAF solid organs could be ascribed to high levels of anti‐pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.
Prolonged survival of fetal pig islet xenografts in mice lacking the capacity for an indirect response
Authors: Koulmanda Maria , Laufer Terri M , Auchincloss Hugh , Neal Smith R
Published: October 2004

Abstract: Background: Xenografts of islets from organ‐cultured fetal pig pancreases transplanted into non‐immunosuppressed mice are rejected within 10 days. Immunosuppression with anti‐T cell (anti‐CD4) monoclonal antibody alone delays rejection of these xenografts for about 28 days, but rejection eventually occurs despite marked depletion of T cells. To determine if the critical CD4+ T cells responsible for xenograft islet rejection function through the direct or indirect pathway, selective class II‐deficient mice that express class II antigens only on their thymic epithelium (not on peripheral cells) with normal numbers of CD4+ T cells, (class II‐, CD4+), were used as recipients of xenograft islets to test if rejection occurs in the absence of an indirect response.

Methods: Control (C57BL/6) or class II‐, CD4+ mice were transplanted under the kidney capsule with cultured fetal pig islets. Class II‐, CD4+ mice have normal numbers of B cells, CD4+,γδT cells, and slightly increased numbers of CD8+ T cells. Additional mice were thymectomized before receiving anti‐CD4 or anti‐CD8 monoclonal antibodies. Islet graft survival was determined histologically as fetal pig islets were too immature to secrete insulin.

Results: Xenograft survival in control animals was 7 to 14 days. In contrast, graft survival in class II‐, CD4+ mice was significantly prolonged to greater than 35 days. Depletion of CD8+ T cells in class II‐, CD4+ mice prolonged graft survival to about 70 days. Depletion of CD4+ T cells from these mice further prolonged xenograft survival to about 100 days.

Conclusions: These results suggest that the rejection of pig islets by mice initially depends on a CD4 dependent indirect response. The CD4 direct response also contributes to graft destruction. CD8+ T cells also participate in graft destruction, albeit weakly.
Anti‐non‐Gal porcine endothelial cell antibodies in acute humoral xenograft rejection of hDAF‐transgenic porcine hearts in cynomolgus monkeys
Authors: Lam Tuan T. , Paniagua Ricardo , Shivaram Giridhar , Schuurman Henk‐Jan , Borie Dominique C. , Morris Randall E.
Published: October 2004

Abstract:  Background:  Anti‐Galα1–3Gal (Gal) antibodies play a major role in hyperacute rejection and acute humoral xenograft rejection (AHXR) in porcine‐to‐nonhuman primate transplantation. The role of anti‐non‐Gal antibodies in AHXR is less well defined.

Methods:  Eleven cynomolgus monkeys received a heterotopic heart transplant from a human decay‐accelerating factor transgenic pig, and maintenance immunosuppression with cyclosporin A or tacrolimus, steroids, mycophenolate sodium or mycophenolate mofetil, and in 10 animals the Gal‐containing soluble glycoconjugate GAS914. Six ended with AHXR (6 to 78 day survival) and five did not show AHXR (9 to 36 day survival). Anti‐Gal antibodies were depleted in vivo with GAS914, or in vitro with Gal‐coated Sepharose beads. IgM‐ and IgG‐class anti‐non‐Gal antibodies in serum depleted of anti‐Gal antibodies were measured by flow cytometry using porcine endothelial target cells.

Results:  Compared with pre‐transplant values, all six recipients with AHXR showed a substantially higher level of anti‐non‐Gal IgM antibodies at rejection; in five animals there was also an increase in IgG‐class antibodies. There was no relevant change in recipients without AHXR. AHXR at time of cessation of heart contraction could be preceeded by a steady increase in antibody level starting 2 to 3 weeks earlier.

Conclusions:  AHXR is invariably associated with increased circulating anti‐non‐Gal antibodies. These antibodies are not observed in recipients without AHXR, and five of six recipients with AHXR were adequately depleted of anti‐Gal antibodies by maintenance GAS914. This indicates that anti‐non‐Gal antibodies play a significant role in the pathogenesis of AHXR. Also, the assessment of these antibodies could be used as an early monitor of AHXR.
Herpesvirus infections in xenotransplantation: pathogenesis and approaches
Authors: Mueller Nicolas J. , Fishman Jay A.
Published: October 2004

Abstract:  Infectious risk remains an important consideration in the clinical application of xenotransplantation. Vascularized xenografts create unique immunological niches in which bidirectional transmission of pathogens between donor and recipient may occur. Enhanced replication of many pathogens is stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Herpesviruses are the prototype viruses that are activated during immunosuppression. Quantitative diagnostic molecular assays have been developed for the known herpesviruses causing infection in pigs. Recent data suggest that some herpesviral infections, such as porcine cytomegalovirus, may be excluded from swine used as source animals by careful breeding, while others will require novel strategies for control. This review focuses on porcine and baboon herpesviruses in pig‐to‐non‐human primate solid organ xenotransplantation including direct effects (tissue damage), indirect effects (coagulopathy, rejection), and possible approaches to these infections.
Assessing the risk potential of porcine circoviruses for xenotransplantation: consensus primer‐PCR‐based search for a human circovirus
Authors: Hattermann Kim , Maerz Alexander , Slanina Heiko , Schmitt Cornelia , Mankertz Annette
Published: October 2004

Abstract:  Background:  An important issue with respect to virus safety in xenotransplantation is the search for human analogues of porcine viruses, because transmission of a porcine virus followed by recombination with a related human virus may lead to a new emerging virus of unknown pathogenicity, host range and virulence. In case of circoviruses, two types of porcine circovirus (PCV1 and PCV2) are described, but the existence of an analogous human circovirus has not yet been investigated.

Methods:  This study describes the analysis of human samples with a consensus primer‐PCR approach designed to amplify conserved regions from the rep gene of circoviruses from the genus Circovirus. DNA from human sera, lymph nodes, blood and urine was extracted and investigated with this method that has led previously to the identification of a new avian circovirus.

Results:  By screening 1101 samples (there of 168 from immunocompromised patients), no evidence for the existence of a human circovirus related to the genus Circovirus was obtained.

Conclusions:  This result renders the existence of a human circovirus related to the porcine circoviruses more unlikely, nevertheless the presence of such a virus cannot be ruled out.
Prevalent human coxsackie B‐5 virus infects porcine islet cells primarily using the coxsackie‐adenovirus receptor
Authors: Myers Suzanne E. , Brewer Laurie , Shaw Daniel P. , Greene Wallace H. , Love Brenda C. , Hering Bernhard , Brad Spiller O. , Kariuki Njenga M.
Published: October 2004

Abstract:  Background:  We have previously demonstrated that transplanting porcine encephalomyocarditis virus (EMCV)‐infected porcine islet cells (PICs) results in transmission of the virus to recipient mice, which is manifested by acute fatal infection within 5 to 8 days. Here, we determined PIC susceptibility to a related and highly prevalent human picornavirus, coxsackie B‐5 virus (CVB‐5).

Methods:  PICs were inoculated with CVB‐5 in vitro for up to 96 hours and infectivity, level of virus replication, and cellular function determined. Subsequently, monoclonal and polyclonal antibody blocking experiments were used to investigate the receptor CVB‐5 uses to enter PICs, and the ability of CVB‐5‐infected islets to reverse diabetes analyzed in mice.

Results:  Adult pig islets inoculated with CVB‐5 in vitro showed a typical picornaviral replication cycle with a 2‐h lag phase followed by a 4‐h exponential phase during which the virus titer increased by 4 logs. However, CVB‐5 was less cytolytic to PICs than EMCV, resulting in a persistent productive infection lasting for up to 96 h, with minimal evidence of cell lysis. Double immunostaining confirmed the presence of CVB‐5 antigens in insulin‐producing islets. Infection of PICs in the presence of antibodies against human coxsackie‐adenovirus receptor (CAR) resulted in near complete blockage in production of infectious virus particles whereas blocking with anti‐porcine decay‐accelerating factor (DAF, also called CD55) or anti‐porcine membrane cofactor protein (MCP, also called CD46) only slightly decreased the number of infectious CVB‐5 particles produced. Immunofluoresence staining showed CAR and MCP expression on the islet surface, but not DAF. Transplanting CVB‐5‐infected PICs into diabetic C57BL/6 mice resulted in reversal of diabetes.

Conclusion:  Although PICs are susceptible to human CVB‐5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.
Factors, fiction and endothelium‐derived hyperpolarizing factor
Authors: Sandow Shaun L
Published: October 2004

1. The principal mediators of vascular tone are neural, endothelial and physical stimuli that result in the initiation of dilator and constrictor responses to facilitate the control of blood pressure. Two primary vasodilatory stimuli produced by the endothelium are nitric oxide (NO) and prostaglandins. An additional endothelium‐dependent vasodilatory mechanism is characterized as the hyperpolarization‐mediated relaxation that remains after the inhibition of the synthesis of NO and prostaglandins. This mechanism is due to the action of a so‐called endothelium‐derived hyperpolarizing factor (EDHF) and is dependent on either the release of diffusible factor(s) and/or to a direct contact‐mediated mechanism.

2. Most evidence supports the concept that ‘EDHF’ activity is dependent on contact‐mediated mechanisms. This involves the transfer of an endothelium‐derived electrical current, as an endothelium‐derived hyperpolarization (EDH), through direct heterocellular coupling of endothelial cells and smooth muscle cells via myoendothelial gap junctions (MEGJ). However, there is a lack of consensus with regard to the nature and mechanism of action of EDHF/EDH (EDH(F)), which has been shown to vary within and between vascular beds, as well as among species, strains, sex and during development, ageing and disease.

3. In addition to actual heterogeneity in EDH(F), further heterogeneity has resulted from the less‐than‐optimal design, analysis and interpretation of data in some key papers in the EDHF literature; with such views being perpetuated in the subsequent literature.

4. The focus of the present brief review is to examine what factors are proposed as EDH(F) and highlight the correlative structural and functional studies from our laboratory that demonstrate an integral role for MEGJ in the conduction of EDH, which account for the heterogeneity in EDH(F), while incorporating the reported diffusible mechanisms in the regulation of this activity. Furthermore, in addition to the reported heterogeneity in the nature and mechanism of action of EDH(F), the contribution of experimental design and technique to this heterogeneity will be examined.
Endothelin‐1‐mediated coronary vasoconstriction deteriorates myocardial depression in hearts isolated from lipopolysaccharide–treated rats: Interaction with nitric oxide
Authors: Tu Jie , Shan Qixian , Jin Hongfeng , Bourreau Jean‐Pierre , Xia Qiang
Published: October 2004

1. The aim of the present study was to evaluate the contribution of disturbance of coronary perfusion to myocardial depression in hearts isolated from lipopolysaccharide (LPS)‐treated rats and to investigate the involvement of endothelin (ET)‐1 and nitric oxide (NO).

2. Rats were treated with LPS (10 mg/kg, i.p.) and, 4 h later, plasma ET‐1 concentrations were measured by radioimmunoassay and hearts were excised for perfusion at a constant perfusion flow. The selective ETA receptor antagonist BQ‐123, in the absence or presence of aminoguanidine, a specific inhibitor of inducible NO synthase, was given 15 min before LPS challenge. Coronary perfusion pressure (CPP) and measures of myocardial contractile function were recorded.

3. In hearts isolated from LPS‐treated rats, there was a marked increase in CPP that was abolished by pretreatment with BQ‐123. In parallel, an increase in plasma ET‐1 concentrations was seen in these rats. Lipopolysaccharide also induced decreases in left ventricular developed pressure (LVDP), the product of LVDP and heart rate and maximal rate of rise/fall of left ventricular pressure (+/– dP/dtmax). Single treatment with BQ‐123 or aminoguanidine attenuated LPS‐induced myocardial depression. However, when these two drugs were given simultaneously, myocardial depression elicited by LPS was blocked significantly.

4. Endothelin‐1‐mediated coronary vasoconstriction, together with NO, contributes to myocardial depression in hearts isolated from LPS‐treated rats.
Effects of caffeic acid phenethyl ester on glycerol‐induced acute renal failure in rats
Authors: Aydogdu Nurettin , Atmaca Gulizar , Yalcin Omer , Batcioglu Kadir , Kaymak Kadir
Published: October 2004

1. Free radicals and nitric oxide (NO) play a crucial role in the pathogenesis of myoglobinuric acute renal failure (ARF). The aim of the present study was to investigate the effects of caffeic acid phenethyl ester (CAPE), an anti‐oxidant, on the myoglobinuric ARF induced by intramusculer hypertonic glycerol injection.

2. Thirty rats were divided equally into three groups. Rats in group 1 were given saline and those in groups 2 and 3 were injected with glycerol (10 mL/kg, i.m.). Concomitant and 24 h after glycerol injection, CAPE (10 µmol/kg, i.p.) was administered to group 3 rats. Forty‐eight hours after glycerol injection, blood samples and kidney tissues of rats were taken under anaesthesia.

3. Plasma concentrations of urea, creatinine, malondialdehyde (MDA) and NO were determined, as were superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities and MDA levels in kidney tissues. Kidney morphology was also investigated.

4. In the group receiving CAPE, although SOD enzyme activity was found to be increased, we failed to find any protective effect of CAPE on other parameters investigated. Moreover, although CAPE significantly decreased NO levels, it increased plasma concentrations of urea and MDA.

5. We suggest that the effect of CAPE in decreasing NO concentrations may further increase the renal ischaemia in this model. Thus, CAPE may have a worsening rather than beneficial effect under these conditions in this model of ARF.
Antinociception induced by stimulating the anterior pretectal nucleus in two models of pain in rats
Authors: Villarreal Cristiane F , Kina Vania Aparecida V , Prado Wiliam A
Published: October 2004

1. This study examined whether different parts of the rat anterior pretectal nucleus (APtN) may be involved in the spinal control of brief (tail flick test) or persistent (surgical incision of the plantar aspect of a hind paw) noxious inputs via activation of descending pathways.

2. We have confirmed that stimulation of the dorsal APtN produces a strong antinociceptive effect in the tail flick test, as opposed to a very weak effect obtained from the ventral APtN. Stimulation at the ventral APtN was the most effective part of the nucleus against a persistent incisional pain.

3. The incisional pain was significantly increased following injection of 1 or 2% lignocaine (0.25 µL) into the nucleus, but the effect was more intense after neural block of the ventral rather than the dorsal APtN. Injection of 2% lignocaine (0.10 µL) into the ventral, but not dorsal, APtN significantly increased the perception of the incisional pain.

4. We conclude that the effect of stimulating the APtN depends on the site of stimulation and model of pain used. Sustained noxious stimuli activate pathways from the ventral APtN to reduce further noxious spinal inputs. The noxious stimulation produced during the tail flick test may be not enough to activate the same circuitry, but electrical stimulation at the dorsal APtN is very effective in inhibiting brief thermal noxious inputs at the spinal level.
Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells
Authors: Wang Yongyu , Hou Rong , Li Ping , Li Jinliang , Yan Jie , Yin Feng , Han Chide , Zhang Youyi
Published: October 2004

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of α1‐ or β‐adrenoceptors, a DNA microarray was used.

2. First, the existence of α1‐ and β‐adrenoceptors in A7r5 aortic smooth muscle cells was confirmed by radioligand binding. Then, the inhibitory effects of phenylephrine (an α1‐adrenoceptor agonist) and isoproterenol (a β‐adrenoceptor agonist) on the proliferation of A7r5 cells were determined by [3H]‐thymidine incorporation.

3. The A7r5 cells were treated with 10 µmol/L phenylephrine or 1 µmol/L isoproterenol for 24 h and changes in gene expression were detected with the DNA microarray. Only 14 and 20 genes were identified after treatment of cells with phenylephrine and isoproterenol, respectively, and most genes displayed decreased expression. The changed genes could be grouped into five major functional categories: cell signalling/communication, cell structure/motility, cell/organism defence, gene/protein expression and metabolism. The gene expression profile in response to the activation of α1‐adrenoceptors was very different from that following activation of β‐adrenoceptors. Interestingly, many phenylephrine‐responsive genes were associated with metabolism, whereas many isoproterenol‐responsive genes encoded cell signalling and structure proteins. This means that adrenoceptors may modulate multiple aspects of biological function in vascular smooth muscle cells.

4. Collectively, the activation of both α1‐adrenoceptors (with phenylephrine) and β‐adrenoceptors (with isoproterenol) inhibited the proliferation of A7r5 cells, but microarray data revealed that the mechanisms may be different: the activation of α1‐adrenoceptors could induce the expression of metabolic genes, resulting in the inhibition of proliferation, whereas activation of β‐adrenoceptors altered the expression of genes that encoded cell signalling and structure proteins to inhibit cell proliferation.
T393C polymorphism of GNAS1 associated with the autonomic nervous system in young, healthy Japanese subjects
Authors: Yasuda Koichiro , Matsunaga Tetsuro , Moritani Toshio , Nishikino Mariko , Gu Ning , Yoshinaga Mariko , Nagasumi Kae , Yamamura Tsubasa , Aoki Norihiko , Tsuda Kinsuke
Published: October 2004

1. T393C polymorphism of the gene encoding the Gs‐protein α‐subunit (GNAS1) has been reported recently to be associated with hypertension in which dysfunctions of the autonomic nervous system (ANS) are closely involved. In the present study, the association of this polymorphism with ANS activity was investigated in young, healthy Japanese males.

2. Four hundred and one subjects were genotyped for the T393C polymorphism of GNAS1 by polymerase chain reaction–restriction fragment length polymorphism. Autonomic nervous system activity during supine rest and when standing was assessed in 137 subjects by electrocardiogram R‐R interval power spectral analysis.

3. One hundred and fifty‐four subjects (38.4%) were homozygous for the T allele (TT), 188 (46.9%) were heterozygous (TC) and 59 (14.7%) were homozygous for the C allele (CC). There were no significant differences as to genotype among the clinical characteristics investigated. In power spectral analysis of heart rate variability, the high‐frequency component and parasympathetic nervous system (PNS) index during supine rest were significantly lower in TT and TC carriers than in CC carriers. Furthermore, the increase in heart rate and the responsiveness of sympathetic nervous system index and PNS index to postural change from supine rest to standing were significantly lower in TT and TC carriers than in CC carriers.

4. These observations suggest that the GNAS1 T393C polymorphism is associated with ANS activity in youth, so that it may be useful as a genetic marker for future pathogenesis of hypertension. Follow‐up studies are necessary to clarify the prevalence rates of hypertension among 393T allele carriers in the present study.
Interactions of tachykinin receptor antagonists with lipopolysaccharide‐induced airway inflammation in mice
Authors: Veron M , Guenon I , Nenan S , Emonds‐Alt X , Advenier C , Lagente V , Boichot E
Published: October 2004

1. Several observations suggest that tachykinins are involved in the pathogenesis of bronchopulmonary alterations. We have investigated the effect of antagonists for tachykinin NK1 (SR 140333), NK2 (SR 48968) or NK3 (SR 142801) receptors on inflammatory cell recruitment, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 release and matrix metalloproteinase (MMP)‐9 activity in the bronchoalveolar lavage fluid (BALF) of mice exposed to lipopolysaccharide (LPS; 100 µg/mL aerosol for 30 min).

2. Treatment of mice with a combination of SR 140333 and SR 48968 (10−6 mol/L, aerosol) significantly reduced the increase in the number of total cells and neutrophils and MMP‐9 activity in the BALF of mice 2.5 h after LPS exposure. Treatment with the NK3 antagonist SR 142801 (10−6 mol/L, aerosol) did not inhibit the influx of neutrophils, but markedly reduced the increase in TNF‐α and IL‐6 levels at 2.5 h and MMP‐9 activity at 20 h.

3. These results show that the three tachykinin receptor antagonists may interfere with the development of airway inflammation, namely neutrophilia, TNF‐α release or MMP‐9 activity in the BALF of mice exposed to LPS and suggest that not only NK1 and NK2 receptors, but also NK3 receptors are involved in the modulation of the inflammatory response and airway remodelling.
Different roles of α1‐adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats
Authors: Zhang Yongzhen , Yan Jie , Chen Kai , Song Yao , Lu Zhizhen , Chen Mingzhe , Han Chide , Zhang Youyi
Published: October 2004

1. Three different α1‐adrenoceptor subtypes, designated α1A, α1B and α1D, have been cloned and identified pharmacologically in cardiomyocytes. In vitro studies have suggested that α1‐adrenoceptors play an important role in facilitating cardiac hypertrophy. However, it remains controversial as to which subtype of α1‐adrenoceptors is involved in this response. In the present study, we investigated the different role of each α1‐adrenoceptor subtype in mediating cardiomyocyte protein synthesis, which is a most important characteristic of cardiac hypertrophy in cultured neonatal rat cardiomyocytes.

2. Cardiomyocyte hypertrophy was monitored by the following characteristic phenotypic changes: (i) an increase in protein synthesis; (ii) an increase in total protein content; and (iii) an increase in cardiomyocyte size.

3. The role of each α1‐adrenoceptor subtype in mediating cardiomyocyte protein synthesis was investigated by the effect of specific α1‐adrenoceptor subtype‐selective antagonists on noradrenaline‐induced [3H]‐leucine incorporation. In addition, pKB values for α1‐adrenoceptor subtype‐selective antagonists were calculated and compared with the corresponding pKi values to further identify their effects.

4. Activation of α1‐adrenoceptors by phenylephrine or noradrenaline in the presence of propranolol significantly increased [3H]‐leucine incorporation, protein content and cell size.

5. Pre‐incubating cardiomyocytes with 5‐methyl‐urapidil, RS 17053 or WB 4101 significantly inhibited noradrenaline‐induced [3H]‐leucine incorporation. However, there was no effect when cardiomyocytes were pre‐incubated with BMY 7378. The correlation coefficients between pKB values for α1‐adrenoceptor subtype‐selective antagonists and pKi values obtained from cloned α1A‐, α1B‐ or α1D‐adrenoceptors were 0.92 (P < 0.01), 0.66 (P > 0.05) and 0.24 (P > 0.05), respectively.

6. Our results suggest that the α1‐adrenoceptor is dominantly responsible for adrenergic hypertrophy of cultured cardiomyocytes in neonatal rats. The efficiency in mediating cardiomyocyte protein synthesis is α1A > α1B ≫ α1D.
Management of hepatitis C virus genotype 4
Authors: ABDO AYMAN A , LEE SAMUEL S
Published: October 2004

Hepatitis C virus (HCV) genotype 4 is predominantly found in the Middle East and North Africa. Because most of the large randomized controlled trials of antiviral therapy for chronic hepatitis C were conducted in North America and Europe, little is known about management of patients with this particular genotype. Based on the available data, sustained virological response rates to interferon‐based therapies appear to be intermediate between the relatively resistant HCV genotype 1 and the readily responsive genotypes 2 and 3. Several large prospective studies of pegylated interferon plus ribavirin combination therapy, the current gold‐standard treatment, have recently been completed and will be reviewed.

© 2004 Blackwell Publishing Asia Pty Ltd
Molecular epidemiologic analysis of hepatitis C virus infection in injecting drug users with acute hepatitis C in Japan
Authors: SATOH YASUHIRO , HINO KEISUKE , KATO TAKANOBU , MIZOKAMI MASASHI , YAMASHITA SATOYOSHI , NAKAMURA HIROKI , OKITA KIWAMU
Published: October 2004

Background and Aim:  The aim of this study was to examine whether particular hepatitis C virus (HCV) subtypes are spreading among injecting drug users (IDUs) in Yamaguchi prefecture, on the south‐western tip of the island of Honshu in Japan, as found in European countries.

Methods:  We prospectively enrolled acute hepatitis C patients from January 2001 to March 2003. E2 gene sequences of HCV isolates from IDUs with acute hepatitis C were phylogenetically compared to those from 30 chronic hepatitis C patients with the same HCV subtypes who had or did not have a history of intravenous drug use.

Results:  Nine of 11 patients (82%) with acute hepatitis C were IDUs. The HCV subtypes were 2a in four and 2b in five, which contrasted with the high prevalence of subtype 1b in patients with chronic liver diseases in Japan. IDUs with acute hepatitis C (22.0 ± 2.4 years old) were significantly younger than those with chronic hepatitis C (49.5 ± 9.5 years old) for subtype 2a (P = 0.0005), but not for subtype 2b (25.6 ± 5.4 vs 28.1 ± 2.4 years old). Some HCV isolates of subtype 2b from IDUs with acute hepatitis C were phylogenetically related to those from IDUs with chronic hepatitis C. By contrast, there was no phylogenetic segregation of HCV in IDUs with subtype 2a. HCV isolates from non‐IDUs were genetically divergent from each other and those from IDUs, irrespective of the HCV subtype.

Conclusion:  Hepatitis C virus of the non‐1b subtype, particularly subtype 2b, seemed to be transmitted between IDUs very recently in Yamaguchi prefecture, Japan.
Telomerase reverse transcriptase gene amplification in hepatocellular carcinoma
Authors: TAKUMA YOSHITAKA , NOUSO KAZUHIRO , KOBAYASHI YOSHIYUKI , NAKAMURA SHINICHIRO , TANAKA HIRONORI , MATSUMOTO EIJI , FUJIKAWA TATSUYA , SUZUKI MAYUMI , HANAFUSA TADASHI , SHIRATORI YASUSHI
Published: October 2004

Background and Aim:  Telomerase activation is essential for the immortality of cancer cells. The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, regulates telomerase activity in human cancers. Amplification of the hTERT gene, located at chromosome 5p, is thought to be a potential genetic event contributing to telomerase activation in sporadic tumors.

Methods:  The amplification of the hTERT gene was examined in 46 surgically resected hepatocellular carcinomas (HCC) by real‐time polymerase chain reaction and the status was compared with the expression of hTERT mRNA and clinicopathological parameters.

Results:  Amplified hTERT genes were found in 21.7% (10/46) of HCC. The incidence of amplified hTERT genes in poorly differentiated HCC (6/12, 50%) was significantly higher than that in highly to moderately differentiated HCC (4/34, 11.8%; P = 0.012). Tumor size in those cases with hTERT gene amplification was larger compared to those cases with no amplification (P = 0.047). Amplification of the hTERT gene was not observed in non‐cancerous tissues. The hTERT mRNA level did not correlate with the number of hTERT genes.

Conclusions:  Based on these results, it is thought that hTERT gene amplification is a cancer‐specific event, and may furthermore contribute to the dedifferentiation and development of HCC. However, hTERT gene overexpression was rarely due to an increased hTERT gene copy number in HCC.

© 2004 Blackwell Publishing Asia Pty Ltd
Liver cell transplantation leads to repopulation and functional correction in a mouse model of Wilson's disease
Authors: ALLEN KATRINA J , CHEAH DAPHNE MY , WRIGHT PAUL FA , GAZEAS SOPHIE , PETTIGREW‐BUCK NICOLE E , DEAL YOLANDA H , MERCER JULIAN FB , WILLIAMSON ROBERT
Published: October 2004

Background and Aim:  The toxic milk (tx) mouse is a non‐fatal animal model for the metabolic liver disorder, Wilson's disease. The tx mouse has a mutated gene for a copper‐transporting protein, causing early copper accumulation in the liver and late accumulation in other tissues. The present study investigated the efficacy of liver cell transplantation (LCT) to correct the tx mouse phenotype.

Methods:  Congenic hepatocytes were isolated and intrasplenically transplanted into 3–4‐month‐old tx mice, which were then placed on various copper‐loaded diets to examine its influence on repopulation by transplanted cells. The control animals were age‐matched untransplanted tx mice. Liver repopulation was determined by comparisons of restriction fragment length polymorphism ratios (DNA and mRNA), and copper levels were measured by atomic absorption spectroscopy.

Results:  Repopulation in recipient tx mice was detected in 11 of 25 animals (44%) at 4 months after LCT. Dietary copper loading (whether given before or after LCT, or both) provided no growth advantage for donor cells, with similar repopulation incidences in all copper treatment groups. Overall, liver copper levels were significantly lower in repopulated animals (538 ± 68 µg/g, n = 11) compared to non‐repopulated animals (866 ± 62 µg/g, n = 14) and untreated controls (910 ± 103 µg/g, n = 6; P < 0.05). This effect was also seen in the kidney and spleen. Brain copper levels remained unchanged.

Conclusion:  Transplanted liver cells can proliferate and correct a non‐fatal metabolic liver disease, with some restoration of hepatic copper homeostasis after 4 months leading to reduced copper levels in the liver and extrahepatic tissues, but not in the brain.
Factors contributing to ribavirin‐induced anemia
Authors: NOMURA HIDEYUKI , TANIMOTO HIRONORI , KAJIWARA EIJI , SHIMONO JUNYA , MARUYAMA TOSHIHIRO , YAMASHITA NOBUYUKI , NAGANO MASANORI , HIGASHI MASASHI , MUKAI TAMOTSU , MATSUI YUTAKA , HAYASHI JUN , KASHIWAGI SEIZABURO , ISHIBASHI HIROMI
Published: October 2004

Background and Aim:  Interferon and ribavirin combination therapy for chronic hepatitis C produces hemolytic anemia. This study was conducted to identify the factors contributing to ribavirin‐induced anemia.

Methods:  Eighty‐eight patients with chronic hepatitis C who received interferon‐α‐2b at a dose of 6 MU administered intramuscularly for 24 weeks in combination with ribavirin administered orally at a dose of 600 mg or 800 mg participated in the study. A hemoglobin concentration of <10 g/dL was defined as ribavirin‐induced anemia.

Results:  Ribavirin‐induced anemia occurred in 18 (20.5%) patients during treatment. A 2 g/dL decrease in hemoglobin concentrations in patients with anemia was observed at week 2 after the start of treatment. The hemoglobin concentration in patients with ≥2 g/dL decrease at week 2 was observed to be significantly lower even after week 2 than in patients with <2 g/dL decrease (P < 0.01). A significant relationship was observed between the rate of reduction of hemoglobin concentrations at week 2 and the severity of anemia (P < 0.01). Such factors as sex (female), age (≥60 years old), and the ribavirin dose by body weight (12 mg/kg or more) were significant by univariate analysis.

Conclusions:  Careful administration is necessary in patients ≥60 years old, in female patients, and in patients receiving a ribavirin dose of 12 mg/kg or more. Patients who experience a fall in hemoglobin concentrations of 2 g/dL or more at week 2 after the start of treatment should be monitored with particular care.
Frequency of gastropathy and gastric varices in children with extrahepatic portal venous obstruction treated with sclerotherapy
Authors: PODDAR UJJAL , THAPA BABU RAM , SINGH KARTAR
Published: October 2004

Background:  There are limited reports of the effect of endoscopic sclerotherapy (EST) on portal hypertensive gastropathy (PHG) and gastric varices (GV) in children with extrahepatic portal venous obstruction (EHPVO). We have studied the prevalence of PHG and GV in children with EHPVO and assessed the effect of EST on them on long‐term follow‐up.

Methods:  From January 1992 to June 2002, consecutive children presenting with variceal bleeding due to EHPVO were included in this study. All children underwent EST at presentation and at 2–3 week intervals thereafter. During each session of endoscopy, gastric mucosa and fundus of the stomach was screened carefully to detect PHG and GV. Gastric varices were classified as gastroesophageal (GOV) and isolated gastric varices (IGV).

Results:  In total, 274 cases of EHPVO were managed during the study period. The mean age was 7.4 ± 3.5 years with a male to female ratio of 2.3:1. Of these 274 cases, 186 completed the EST program (study population), 60 were lost to follow‐up, five died and 23 underwent surgery. At presentation (n = 274) 27% cases had PHG (3.6% severe) and 68.6% had GV (GOV 66.8%, IGV 1.8%). Following EST (n = 186) there was a significant (P < 0.001) decrease in GOV (45% from 64%) but an increase in IGV (14% from 1%) and PHG (51.6% from 24.7%).There was also a significant increase in severe PHG (15.6% from 3.2%, P < 0.05). On follow‐up (mean follow‐up 38 ± 30 months) 19% children with IGV bled while none with PHG bled.

Conclusions:  Portal hypertensive gastropathy and gastric varices are quite common in children with EHPVO. Following EST, there is a chance of developing isolated gastric varices.
Gastrointestinal luminal tuberculosis: Establishing the diagnosis
Authors: PATEL NIKHIL , AMARAPURKAR DEEPAK , AGAL SUBHASH , BAIJAL RAJIV , KULSHRESTHA PRAMOD , PRAMANIK SNEHANSU , GUPTE PARIJAT
Published: October 2004

Background and Aim:  To study the profile of gastrointestinal luminal tuberculosis (GITB) patients who have been treated on a confirmed and a presumptive diagnosis.

Methods:  A total of 260 patients who had an initial diagnosis of GITB were included in this retrospective analysis. Clinical, radiologic, endoscopic, histopathologic and microbiologic features of these patients were studied in detail.

Results:  GITB was confirmed in 66.5% patients (cGITB), while 29.5% had presumed GITB (pGITB). In 3.9% patients, Crohn's disease (CD) was misdiagnosed initially as GITB. There was no significant difference in the clinical and radiologic features except a higher incidence of a radiologically abnormal ileocecal region in cGITB patients. Endoscopic biopsies from ulcerated masses and ulcers had the highest yield of confirmation (100% and 68%, respectively) when subjected to histopathology, acid‐fast bacilli smear and culture studies. Confirmed diagnosis was obtained in 100% of cases occurring in the upper gastrointestinal tract, 66% of cases in the ileocecal region/colon and 40% of cases that had small bowel involvement. In 21% of cases, extraluminal sites helped to confirm the diagnosis. As the presence of diarrhea, bleeding, fistulae, perianal disease and extraintestinal manifestations favored a diagnosis of CD, the presence of these features initially or on subsequent follow up helped to minimize the misdiagnosis of GITB.

Conclusions:  As a differential diagnosis, CD must be ruled out before starting treatment for GITB. In our study, an intense search for histologic and microbiologic proof of the presence of TB from luminal and extraluminal sites established the diagnosis in 66.5% of cases. Surgery for establishing the diagnosis should be reserved for complicated cases.
Severe ulcerative colitis: Prospective study of parameters determining outcome
Authors: KUMAR SANJAY , GHOSHAL UDAY C , AGGARWAL RAKESH , SARASWAT VIVEK A , CHOUDHURI GOURDAS
Published: October 2004

Background:  One‐third of patients with exacerbations of ulcerative colitis (UC) require colectomy and 1% die. Although several studies have reported parameters associated with failure of medical treatment, studies using multivariate analysis of various predictors are scanty.

Methods:  Demographic, clinical and laboratory parameters within 48 h of admission of 50 consecutive patients (55 episodes) were prospectively recorded. Each episode was treated with intravenous hydrocortisone, antibiotics and fluid administration. The data of patients who failed to respond, deteriorated, developed complications requiring emergency surgery or died within 7 days of hospitalization, were compared with the data of those who responded during this period using Mann–Whitney U‐test or chi‐squared tests. Variables found to be significant on univariate analysis were entered into a multivariate model.

Results:  Medical treatment failed in 10 of 55 episodes; eight had surgery (one died), another died with bowel perforation and peritonitis and one left hospital in a moribund state. Younger age, pedal edema, transverse colonic diameter >5 cm on abdominal radiograph, low hemoglobin, low serum albumin, high C‐reactive protein, presence of fibrin degradation product (FDP) in serum, low serum fibrinogen, prolonged prothrombin time, Truelove‐Witt's fulminant disease, and high clinical activity index were associated with failure of medical treatment on univariate analysis. All the above variables were significant by multivariate analysis except age, serum albumin level and presence of FDP.

Conclusion:  It may be possible to predict the failure of medical treatment in exacerbations of UC using a scoring system based on clinical parameters and simple laboratory tests.
Normal colon transit time in healthy Chinese adults in Hong Kong
Authors: CHAN YIU‐KAY , KWAN AMBROSE CHI‐PONG , YUEN HON , YEUNG YAT‐WAH , LAI KAM‐CHUEN , WU JUSTIN , WONG GRACE SAU‐WAI , LEUNG CHI‐MAN , CHEUNG WAI‐CHING , WONG CHI‐KIN
Published: October 2004

Background and Aim:  Colon transit study with radio‐opaque markers is helpful in the evaluation of chronic constipation. Normal reference values, based on radio‐opaque markers, have not been reported from Chinese adults in Hong Kong.

Methods:  Fifty‐one healthy Chinese (27 men, age 42 ± 12 years) were enrolled through a newspaper advertisement. They were requested to fill in detailed dietary records and a gastrointestinal symptom questionnaire. Each subject swallowed, under supervision, a total of three Sitzmarks capsules, one capsule at the same time on each of day 1, 2 and 3. Abdominal films were taken on days 4 and 7, also at the same time of the day. Total and segmental transit times were calculated using Metcalf's method.

Results:  The upper limit of normal total colonic transit time (CTT) was 62 h, while right, left and rectosigmoid CTT were 16 h, 31 h and 32 h, respectively. Women of age 18–35 years had a longer rectosigmoid and total CTT compared with their male counterparts (mean total CTT: 39 h vs 18 h, P = 0.05). The CTT appeared to be unaffected by the usual dietary components.

Conclusion:  A single normal reference value for CTT appeared to be applicable to Chinese adults in Hong Kong, irrespective of age, sex and dietary content. However, young Chinese women (age 18–35 years) appeared to have longer total and rectosigmoid CTT.
Changes in population structure of the soilborne fungus Gaeumannomyces graminis var. tritici during continuous wheat cropping
Authors: Lebreton Lionel , Lucas Philippe , Dugas Françoise , Guillerm Anne‐Yvonne , Schoeny Alexandra , Sarniguet Alain
Published: October 2004

A method was developed to assess the genetic structure of Gaeumannomyces graminis var. tritici (Ggt) populations and test the hypothesis of an association between disease level in the field with changes in pathogen populations. A long‐term wheat monoculture experiment, established since 1994, generated different take‐all epidemics with varying the number of wheat crop successions in the 1999–2000 cropping season. Genetic polymorphism in Ggt populations was investigated over natural, local epidemics. Four populations of 30 isolates were isolated from necrotic wheat roots in a first, third, fourth, and sixth wheat crop in the same year. Each Ggt isolate was characterized with RAPD (Random Amplification Polymorphism DNA) markers and AFLP (Amplified Fragment Length Polymorphism) fingerprinting. Seventeen multilocus genotypes based on the combination of RAPD and AFLP markers were identified among all these populations. The 120 isolates were divided into two main groups, G1 and G2, according to bootstrap values higher than 86%, except for an unique isolate from the third wheat crop. Within each group, populations ranged between 93 and 100% similarity. Both groups included isolates collected from the first, third, fourth or sixth wheat crop. However, G1 group profiles dominated amongst isolates sampled in the first and the sixth wheat crops, whereas G2 group profiles largely dominated amongst isolates collected from the third and fourth wheat crops. Aggressiveness of group G2 (38%) was significantly greater than that of G1 (29.5%). These results suggest that changes in Ggt population structure occur during continuous wheat cropping. The distinction of two Ggt groups provides a simple basis for further spatio‐temporal analysis of Ggt population during polyetic take‐all decline.
Design of new promoters and of a dual‐bioreporter based on cross‐activation by the two regulatory proteins XylR and HbpR
Authors: Tropel David , Bähler Alexandra , Globig Kathrin , Van Der Meer Jan Roelof
Published: October 2004

The HbpR protein is the σ54‐dependent transcription activator for 2‐hydroxybiphenyl degradation in Pseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross‐activate the PhbpC and Pu promoters was investigated by determining HbpR‐ or XylR‐mediated luciferase expression and by DNA binding assays. XylR measurably activated the PhbpC promoter in the presence of the effector m‐xylene, both in Escherichia coli and Pseudomonas putida. HbpR weakly stimulated the Pu promoter in E. coli but not in P. azelaica. Poor HbpR‐dependent activation from Pu was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on PhbpC were gradually changed into the XylR binding sites of Pu by site‐directed mutagenesis. Inducible luciferase expression from mutated promoters was tested in E. coli on a two plasmid system, and from mono copy gene fusions in P. azelaica and P. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR‐dependent transcription than from Pu itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild‐type PhbpC, while keeping the same maximal induction level. On the basis of these results, a dual‐responsive bioreporter strain of P. azelaica was created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently with m‐xylene and 2‐hydroxybiphenyl.
Widespread association of a Rickettsiales‐like bacterium with reef‐building corals
Authors: Casas Veronica , Kline David I. , Wegley Linda , Yu Yanan , Breitbart Mya , Rohwer Forest
Published: October 2004

White band disease type I (WBD I) has been a major cause of the dramatic decline of Acroporid coral populations throughout the Caribbean during the last two decades, yet the aetiological agent of this disease is unknown. In this study, the bacterial communities associated with both healthy and diseased Acropora species were compared by 16S rDNA analyses. The bacterial communities of both healthy and diseased Acropora spp. were dominated by a single ribotype with 90% identity to a bacterium in the order Rickettsiales. Screening by nested PCR specific to the coral‐associated Rickettsiales 1 (CAR1) bacterium showed that this microbe was widespread in both healthy and diseased A. cervicornis and A. palmata corals from ‘healthy’ (i.e. low WBD I incidence) and ‘stressed’ reefs (i.e. high WBD I incidence). These results indicate that there were no dramatic changes in the composition of the microbial community associated with WBD I. CAR1 was also associated with non‐Acroporid corals of the Caribbean, as well as with two Acroporid corals native to the Pacific. CAR1 was not present in the water column. This bacterium was also absent from preserved Caribbean Acroporid samples collected between 1937 and 1980 before the outbreak of WBD I. These results suggest CAR1 is a relatively new bacterial associate of Acroporids and that a non‐bacterial pathogen might be the cause of WBD I.