bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomal region 15q21.1 is a frequent target of allelic imbalance in advanced breast carcinomas
Authors: Rhiem Kerstin , Klein Annette , Münch Miriam , Kreutzfeld Rene , Ramser Juliane , Wardelmann Eva , Schackert Gabriele , von Deimling Andreas , Wiestler Otmar D. , Schmutzler Rita K.
Published: June 2003

Allelic imbalance constitutes a major mechanism of genetic aberrations in breast cancer and strongly indicates the involvement of tumor associated genes in the affected chromosomal regions. Preliminary results from our study indicated the existence of a tumor suppressor gene located on chromosomal arm 15q which may be involved in breast cancer progression.1 In the present study, 210 primary breast carcinomas, 30 metastases and 26 local recurrences from primary breast carcinomas have been analyzed with a panel of 18 highly polymorphic microsatellite markers spanning the chromosomal region 15q11‐21.3. Allelic imbalance at 15q with at least 1 marker was seen in 36 of 56 (64.3%) metastases and recurrences, but only in 58 of 210 (27.6%) primary tumors (p<0.0001). We identified a subregion defined by microsatellite marker CYP19 (15q21.1) that showed significantly higher frequencies of allelic imbalance in metastases and recurrences (57.6%) when compared to primary carcinomas (8.9%; p<0.0001). Allelic imbalance at 15q was correlated with histopathologic parameters of the patients with primary breast carcinomas. We detected a significant association with established predictors of poor prognosis, i.e., negative estrogen receptor status (p=0.003), negative progesterone receptor status (p=0.028), high grade (p=0.014) and positive axillary lymph nodes (p=0.013). In summary, our data provide further evidence for a novel prognostic marker in breast carcinomas located in the chromosomal region 15q21. © 2003 Wiley‐Liss, Inc.
Cytotoxicity, apoptosis induction and downregulation of MDR‐1 expression by the anti‐topoisomerase II agent, salvicine, in multidrug‐resistant tumor cells
Authors: Miao Ze‐Hong , Tang Tao , Zhang Yi‐Xiang , Zhang Jin‐Sheng , Ding Jian
Published: June 2003

Salvicine, a novel topoisomerase II inhibitor and a diterpenoid quinone compound, exerts potent in vitro and in vivo antitumor effects. In our study, we show that salvicine effectively kills multidrug‐resistant (MDR) sublines, such as K562/A02, KB/VCR and MCF‐7/ADR, and parental K562, KB and MCF‐7 cell lines to an equivalent degree. These cytotoxic activities of salvicine were much more potent than those of several classical anticancer drugs (average resistance factor: 1.42 for salvicine vs. 344.35, 233.19 and 71.22 for vincristine, doxorubicin and etoposide, respectively). Flow cytometry and DNA agarose gel electrophoresis demonstrated that salvicine induced similar levels of apoptosis in MDR K562/A02 and parental cells. The compound activated caspase‐1 and ‐3 (but not caspase‐8) and increased the ratio of bax to bcl‐2 mRNA via reduction of bcl‐2 mRNA expression in the same cells. Furthermore, salvicine induced the downregulation of mdr‐1 gene and P‐gp expression but had no effect on MRP and LRP gene expression in MDR K562/A02 cells. These results suggest that the reduction of mdr‐1 and bcl‐2 expression by salvicine possibly contributes to its cytotoxicity and apoptotic induction in this system. The effectiveness, broad‐spectrum activity and possibly novel mechanism of killing MDR tumor cells in vitro of salvicine signify promising in vivo and clinical activity. The novel chemical structure of this compound further implies a role for salvicine in future MDR tumor therapy. © 2003 Wiley‐Liss, Inc.
Role of hMLH1 promoter hypermethylation in drug resistance to 5‐fluorouracil in colorectal cancer cell lines
Authors: Arnold Christian N. , Goel Ajay , Boland C. Richard
Published: June 2003

Loss of DNA mismatch repair (MMR) occurs in 10–15% of sporadic colorectal cancer, is usually caused by hMLH1 hypermethylation, and has been shown to confer resistance to various chemotherapeutic reagents, including 5‐fluorouracil (5‐FU). We tested the hypothesis that demethylation of the hMLH1 promoter in hypermethylated colorectal cancer cells would restore MMR proficiency and drug sensitivity to 5‐FU. We used the MMR‐deficient cell lines SW48, HCT116, HCT116+chr2 and the ‐proficient cell line HCT116+chr3. After treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (5 aza‐dC), hMLH1 mRNA and protein expression were determined by RT‐PCR and immunoblots. The methylation status for hMLH1 was investigated by methylation‐specific PCR. Cells were subsequently treated with 5‐FU and the growth characteristics ascertained by clonogenic assays. hMLH1 hypermethylation was reverted in SW48 cells 24 hr after treatment with 5 aza‐dC and was accompanied by hMLH1 mRNA and protein reexpression. While 5 aza‐dC alone did not affect the growth of SW48 cells, all other cell lines responded with a pronounced growth inhibition. 5‐FU treatment strongly reduced the colony formation of HCT116+chr3 cells. These effects were significantly less in the MMR‐deficient cells. Combined treatment of SW48 cells resulted in a similar growth pattern as seen in 5‐FU only treated HCT116+chr3 cells. We demonstrate that in vitro resistance to 5‐FU can be overcome by reexpression of hMLH1 protein through 5 aza‐dC‐induced demethylation in hypermethylated cell lines. Induction of the expression of methylated tumor suppressor or MMR genes could have a significant impact on the development of future chemotherapy strategies. © 2003 Wiley‐Liss, Inc.
Prospective study of screening for stomach cancer in Japan
Authors: Mizoue Tetsuya , Yoshimura Takesumi , Tokui Noritaka , Hoshiyama Yoshiharu , Yatsuya Hiroshi , Sakata Kiyomi , Kondo Takaaki , Kikuchi Shogo , Toyoshima Hideaki , Hayakawa Norihiko , Tamakoshi Akiko , Ohno Yoshiyuki , Fujino Yoshihisa , Kaneko Satoshi ,   the Japan Collaborative Cohort Study Group
Published: June 2003

Although screening for stomach cancer is a widespread community service in Japan, the benefits of the screening program remain unclear. Our study investigated prospectively the relation between participation in stomach‐cancer screening during the past 12 months and subsequent deaths. Data was derived from the Japan Collaborative Cohort Study, in which 480 stomach‐cancer deaths were identified during an 8‐year follow‐up period. Cox proportional hazard regression was used to estimate the relative risk of death from stomach cancer and that from other causes while adjusting for potential confounding factors. In men, screening participation was associated significantly with a reduced risk of death from stomach cancer (relative risk [RR] = 0.54; 95% confidence interval [CI] = 0.41–0.70). The extent of the risk reduction was greater than potential health selection (for deaths other than stomach, RR = 0.71). The adjustment for potential confounding variables, however, attenuated the difference in risk of death (stomach cancer, RR = 0.65; other causes, RR = 0.71). In women, the magnitude of the association between screening participation and death from stomach cancer (RR = 0.74; 95% CI = 0.52–1.07) was equal to that for deaths from non‐stomach cancers (RR = 0.74). Subgroup analysis showed that women with a parental history of stomach cancer had a reduced risk of death from stomach cancer associated with screening (RR = 0.32; 95% CI = 0.12–0.87). The present results underline the potential for selection bias in observational studies, and thus it remains an open question whether screening for stomach cancer is effective. © 2003 Wiley‐Liss, Inc.
RASSF1A gene inactivation in non‐small cell lung cancer and its clinical implication
Authors: Endoh Hideki , Yatabe Yasushi , Shimizu Shigeki , Tajima Kohei , Kuwano Hiroyuki , Takahashi Takashi , Mitsudomi Tetsuya
Published: June 2003

Methylation‐associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, is reported in several cancers. We examined RASSF1 in non‐small lung cancer (NSCLC) to search for clinical implications. RT‐PCR analysis showed no expression of RASSF1A in 12 of 20 lung cancer cell lines. Loss of expression correlated well with promoter methylation status of these lines. Sequence analysis revealed 2 polymorphisms (codons 21 and 133) in RASSF1A transcripts, but not in RASSF1C transcripts. No somatic mutations were found. Of 7 cell lines with K‐ras mutations at codon 12 or 61, 2 lost expression of RASSF1A, whereas in 13 cell lines with wild‐type K‐ras gene, 10 lost RASSF1A gene expression (p = 0.0521). We investigated methylation status of this putative tumor suppressor gene in 100 primary NSCLCs to determine whether there is a clinical significance. Forty‐two of primary NSCLCs demonstrated methylated allele. There is no correlation between promoter methylation of RASSF1A and clinicopathological findings, including histological type or grade, tumor staging, p53 and K‐ras mutational status, or patients' survival. In the cases of Stage I and II disease, however, RASSF1A methylation was associated with earlier recurrence (p = 0.0247). Epigenetic silencing of RASSF1A is a frequent event in non‐small lung cancer and will provide novel opportunities to develop diagnosis and therapy of NSCLC. © 2003 Wiley‐Liss, Inc.
Signaling pathway for aloe‐emodin‐induced apoptosis in human H460 lung nonsmall carcinoma cell
Authors: Yeh Feng‐Tsgh , Wu Chun‐Hsiung , Lee Hong‐Zin
Published: June 2003

Aloe‐emodin (1,8‐dihydroxy‐3‐(hydroxymethyl)‐anthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumor effects through an unknown mechanism. Our study investigated the mechanisms of aloe‐emodin‐induced cell death in the human lung nonsmall cell carcinoma cell line H460. Aloe‐emodin (40 μM)‐induced apoptosis of H460 cells involves modulation of cAMP‐dependent protein kinase, protein kinase C, Bcl‐2, caspase‐3 and p38 protein expression. The relationship of various signals involved in cell death, such as cAMP‐dependent protein kinase, protein kinase C, Bcl‐2, caspase‐3 and p38, has been investigated in the regulation of apoptotic cell death of aloe‐emodin. We demonstrated that the expression of p38 is an important determinant of apoptotic death induced by aloe‐emodin. © 2003 Wiley‐Liss, Inc.
Effect of family history, obesity and exercise on breast cancer risk among postmenopausal women
Authors: Carpenter Catherine L. , Ross Ronald K. , Paganini‐Hill Annlia , Bernstein Leslie
Published: June 2003

We examined effects of obesity and lifetime exercise patterns on postmenopausal breast cancer risk according to family history in a large population‐based case control study conducted in Los Angeles County, California, because we hypothesized that both factors would affect risk through similar mechanistic pathways, and that their effects would be stronger among women with a family history. We studied 1,883 postmenopausal breast cancer case subjects and 1,628 postmenopausal control subjects ranging in age from 55–72 years. Cases were diagnosed with incident breast cancer in the late 1980s and 1990s. Controls were individually matched to case subjects on age, ethnic origin and neighborhood. In‐person interviews determined known breast cancer risk factors including: height, weight, lifetime exercise, and family history of breast and other cancers. Breast cancer risk was raised among women who had at least 1 first‐degree relative with breast cancer (odds ratio [OR] = 1.68; 95% confidence interval [CI] = 1.36–2.08). Risk increased with increasing levels of body‐mass index (wt‐kg/ht‐m2) (p‐trend = 0.005). Breast cancer risk was reduced among women who maintained, on average, 17.6 metabolic equivalent of energy expenditure (MET)‐hr of activity/week from menarche onward (OR = 0.66; 95% CI = 0.48–0.90). Body‐mass index, adjusted for lifetime exercise, was strongly associated with breast cancer risk among women with a positive family history of breast cancer (p‐trend < 0.0001), but only weakly associated among women with no family history (p‐trend = 0.08; homogeneity of trends p = 0.0005). In contrast, the risk reduction associated with exercise activity, adjusting for body‐mass index, was limited to women without a family history of breast cancer (p‐trend = 0.001; homogeneity of trends p = 0.005). Body‐mass index and exercise activity, both modifiable risk factors for breast cancer, seem to have differential effects depending on a woman's family history of breast cancer, and may impact risk through different biological mechanisms. © 2003 Wiley‐Liss, Inc.
Expression profiles of angiogenic growth factors in squamous cell carcinomas of the head and neck
Authors: Ninck Simon , Reisser Christoph , Dyckhoff Gerhard , Helmke Burkhard , Bauer Harald , Herold‐Mende Christel
Published: June 2003

Inhibition of angiogenesis by blocking angiogenic cytokines or their pathways has become a major target in experimental cancer therapies. This therapeutical approach requires a profound knowledge of growth factor profiles that contribute to tumor growth and progression. The respective knowledge is presently rather incomplete for head and neck squamous cell carcinomas (HNSCC). Therefore we studied expression of several angiogenic cytokines including VEGF, bFGF, PDGF‐AB, PDGF‐BB, G‐CSF and GM‐CSF in HNSCC in vivo and in vitro. In tumor tissues expression of all cytokines was observed albeit with marked differences concerning intensity and distribution pattern. Quantification of the cytokines in the supernatant of 15 tissue‐corresponding HNSCC cultures revealed that VEGF, PDGF‐AB and less frequently GM‐CSF were secreted in high amounts of up to 13 ng/ml/106 cells. Twenty percent of the HNSCC cultures expressed only 1 cytokine in biologically active amounts, 60% 2 or 3 and 20% expressed the maximum of 4 cytokines simultaneously. Interestingly, we observed a distinct cytokine pattern: HNSCC cells secreting only 1 or 2 cytokines presented always with either VEGF and/or PDGF‐AB, while G‐CSF and GM‐CSF were secreted primarily together with VEGF and PDGF‐AB. The number of cytokines expressed by HNSCC cells correlated with the microvessel density of the original tumor and with the clinical outcome: tumors producing at least 3 cytokines revealed a significantly poorer patient prognosis. Our data indicate a major role for VEGF and PDGF‐AB in HNSCC and that the additional secretion of G‐CSF or GM‐CSF might contribute to a poorer prognosis in patients suffering from these tumors. © 2003 Wiley‐Liss, Inc.
Circulating levels of insulin‐like growth factor I, its binding proteins ‐1,‐2, ‐3, C‐peptide and risk of postmenopausal breast cancer
Authors: Keinan‐Boker Lital , Bueno de Mesquita H. Bas , Kaaks Rudolf , van Gils Carla H. , van Noord Paul A.H. , Rinaldi Sabina , Riboli Elio , Seidell Jaap C. , Grobbee Diederick E. , Peeters Petra H.M.
Published: June 2003

Higher levels of circulating Insulin‐like Growth Factor (IGF)‐I may be associated with higher risks for premenopausal breast cancer. We investigate the associations between circulating levels of IGF‐I, its binding proteins (IGFBPs) ‐1, ‐2, ‐3, C‐peptide and postmenopausal breast cancer. This is a prospective study nested in 2 Dutch cohorts. The study population included women who were postmenopausal at baseline. Breast cancer cases were identified through linkage with cancer registries. Controls were matched to cases by cohort, age, date of blood donation and place of residence. In total, 149 breast cancer cases and 333 healthy controls were included. Plasma levels of IGF‐I, IGFBP‐1, ‐2, ‐3 and C‐peptide were measured by radioimmunoassays. Estimates of the relative risk for breast cancer associated with the quartiles of the peptides' circulating levels were obtained by conditional logistic regression. Models were adjusted for BMI, age at menarche and age at first full‐term delivery. For IGF‐I, the adjusted OR (95% CI) of the top vs. bottom quartile was 1.1 (0.6; 2.1); for IGFBP‐1 it was 0.7 (0.3; 1.3); for IGFBP‐2, 1.1 (0.5; 2.4); for IGFBP‐3, 1.6 (0.7; 3.5), for C‐peptide, 1.3 (0.7; 2.7) and for IGF‐I/IGFBP‐3 ratio, 1.0 (0.5; 1.8). Our data do not support an association between postmenopausal circulating levels of IGF‐I, IGFBP‐1, ‐2, ‐3, C‐peptide and postmenopausal breast cancer. © 2003 Wiley‐Liss, Inc.
Target selection for antisense oligonucleotide induced exon skipping in the dystrophin gene
Authors: Errington Stephen J. , Mann Christopher J. , Fletcher Sue , Wilton Stephen D.
Published: June 2003

Background

Duchenne muscular dystrophy (DMD) is an X‐linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re‐direct dystrophin pre‐mRNA processing by blocking sequences crucial to pre‐mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons.

Methods

Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre‐mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping.

Results

Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments.

Conclusions

Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre‐mRNA. Copyright © 2003 John Wiley & Sons, Ltd.
Sustained tetracycline‐regulated transgene expression in vivo in rat retinal ganglion cells using a single type 2 adeno‐associated viral vector
Authors: Folliot Sebastien , Briot Delphine , Conrath Hervé , Provost Nathalie , Cherel Yan , Moullier Philippe , Rolling Fabienne
Published: June 2003

Background

Viral vector delivery of neurotrophic‐expressing transgenes in the retina may retard or prevent the onset of blindness associated with photoreceptor degeneration. A key safety issue is to achieve regulated expression of these genes in the retina. The purpose of our study was to evaluate whether a single recombinant AAV‐2 (rAAV) encoding for a tetracycline (Tet)‐regulated destabilized reporter gene could provide quantitative profiles of gene regulation targeted to the rat neuroretina.

Methods

A rAAV vector carrying a destabilized green fluorescent protein (dgfp) under a tet‐regulatable promoter and the tetracycline‐repressed transactivator (tTA) was generated (rAAVtetoff.dgfp) and administered intravitreally in nine Wistar rats. Retinas were monitored for 6 months using noninvasive fluorescence imaging and the animals were subjected to two cycles of doxycycline (Dox), a tetracycline analog. Eyes were ultimately examined by histology.

Results

Intravitreal injection of rAAVtetoff.dgfp resulted in effective transduction of ganglion cells. Following full expression of the transgene in the absence of Dox, 95% of the GFP signal was shut down 48 h post Dox administration and the signal was undetectable 7 days later. Initial levels of GFP expression were restored 21 days after Dox administration ceased. This pattern of expression was repeated twice over a period of 6 months.

Conclusions

This report demonstrates that rAAVtetoff.dgfp intravitreally injected rats displayed tight and sustained long‐term regulation of the reporter gene in ganglion cells. These findings may have important implications regarding rAAV‐mediated gene therapy using neuroprotective approaches for retinitis pigmentosa and glaucoma. Copyright © 2003 John Wiley & Sons, Ltd.
Sustained transgene expression in cartilage defects in vivo after transplantation of articular chondrocytes modified by lipid‐mediated gene transfer in a gel suspension delivery system
Authors: Madry Henning , Cucchiarini Magali , Stein Ute , Remberger Klaus , Menger Michael D. , Kohn Dieter , Trippel Stephen B.
Published: June 2003

Background

Genetically modified chondrocytes may be able to modulate articular cartilage repair. To date, transplantation of modified chondrocytes into cartilage defects has been restricted to viral vectors. We tested the hypothesis that a recombinant gene can be delivered to sites of cartilage damage in vivo using chondrocytes transfected by a lipid‐mediated gene transfer method.

Methods

Isolated lapine articular chondrocytes were transfected with an expression plasmid vector carrying the P. pyralis luciferase gene using the reagent FuGENE 6. Transfected chondrocytes were encapsulated in alginate spheres and implanted into osteochondral defects in the knee joints of rabbits.

Results

In vitro, luciferase activity in pCMVLuc‐transfected spheres showed an early peak at day 2 post‐transfection and remained elevated at day 32, the longest time point evaluated. The number of viable chondrocytes in non‐transfected and transfected spheres increased over the period of cultivation. In vivo, luciferase activity was maximal at day 5 post‐transfection, declined by day 16, but was still present at day 32. On histological analysis, the alginate‐chondrocyte spheres filled the cartilage defects and were surrounded by a fibrous repair tissue composed of spindle‐shaped cells.

Conclusions

These data demonstrate the successful introduction of articular chondrocytes modified by lipid‐mediated gene transfer in a gel suspension delivery system into osteochondral defects and the sustained expression of the transgene in vivo. This method may be used to define the effects of genes involved in cartilage repair and may provide alternative treatments for articular cartilage defects. Copyright © 2003 John Wiley & Sons, Ltd.
Augmented antitumor activity of a secondary lymphoid‐tissue chemokine (SLC)‐interleukin (IL) 2 fusion protein in mouse
Authors: Nakahara Koichiro , Sakata Tsuneaki
Published: June 2003

Background

To enhance the antitumor efficacy of IL2 gene therapy, combinations of several other genes, such as p53, a tumor suppressor gene, or lymphotactin, a C‐chemokine, and the IL2 gene are attempted, and synergistic effects are observed. We report here on the enhanced antitumor activity of a fusion protein (mSLC‐IL2) comprised of a newly identified member of the CC‐chemokine family, mouse SLC (mSLC), and mouse IL2 (mIL2).

Methods

We constructed mSLC‐IL2 by connecting the N‐terminus of mIL‐2 to the C‐terminus of mSLC using a two‐amino‐acid linker. The resultant fusion protein retained both mIL2 activity, as measured in a standard proliferation assay using a mouse IL‐2 dependent cell line, and chemokine activity, as measured in a chemotaxis assay using a preB cell line expressing mSLC‐specific receptor, CCR7. The gene encoding mSLC‐IL2 was retrovirally transduced into fibroblast CL.7 cells, derived from Balb/c mice.

Results

Intradermal transplantation of fibroblasts expressing mSLC‐IL2 into syngenic mice induced a dense accumulation of CD4+ and CD8+ cells at the sites of transplantation. Moreover, when CT‐26 cells, derived from colon adenocarcinoma cells, were co‐transplanted with mSLC‐IL2‐transduced fibroblasts, the CT‐26 cell exhibited significantly lower tumorigenicity than CT‐26 cells co‐transplanted with mIL2‐transduced fibroblasts.

Conclusions

These findings, obtained from both in vitro and in vivo data, suggest that the gene encoding mSLC‐IL2 may be a good candidate for inclusion as part of an anticancer gene therapy protocol. Copyright © 2003 John Wiley & Sons, Ltd.
Electroporation‐mediated ex vivo gene transfer into graft not requiring injection pressure in orthotopic liver transplantation
Authors: Kobayashi Shogo , Dono Keizo , Takahara Shiro , Isaka Yoshitaka , Imai Enyu , Zhenhui Lu , Nagano Hiroaki , Tomoaki Kato , Umeshita Koji , Nakamori Shoji , Sakon Masato , Monden Morito
Published: June 2003

Background

We investigated optimum conditions for ex vivo gene transfer into liver grafts by plasmid injection via the portal vein combined with electroporation in rat liver transplantation.

Methods

Anesthetized 9‐week‐old male Shionogi‐Wistar rats were used as donors and recipients. After harvest of the liver graft from the donor rat, a tapered 3Fr. catheter was inserted into the portal vein of the liver graft ex vivo. After clamping the afferent vessels around the right and caudal liver lobes, pCAGGS‐luciferase, which was diluted with one of several osmotic pressure solutions, or pCAGGS‐green fluorescence protein (GFP) plasmid was injected into these lobes to keep the efferent vessels patent. Electrical pulses were applied to the liver graft during cold preservation in lactated Ringer's solution, University of Wisconsin solution, and histidine‐tryptophan‐ketoglutarate solution.

Results

Transfection efficacy was estimated by measurement of luciferase activity. Luciferase activity in the liver was dependent on both the voltage and electric current of the electrical pulse, and also on the type of preservation solution and plasmid osmotic pressure. Luciferase activity was noted only in plasmid‐injected lobes of the liver graft. GFP‐transfected cells were identified by GFP fluorescence. GFP was observed predominantly in perivascular cells, including hepatocytes.

Conclusions

We have demonstrated successful ex vivo gene transfection into liver grafts without injection pressure by using a non‐viral method. Copyright © 2003 John Wiley & Sons, Ltd.
Effects of adenovirus‐mediated SV5 fusogenic glycoprotein expression on tumor cells
Authors: Gómez‐Treviño A. , Castel S. , López‐Iglesias C. , Cortadellas N. , Comas‐Riu J. , Mercadé E.
Published: June 2003

Background

The fusogenic (F) membrane glycoprotein of the paramyxovirus SV5 allows virus to enter host cells and mediates fusion between neighboring cells, which leads to cell death. F glycoprotein is synthesized as an inactive precursor (F0) that is cleaved by cellular protease furine to form the active heterodimer F1 + F2. The active protein can induce syncytium formation in the absence of another integral glycoprotein (HN), a property that appears to be unique among paramyxoviruses.

Methodology

We constructed a non‐replicative adenovirus to express SV5 F protein in tumor cells, and its fusion capacity was analyzed by fluorescent and confocal microscopy. Cell viability and bystander effect were compared with the thymidine kinase/ganciclovir suicide gene therapy. The structure of F‐expressing cells was studied using electron microscopy.

Results

F glycoprotein expression induced syncytium formation to a maximum at 72 h, after which syncytia progressively lost viability and detached. The cell membrane was disrupted while nuclear structure was preserved. Over‐expression of SV5 F protein in tumor cells led to high cytotoxicity comparable with that associated with the thymidine kinase/ganciclovir. A potent bystander killing effect was detected until the ratio of F‐transduced to non‐transduced cells was 1 : 100.

Conclusions

These results indicate that the fusogenic glycoprotein of the paramyxovirus SV5 could be used to eliminate tumor cells and may encourage studies aimed at modifying its selectivity and combining its expression with other cytotoxic strategies to improve their efficacy. Copyright © 2003 John Wiley & Sons, Ltd.
Aspartylglucosaminidase (AGA) is efficiently produced and endocytosed by glial cells: implication for the therapy of a lysosomal storage disorder
Authors: Harkke Salli , Laine Minna , Jalanko Anu
Published: June 2003

Background

Aspartylglucosaminuria (AGU) represents diseases affecting the central nervous system and is caused by a deficiency of a lysosomal enzyme, aspartylglucosaminidase (AGA). AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose‐6‐phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies.

Methods

Adenoviruses containing tissue‐specific glial fibrillary acidic protein (GFAP) promoter and neuron‐specific enolase (NSE) promoter were generated to target expression of AGA in Aga‐deficient mouse primary glial and neuronal cell cultures. In addition an endogenous AGA promoter was used. The experimental design was planned to measure the enzymatic activities in the cells and media of neurons and glia infected with each specific virus. The endocytosis of AGA was analyzed by incubating neuronal and glial cells with media produced by each virus‐cell combination.

Results

AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell‐specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA.

Conclusions

These data indicate the importance of glial cells in the expression and transport of AGA. Subsequently, new approaches can be developed for therapeutic intervention. Copyright © 2003 John Wiley & Sons, Ltd.
Antisense downregulation of mutant huntingtin in a cell model
Authors: Hasholt Lis , Abell Kathrine , Nørremølle Anne , Nellemann Christine , Fenger Kirsten , Sørensen Sven Asger
Published: June 2003

Background

Huntington's disease (HD) is an inherited neurodegenerative disorder which is caused by an expansion of a CAG repeat sequence in the HD gene. The repeat encodes an expanded polyglutamine tract in the protein huntingtin. The still unknown pathological mechanisms leading to death of specific neurons in the brains of HD patients correlate with the expression of mutant huntingtin. Therefore, we have studied whether mutant huntingtin expression can be downregulated by antisense technique.

Methods

NT2 precursor cells and differentiated postmitotic NT2‐N neurons, respectively, were transfected with plasmid constructs containing exon 1 of the HD gene with expanded CAG repeats in frame with the reporter protein EGFP. The transfected cell cultures were treated with a phosphorothioated antisense oligonucleotide (PS‐ASHD/20+) or a control oligonucleotide either by cotransfection or by addition to the culture medium.

Results

Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2‐N neurons. We obtained antisense sequence‐specific inhibition of expression of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo.

Conclusions

The PS‐antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems. Copyright © 2003 John Wiley & Sons, Ltd.
Adenovirus endocytosis
Authors: Meier Oliver , Greber Urs F.
Published: June 2003

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin‐mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR‐docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin‐mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F‐actin, protein kinase C and small G‐proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high‐affinity Fcγ receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest. Copyright © 2003 John Wiley & Sons, Ltd.
Immunological recovery despite virological failure is independent of human immunodeficiency virus‐type 1 resistant mutants in children receiving highly active antiretroviral therapy
Authors: Chiappini Elena , Galli Luisa , Zazzi Maurizio , de Martino Maurizio
Published: June 2003

Relationships between human immunodeficiency virus‐type‐1 (HIV‐1) drug‐resistant mutants and immunological recovery were investigated after 12‐week antiretroviral therapy in 43 children infected perinatally with virological failure. Twenty‐two children received highly active antiretroviral therapy (HAART) and 21 received double therapy with reverse transcriptase inhibitors. At baseline, no difference in reverse transcriptase or protease inhibitors resistant mutants was present among the groups. After 12 weeks, the two groups were similar regarding proportion of children with reverse transcriptase resistant mutants. Sixteen (73%) HAART‐treated children, but no child receiving double therapy had HIV‐1 with primary resistance mutations to protease inhibitors. Secondary protease mutations were found in all HAART‐treated and in 17/21 (81%) children receiving double therapy. The mutation numbers in reverse transcriptase or protease genes were significantly higher after HAART than after double therapy. Nevertheless, 12 (55%) of HAART‐treated children (but no child receiving double therapy) showed immunological recovery. The frequency and number of mutations were similar in HAART‐treated children with or without immunological recovery both at baseline and after 12 weeks. The findings suggest, immunological recovery notwithstanding, virological failure is independent of drug‐resistant mutations and consequent possible changes in viral fitness. J. Med. Virol. 70:506–512, 2003. © 2003 Wiley‐Liss, Inc.
Molecular epidemiology and transmission of hepatitis B virus in close family contacts of HBV‐related chronic liver disease patients
Authors: Thakur Varsha , Kazim Seyed N. , Guptan Rajkumar C. , Malhotra Veena , Sarin Shiv K.
Published: June 2003

There is limited data on the patterns of HBV mutation in family contacts of chronic liver disease (CLD) patients in India. DNA sequence analysis is an important tool to study this viral epidemiology. Transmission and prevalence of mutations in the S and pre‐C gene region in HBV infected close family contacts of HBV‐related CLD patients were studied. Twelve HBsAg+ index patients and their 20 HBV DNA contacts were studied in detail. The S ORF and the pre‐C region were sequenced using direct PCR products. S‐gene sequencing included 32 specimens (12 index cases and all 20 contacts). Pre‐C gene sequencing included 26 specimens (12 index cases and all the 14 HBsAg+ contacts irrespective of their HBeAg status). More than 98% sequence homology was found between the index patients and their contacts. The in‐depth study of 12 families revealed that the transmission pattern was primarily horizontal in 6 (50%) and vertical in 2 (17%) families (P < 0.05). The remaining four families had evidence of both horizontal and vertical transmission. Mutations in the S‐gene were found in 80% of HBsAg+ and 17% HBsAg− subjects (P < 0.05). A total of 22‐point mutations at different nucleotide positions were found. In these, 16 (72%) were mutation of the “a” determinant region and 14 (64%) resulted in missense mutations. The commonest S‐gene mutations were T118V and A128V, present in 44 and 38% specimens, respectively. T143M and G145R mutations in the second loop of the “a” determinant were found in 9% of the specimens. Novel mutations, C137stp and C138stp were found in only one HBsAg− subject. Mutations in the pre‐C gene were common (91%) in patients with HBeAg− phenotype. G1896A mutation was found in 7 of 11 (64%) specimens changing amino acid tryptophane (W) to stop codon. Other mutations were at codons 25 and 29. The results of the study, demonstrate (1) clustering of Pre‐C and S‐gene mutations in the families, (2) horizontal mode of transmission and a common source infection appears to be frequent as evidenced by sequence homology and detailed history, (3) T118V and A128V were the commonest mutations in the S‐gene region, while (4) M2 (G1896A) was the commonest pre‐C gene mutation, and (5) long‐term follow‐up evaluation of these mutations suggested. J. Med. Virol. 70:520–528, 2003. © 2003 Wiley‐Liss, Inc.
Genotypes and phylogenetic characterization of hepatitis B and delta viruses in Egypt
Authors: Saudy Niveen , Sugauchi Fuminaka , Tanaka Yasuhito , Suzuki Seiji , Aal Amina Abdel , Zaid Mostafa Abo , Agha Salah , Mizokami Masashi
Published: June 2003

Hepatitis B virus (HBV) and hepatitis D virus (HDV) sequences among HBV carriers from Egypt have not been evaluated sufficiently. The genotypes of HBV isolated from 105 serum samples from Egyptian carriers were determined. Four complete genomes and 11 entire preS1/S2/S genes were sequenced and evaluated. All serum samples were classified into HBV genotype D using serologic and genetic methods. The length of four complete nucleotide sequences was 3,182 bp. In all 15 samples, the common 33 nucleotides (11 amino acids) deletions in the preS1 region specific for HBV genotype D were observed. In the phylogenetic analysis based on the complete nucleotide sequences, all samples were clustered with the HBV isolates reported from previously Western and Mediterranean countries with nucleotide homology ranging from 96.0–98.0%. Of 75 HBsAg positive samples, anti‐HDV was found in 15 (20%), and HDV RNA was detected in 9 of 15 (60%). The proportion of the patients with liver disease was higher in HBV carriers of anti‐HDV positive with HDV RNA than in HBV carriers of anti‐HDV positive without HDV RNA (P < 0.05). In the phylogenetic analysis based on the sequences in nucleotide position 853–1267 of HDV, nine samples were classified into HDV genotype I with the nucleotide homology ranging from 88.3–92.1% (mean; 90.5%) and clustered with HDV strains reported previously from Ethiopia, Somalia, Egypt, and Lebanon. These results indicate that HBV genotype D and HDV genotype I are most prevalent in Egypt, and HDV co‐infection in HBV carriers is related to severity of liver disease. J. Med. Virol. 70:529–536, 2003. © 2003 Wiley‐Liss, Inc.
A case‐control study for early prediction of hepatitis B e antigen seroconversion by hepatitis B virus DNA levels and mutations in the precore region and core promoter
Authors: Yamaura Takahiro , Tanaka Eiji , Matsumoto Akihiro , Rokuhara Akinori , Orii Koji , Yoshizawa Kaname , Miyakawa Yuzo , Kiyosawa Kendo
Published: June 2003

Factors influencing and predictive of seroconversion from hepatitis B e antigen (HBeAg) to antibody (anti‐HBe) were sought in a case‐control study of 61 patients with chronic hepatitis B who had been observed from 5 years before to 1 year after seroconversion, and 32 patients who did not seroconvert during the entire 6‐year period. Almost all of the patients (96%) were infected with HBV genotype C. HBV DNA levels began to decrease 3 years before seroconversion in the seroconverters, while they remained high in the non‐converters. The frequency of precore mutation and the loss of HBeAg (A1896) started to increase 1 year before in the converters, and became significantly higher at seroconversion (23 vs. 3%, P = 0.030) than that in the non‐converters. Double mutation in the core promoter (T1762/A1764) was more common in the seroconverters than in the non‐converters 5 years before seroconversion (48 vs. 28%), and became significantly more frequent at seroconversion (65 vs. 41%, P = 0.046). Seroconversion occurred in 75% of the patients with at least HBV DNA levels <5.5 logarithmic equivalents/mL; precore mutation in 20% or more of HBV DNA; or core promoter mutation. Seroconversion occurred in 50% of those patients within 1 year, 88% within 2 years, and 93% within 5 years. These results indicate that a decrease in HBV DNA levels and mutations in the precore region and the core promoter were associated significantly and complementarily with seroconversion, and each of them or a combination thereof was predictive of seroconversion years ahead. J. Med. Virol. 70:545–552, 2003. © 2003 Wiley‐Liss, Inc.
Hepatitis B virus reactivation in breast cancer patients receiving cytotoxic chemotherapy: A prospective study
Authors: Yeo Winnie , Chan Paul K.S. , Hui Pun , Ho Wing M. , Lam Kwok C. , Kwan Wing H. , Zhong Sheng , Johnson Philip J.
Published: June 2003

Breast cancer is a rapidly increasing problem in many developing countries, and cytotoxic chemotherapy is now an integral part of its management. In several developing countries, the carriage of hepatitis B virus (HBV) in cancer patients may be as high as 12%, and such patients are at risk of developing fatal HBV reactivation during chemotherapy. HBV reactivation is well recognized in patients with hematological malignancies, but limited data are available on patients with other, more common, cancers, such as breast cancer. Recent data have suggested that increased viral replication, an indication of HBV reactivation, may precede clinical hepatitis. In the absence of serial HBV DNA monitoring, HBV reactivation during chemotherapy may have been underestimated. In this prospective study, breast cancer patients who were hepatitis B surface antigen (HBsAg) seropositive were followed up during chemotherapy. The main objectives were to determine the incidence of HBV reactivation in breast cancer patients undergoing conventional chemotherapy; to investigate whether “serial HBV DNA monitoring” improves the accuracy of diagnosing HBV reactivation when compared with previous schema that only measured HBV DNA at the time of clinical hepatitis (“conventional monitoring”); and to assess the clinical consequences as a result of developing the condition. The secondary objective was to identify risk factors associated with this condition. Over an 18‐month period, 41 patients were studied. Ten developed HBV reactivation by conventional monitoring criteria, but with serial HBV DNA monitoring, seven additional patients were diagnosed when increased HBV DNA levels were detected before, but not concomitant with, clinical hepatitis. Thus, a total of 17 patients (41%) developed HBV reactivation. Premature termination of chemotherapy or delay in treatment schedules occurred in 71% of the patients who developed viral reactivation, as compared with 33% in those who did not develop the condition (P = 0.019). No risk factors associated with the development of HBV reactivation could be identified. Serial monitoring of HBV DNA, in addition to liver function, increases the sensitivity of diagnosing of HBV reactivation, and helps explain some cases that would otherwise be labeled as “cryptogenic hepatitis,” for which concomitant HBV DNA measured at the time of hepatitis was undetectable. The present study highlights the importance of monitoring HBsAg‐seropositive patients who are receiving chemotherapy for common solid tumors such as breast cancer. J. Med. Virol. 70:553–561, 2003. © 2003 Wiley‐Liss, Inc.
Endogenous levels of mRNA for IFNs and IFN‐related genes in hepatic biopsies of chronic HCV‐infected and non‐alcoholic steatohepatitis patients
Authors: Abbate Isabella , Romano Mario , Longo Roberta , Cappiello Giuseppina , Lo Iacono Oreste , Di Marco Vito , Paparella Concetta , Spanǒ Alberto , Capobianchi Maria Rosaria
Published: June 2003

To investigate the intra‐hepatic activation of the IFN system in patients affected by chronic HCV‐infection in comparison with that observed in a non‐infectious liver disease such as non‐alcoholic steatohepatitis, we measured the liver steady state mRNA levels of interferon‐α, interferon‐β and interferon‐γ as well as of IFN‐related genes (IFNAR‐1, STAT1α, PKR, 2‐5 AS, IRF‐1, ICE and IL‐18). In HCV‐infected subjects, possible correlations of these parameters with viral load and liver injury were also analyzed. Twenty‐four chronic untreated HCV‐infected subjects and seven patients with non‐alcoholic steatohepatitis were enrolled in the study. Liver biopsies were graded according to Knodell scores. Intra‐hepatic mRNA levels of IFNs and related genes were assessed by semi‐quantitative RT‐PCR. In comparison with non‐alcoholic steatohepatitis, in HCV‐infected subjects IFN‐α and ‐β mRNA levels were significantly lower, whereas IFN‐γ, IFNAR‐1, STAT1α IRF‐1, and IL‐18 mRNA were upregulated. Moreover, IFN‐γ mRNA steady state levels were correlated positively with those of IFNAR‐1, IRF‐1, and IL‐18, suggesting a coordinated induction of these genes. Although plasma viral load was correlated inversely with IL‐18‐specific mRNA, viral load was not related to liver injury. IFN‐γ and IRF‐1 mRNA levels were correlated positively with ALT, but not with the grading or staging. Conversely, IFN‐α and ‐β mRNA levels were higher in livers with lower staging scores. These findings support the hypothesis that in chronic HCV infection there is an imbalance between an upregulated IFN‐γ system and a downregulated IFN‐α and ‐β system, probably due to a mixed effect exerted by HCV‐specific and inflammatory non‐specific factors. J. Med. Virol. 70:581–587, 2003. © 2003 Wiley‐Liss, Inc.
Production of interleukin‐18 and interleukin‐12 in patients suffering from chronic hepatitis C virus infection before antiviral therapy
Authors: Schvoerer E. , Navas M.C. , Thumann C. , Fuchs A. , Meyer N. , Habersetzer F. , Stoll‐Keller F.
Published: June 2003

Interleukin‐12 and ‐18 (IL‐12 and IL‐18) are known to enhance the cytotoxic T‐lymphocyte response synergistically, but their precise involvement in hepatitis C virus (HCV) infection is not well known, especially for IL‐18. Enzyme‐linked immunosorbent assay (ELISA) was used to study the production of these cytokines in plasma and peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) of patients infected chronically with HCV before initiation of antiviral therapy. Fifty‐six patients and 40 healthy controls were evaluated. Patients infected with genotype 1 or with genotype other than genotype 1 HCV had significantly a high production of plasma IL‐12 compared with controls (P < 0.05). However, patients infected with genotype 1 HCV had lower levels of PBMC IL‐18 than were founded in the controls (P < 0.05); plasma IL‐18 also tended to be lower in this group of patients than in the controls, although nonsignificantly. Plasma IL‐18 was related to hepatic histological activity (P < 0.05). The data suggest a relationship between these two cytokines and some features of HCV infection, so that their respective production in relation to the outcome of the infection deserves further study. J. Med. Virol. 70:588–593, 2003. © 2003 Wiley‐Liss, Inc.