bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Identification in Asthma and Allergy
Authors: Moffatt Miriam F. , Cookson William O.C.M.
Published: April 2003

Asthma and atopy are due to the interaction between genetic and environmental factors. The genetic basis of asthma and atopy is becoming more certain, with the promise of improvements in the diagnosis and treatment of these disorders. A combination of candidate gene and positional cloning studies has already identified several genes which influence allergic disease. These genes predispose in general to atopy, or influence the specific IgE response to individual allergens, or enhance inflammation independently of atopic mechanisms. In addition to these known factors, the chromosomal localisation of other putative genes is becoming clear.
Allergy to Bovine â–Lactoglobulin: Specificity of Human IgE Using Cyanogen Bromide–Derived Peptides
Authors: Sélo Isabelle , Négroni Luc , Créminon Christophe , Yvon Mireille , Peltre Gabriel , Wal Jean-Michel
Published: April 2003

Background: Bovine β–Lactoglobulin (Blg) is a major allergen involved in allergy to cow's milk proteins. Hydrolyzing Blg did not totally suppress its allergenicity; moreover its immunoreactivity may be increased. The aim of this work was to evaluate the specificity of serum IgE to different fragments of Blg in a group of 19 individuals allergic to cow's milk. Methods: This study was performed using both direct and competitive inhibition ELISA involving immobilized native protein or peptides derived from Blg cyanogen bromide cleavage. Results: Analyses of responses to each peptide revealed a large number of epitopes recognized by specific IgE of human allergic sera. However, there were differences in the specific determinants recognized, depending on the serum. Generally, peptides (25–107) and (108–145) retained substantial proportions of the immunoreactivity of the whole protein. Two other peptides, i.e. (8–24) and (146–162), were less recognized but were not inert. Conclusion: The main conclusion is that many epitopes were identified all along the Blg sequence by specific anti–Blg IgE from allergic humans.
Lack of Cross–Reactivity between Casein and Gliadin in Sera from Coeliac Disease Patients
Authors: Paranos Svetlana , Nikolic Goran
Published: April 2003

Background: While searching for antigliadin and anticasein antibodies in sera from coeliac patients we investigated more than 20 persons suffering from this enteropathy. In some of sera, we registered antibodies against gliadin and in parallel the appearance of anticasein antibodies. A high percentage of glutamic acid has been proven by amino acid analysis in both these proteins. Therefore we try to demonstrate the existence of cross–reactivity between them in sera from coeliac patients. Methods: We analyzed sera from 5 adult patients by using Western blot (SDS–PAGE and immunoblot assay). All of them had previously proven serum antibodies against casein and gliadin. In the procedure, antigliadin–antibodies were removed from sera by adsorption with gliadin. Adsorbed antibodies were then eluted from the adsorbent. Results: Adsorbed samples retained anticasein activity. Eluates had a strong reactivity to gliadin but not to casein. Conclusion: No cross–reactivity restricted to any of the immunoglobulin isotypes (IgG, IgM, IgA) had been proven between these two proteins. Before the final conclusion, we believe the study has to be completed by using partly of totally disassembled proteins by intestinal enzymes. It might be that enzymatic digestion could open some epitopes shared by both these proteins.
Expression of a Human IgG4 Antibody, BAB2, with Specificity for the Major Birch Pollen Allergen, Bet v 1 in Escherichia coli: Recombinant BAB2 Fabs Enhance the Allergic Reaction
Authors: Eibensteiner Petra , Denépoux Stéphane , Steinberger Peter , Kraft Dietrich , Visco Vincenzo , Banchereau Jacques , Lebecque Serge , Valenta Rudolf
Published: April 2003

Background: Antigen recognition by antibodies of different isotypes can result in completely different effects as exemplified by Type I allergy. While the IgE–antibody–mediated release of biological mediators constitutes the immunopathological basis for the immediate symptoms observed in allergic patients, allergen–specific IgG antibodies are thought to have protective effects. Methods: Cell lines secreting five human monoclonal IgG antibodies (BAB1–BAB5) with specificity for the major birch pollen allergen Bet v 1 were established from a birch–pollen–allergic patient who had received birch– pollen–specific immunotherapy. The influence of the Bet v 1–specific IgG antibodies on IgE binding to Bet v 1 was investigated. BAB2 was expressed in Escherichia coli as recombinant Fab, purified and tested for its ability to modulate Bet v 1–induced immediate–type skin reactions. Results: The BAB antibodies belonged to different IgG subclasses (BAB1: IgG1; BAB2, BAB3, BAB5: IgG4; and BAB4: IgG2) reflecting a tendency towards Th2. BAB1 represented the only antibody which strongly blocked IgE binding to Bet v 1, whereas BAB 3–BAB5 had little effect on IgE binding. Surprisingly, natural BAB2 antibodies as well as recombinant BAB2 Fabs strongly enhanced IgE binding to Bet v 1 and Bet v 1–induced immediate–type skin reactions and thus represent ‘enhancing antibodies’. Conclusion: The demonstration that anti–allergen IgG antibodies can also enhance IgE binding to a given allergen explains the unpredictability of specific immunotherapy as well as the controversy on the role of IgG in atopy.
Expansion and Immunological Study of Human Tumor Infiltrating Gamma/Delta T Lymphocytes in vitro1
Authors: Yu Songtao , He Wei , Chen Juan , Zhang Fang , Ba Denian
Published: April 2003

γ/δ T cells have stimulated a lot of interest because of their unique features in antigen recognition and cytotoxicities to many autologous and/or allogeneic tumor cells. We have developed a novel method to selectively expand larger amounts of human tumor–infiltrating γ/δ T lymphocytes (γ/δ TILs) ex vivo by immobilized pan– anti–TCRγ/δ monoclonal antibody in the presence of exogenous IL–2. The expanded γ/δ TILs mainly expressed CD45RO and HLA–DR molecules and did not express CD4. CD8+ γ/δ TILs accounted for 19% of γ/δ TILs. The expression of CD25 molecule on expanded γ/δ T cells was inducible and downregulated following a time course. The Vδ1 and Vδ2 subsets amount to 37 and 58%, respectively. The expanded γ/δ TILs show an IL–2–dependent proliferation, MHC class I–unrestricted and TCRγ/δ–related cytotoxicities to two MHC class I+ and two MHC class I+ allogeneic tumor cell lines in vitro.
Tropomyosin: An Invertebrate Pan–Allergen
Authors: Reese G. , Ayuso R. , Lehrer Samuel B.
Published: April 2003

Among food allergens, crustaceans, such as shrimp, crab, crawfish and lobster, are a frequent cause of adverse food reactions in allergic individuals. The major allergen has been identified as the muscle protein tropomyosin. This molecule belongs to a family of highly conserved proteins with multiple isoforms found in both muscle and nonmuscle cells of all species of vertebrates and invertebrates. Its native structure consists of two parallel alpha–helical tropomyosin molecules that are wound around each other forming a coiled–coil dimer. Allergenic tropomyosins are found in invertebrates such as crustaceans (shrimp, lobster, crab, crawfish), arachnids (house dust mites), insects (cockroaches), and mollusks (e.g. squid), whereas vertebrate tropomyosins are nonallergenic. Studies of cross–reactivities among crustaceans and the high degree of sequence identity among them suggest that tropomyosin is probably the common major allergen in crustaceans. Furthermore, immunological relationships between crustaceans, cockroaches and housedust mites have been established and may suggest tropomyosin as an important cross–sensitizing pan allergen.
Immunogenicity of Neutralizing Epitopes on Multiple–Epitope Vaccines against HIV–1
Authors: Lu Yun , Xiao Yi , Ding Jian , Dierich Manfred P. , Chen Ying-Hua
Published: April 2003

Based on our hypothesis that epitope vaccine may be a new strategy to induce high levels of neutralization antibodies against HIV–1, we prepared multiple–epitope vaccines using three neutralizing epitopes (GPGRAFY, RILAVERYLKD and ELDKWA) of HIV–1 gp160, and characterized their immunogenicity. Peptide 1 [C–G–(ELDKWA–GPGRAFY)2–K] and peptide 2 (CG–GPGRAFY–ELDKWA–G–RILAVERYLKD) were synthesized and conjugated with carrier protein bovine serum albumin (BSA). After vaccination antibody responses to these immunogens were induced and evaluated by ELISA. The C–G–(ELDKWA–GPGRAFY)2–K–BSA (BSA: carrier protein) multiple–epitope vaccine induced a strong antibody response to the C–G–(ELDKWA–GPGRAFY)2–K peptide (antibody titer: 1:25,600) and C–(ELDKWAG)4 peptide (antibody titer: 1:12,800), but a weak antibody response to the C–(GPCGRAFY)4 peptide. The CG–GPGRAFY–ELDKWA–G–RILAVERYLKD–K–BSA (BSA: carrier protein) multiple–epitope vaccine also induced strong antibody response to the CG–GPGRAFY–ELDKWA–G–RILAVERYLKD–K peptide (antibody titer: 1:25,600) and C–(ELLDKWAG)4 peptide (antibody titer: 1:6,400), a very strong response to C–(RIVALVERYLKD–G)2–K peptide (dilution: 1:102,400), and a very weak response to the C–(GPGRAFY)4 peptide (dilution: 1:400) in mice. Both antisera induced by both multiple–epitope vaccines interacted with the recombinant soluble gp41 (rgp41), but did not bind two control peptides. In comparison with both epitope vaccines, the rgp160 subunit vaccine could induce weak epitope–specific antibody response to these three epitopes on the three epitope peptides and V3, N–domain and C–domain peptides (dilution: 1:400–1:1,600). These results indicate that both multiple–epitope vaccines could induce high levels of antibodies to both neutralizing epitopes RILAVERYLKD and ELDKWA, while the GPGRAFY epitope on both vaccines appeared to have weak immunogenicity. Both multiple–epitope vaccines showed significant potency on inducing high levels of epitope–specific neutralization antibodies in comparison with rgp160 subunit vaccine.
CpG DNA as a Th1 Trigger
Authors: Heeg Klaus , Zimmermann Stefan
Published: April 2003

Over the last few years, it has been recognized that along with structural components and products of bacteria, bacterial DNA is also capable of signaling infectious danger to cells of the innate immune system. Particular DNA sequences (CpG motifs), which are abundant in prokaryotic (bacterial) but not in mammalian DNA, cause the activation and stimulation of immune cells. Research has been catalyzed by the finding that certain synthetic oligodeoxynucleotides mimic the action of bacterial DNA. Immunostimulation induced by bacterial DNA or synthetic oligonucleotides not only contributes to our knowledge of the pathogen-host interrelationship during infection, but can also be used therapeutically to condition or modify ongoing immune responses of the adaptive immune system. Accordingly, CpG motifs have been used as vaccine adjuvants as well as instructing agents to selectively induce Th1-dominated immune responses. Hence, CpG motifs might be used in the future as adjuvants and/or immunomodulatory agents to treat or prevent undesired Th2-dominated immune responses, such as allergy.
IgE-Mediated Sensitization to Mould Allergens among Patients with Allergic Respiratory Diseases in a Desert Environment
Authors: Ezeamuzie C.I. , Al-Ali S. , Khan M. , Hijazi Z. , Dowaisan A. , Thomson M.S. , Georgi J.
Published: April 2003

Background: The importance of fungal allergens in the development of allergic diseases in a desert environment is uncertain. This study evaluated the prevalence of IgE sensitization to moulds among patients with allergic respiratory diseases in Kuwait – a desert country. Methods: A total of 810 patients (male:female ratio 1.4) with a mean age of 32.3 years (range 2–76 years) with extrinsic asthma or allergic rhinitis were studied. Sera from the patients were tested by the CAP-RAST method for specific IgE to 6 fungi (Penicillium, Cladosporium, Aspergillus, Candida, Alternaria and Helminthosporium). For comparison house dust mite and Bermuda grass were also assessed. Results: The overall positivity to at least one mould was 20.9%. Among 120 matched control subjects, the value was 5.8%. The value was much higher among patients with asthma alone (45.8%) or both asthma and rhinitis (28.3%) than those with rhinitis alone (11.8%; p < 0.001). Asthmatic children had the highest sensitization rate (66.0% in the 7- to 12-year age group), which declined sharply with age. Among asthmatics, Candida and Aspergillus had the highest sensitization rates (23.1 and 21.3%, respectively), followed by Helminthosporium (18.8%), Cladosporium (15.9%), Alternaria (14.6%) and Penicillium (13.9%). The values for mite and Bermuda grass were 41.2 and 54.6%, respectively. Among asthmatic children, severe asthma was significantly more frequent among mould-positive (51.6%) than mould-negative patients (17.5%; p < 0.0001). Conclusions: Even in this desert environment, sensitization to moulds is quite common among patients with allergic respiratory diseases, with a striking preponderance among children with asthma. Mould allergy could also be an important factor determining asthma severity in this environment.
IgG Immune Complex Binding to and Activation of Liver Cells
Authors: Johansson Anders G. , Sundqvist Tommy , Skogh Thomas
Published: April 2003

Background/Aims: The aim was to study IgG immune complex (IC) binding to isolated hepatocytes, Kupffer cells (KCs) and sinusoidal endothelial cells (SECs). Further, we wished to analyze the capacity of IgG ICs to induce release of reactive oxygen metabolites by the IC-binding liver cells. Methods: ICs were formed between 125I-tyramine-cellobiose-labelled dinitrophenyl-conjugated human serum albumin (125I-TC-DNP10HSA) and polyclonal rabbit IgG antibodies. Binding of ICs to different rat liver cells in suspension was studied at 4°C. Production of reactive oxygen metabolites was measured by luminol-enhanced chemiluminescence at 37°C. Results: IgG mediated binding of 125I-TC-DNP10HSA to both KCs and SECs, but not to hepatocytes. The binding showed saturation kinetics and was blocked by an excess of unlabelled IgG-ICs. IgG-ICs activated KCs, but not SECs, to a chemiluminescence response. Conclusions: Both KCs and SECs bind IgG-ICs in vitro, probably via Fc receptor interaction. IgG-ICs activate KCs to produce reactive oxygen metabolites. The binding of IgG-ICs to isolated hepatocytes is small.
Expression of FasL and Fas Protein and Their Soluble Form in Patients with Hypersensitivity Pneumonitis
Authors: Kuwano Kazuyoshi , Hagimoto Naoki , Kawasaki Masayuki , Nakamura Norio , Shirakawa Kamon , Maeyama Takashige , Hara Nobuyuki
Published: April 2003

Background: Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis and loosely formed granulomas in lung biopsy specimens. HP improves or disappears altogether after cessation of antigen exposure. The Fas-Fas ligand (FasL) system is one of the representative systems of apoptosis-signaling receptor molecules, and is involved in various inflammatory diseases. We hypothesized that the Fas-FasL system may be associated with this disorder. Methods: We examined the expression of FasL and Fas proteins in lung tissues from patients with HP using immunohistochemistry. We also measured the soluble form of FasL (sFasL) and sFas levels in serum and bronchoalveolar lavage fluid (BALF) from patients with HP using enzyme-linked immunosorbent assay (ELISA). Furthermore, we also measured the cytotoxic activity of BALF sFasL in vitro. Results: FasL was detected in infiltrating mononuclear cells, and Fas was detected in infiltrating mononuclear cells, alveolar macrophages, and epithelioid cells in HP, whereas FasL was not detected and Fas was detected in few alveolar macrophages in controls. The levels of sFasL and sFas in BALF, but not in serum, were significantly increased in HP compared with controls. BALF of HP that included high levels of sFasL had no cytotoxic activity for bronchiolar epithelial cells in vitro. Conclusions: In HP, there is an upregulation of FasL and Fas in lung tissues. Since there is no incidence of apoptosis and no cytotoxic activity for lung epithelial cells in BALF from patients with HP, the increased levels of BALF sFasL and sFas may reflect the activation and sequestration of inflammatory cells rather than apoptosis.
A Method for Measuring Specific IgE in Sera by Direct ELISA without Interference by IgG Competition or IgG Autoantibodies to IgE
Authors: Kadooka Yukio , Idota Tadashi , Gunji Hiromi , Shimatani Masaharu , Kawakami Hiroshi , Dosako Shun-ichi , Samori Tomoji
Published: April 2003

Background: In measuring specific IgE levels in sera by direct ELISA, competition with coexisting IgG often impedes an exact IgE determination; additionally, IgG autoantibodies to IgE (IgG-IgE) in sera affect the assay. In this paper, we attempt to determine accurate specific IgE levels by selective removal of IgG with a protein G-immobilized gel (PG) and by acid treatment of the PG to compensate for the unintended removal of IgE, probably due to the PG binding IgG-IgE. Methods: IgG in sera was removed using PG at pH 7.0. Then, the PG was treated with citrate buffer at pH 3.0 for 5 min to liberate IgE from IgG-IgE complexes, after IgG-binding sites on the PG were saturated with bovine IgG, since PG came to bind IgE at acidic pHs. IgE levels were then measured by ELISA. Results: The PG treatment of sera removed the effect of inhibitory competition by coexisting IgG, especially at higher concentrations of sera, to improve specific IgE detection by direct ELISA. However, PG treatment alone sometimes reduced IgE levels (39% of sera tested), even though PG does not bind IgE at pH 7.0, which indicated the presence of IgG-IgE complexes. The reduction in IgE returned almost to their original levels in the sera by acid treatment of the PG. By combining the PG treatment with acid treatment, specific IgE measurement in sera was improved significantly (p < 0.01, Wilcoxon signed rank test). Conclusion: Measurement of specific IgE in sera by direct ELISA was improved by using the PG and acid treatment technique.
Anti-Tetanus Toxoid Antibody Production and Protection against Lethal Doses of Tetanus Toxin in hu-PBL-SCID Mice
Authors: Naito Seishiro , Okada Yoshiaki , Takahashi Motohide , Kato Hiroshi , Taneichi Maiko , Ami Yasushi , Suzaki Yuriko , Oka Tetsuya , Okuma Kunio , Morokuma Kazunori , Onodera Hideki , Inoue Masayoshi , Takahashi Yoshiharu , Yamazaki Setuko , Kimura Hitoshi , Komuro Katsutoshi , Uchida Tetsuya
Published: April 2003

Background: In this study, severe combined immunodeficiency (SCID) mice, which permit the survival of lymphoid cells of human origin, were used to study the human anti-tetanus immune response. Methods: Human peripheral blood lymphocytes (hu-PBL) obtained from 88 healthy donors (aged from 18 to 62) were transplanted into SCID mice, and anti-tetanus toxoid (Ttd) antibody production and protection against lethal doses of tetanus toxin (Ttx) were investigated in the hu-PBL-SCID mice. Results: The transfer of human PBL evoked significant human anti-Ttd IgG antibody production for 37.5% of the donors. After in vivo immunization, the percentage of donors with PBL exhibiting positive anti-TtD IgG production in the mice increased to 54.5%. Mean anti-Ttd IgG levels in the sera were also significantly elevated in response to immunization. The mean IgG titer for the mice injected with PBL from donors under the age of 40 was significantly higher than that of the mice injected with PBL from donors aged 40 or older. Four weeks after the cell transfer, the mice were challenged with Ttx. The induction of protection against Ttx challenge was observed mostly in mice with PBL transferred from donors under the age of 40. In vivo immunization in SCID mice with Ttd increased the number of cases of resistance to Ttx. Conclusions: These results suggest that hu-PBL-SCID mice might serve as a tool for predicting the protective ability against pathogens in PBL donors and also for evaluating vaccine efficacy.
Mechanisms of Downregulation of Transfected E-Cadherin cDNA during Formation of Invasive Tumors inSyngeneic Mice
Authors: Keirsebilck Annick , Hoorde LeenVan , Gao Yan , Bruyne GeorgesDe , Bruyneel Erik , Vermassen Petra , Mareel Marc , vanRoy Frans
Published: April 2003

Loss of E-cadherin expression has been observed both in experimental tumors and in human cancers and is related to invasiveness and poor differentiation. The E-cadherin-negative mouse mesenchymal tumor cell line MO4 was transfected with several plasmids expressing mouse E-cadherin cDNA. These plasmids differed from each other by the extent of E-cadherin-specific 3′ untranslated region (UTR) sequences and by the use of different constitutive promoters. Transfectants were isolated that expressed functional E-cadherin in a homogeneous way. In syngeneic mice, such MO4-Ecad transfectants invariably produced malignant fibrosarcoma-like tumors, which were completely E-cadherin-negative at the protein level. Northern blotting revealed that E-cadherin mRNA expression was downregulated in some but not all MO4-Ecad tumors. Downregulation was caused by mRNA instability triggered by particular 3′ UTR sequences. This in vivo downregulation of E-cadherin in malignant MO4-Ecad tumors turned out to be reversible and is likely to be mediated by host factors to be further identified.
Expression of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases in Non-Small-Cell Lung Cancer
Authors: Suzuki Makoto , Iizasa Toshihiko , Fujisawa Takehiko , Baba Masayuki , Yamaguchi Yutaka , Kimura Hideki , Suzuki Hiroto
Published: April 2003

Expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied in non-small-cell lung cancer (NSCLC). Activity of MMP-2 and MMP-9 by gelatin zymography and expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were examined in 11 lung cancer cell lines which included six small-cell lung cancer (SCLC) cell lines. Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 was examined by immunohistochemistry in 43 resected NSCLC (22 adenocarcinomas, 17 squamous cell carcinomas, 4 large cell carcinomas) using specific anti-human monoclonal antibodies. Expression of MMP-2 mRNA was detected in 5 (100%), MMP-9 in 1 (20%), TIMP-1 in 4 (80%), and TIMP-2 in 5 (100%) of 5 NSCLC cell lines examined. MMP-2 gelatinolytic activity also was detected in all five NSCLC cell lines, whereas MMP-9 activity was detected in only one cell line. In 43 patients, MMP-2, MMP-9, TIMP-1, and TIMP-2 immunoreactivity was demonstrated in 19 (44%), 9 (21%), 15 (35%), and 29 (67%) excised tumors, respectively. All stromal fibroblasts in tumor samples stained positive for MMP-2. There was a correlation between TIMP-2 immunoreactivity and disease stage (42% stage I versus 88% stages II, III, and IV) (p = 0.0024). Both cancer cell lines and NSCLC tumor samples frequently expressed MMP-2, MMP-9, TIMP-1, and TIMP-2; MMP-2 in particular was highly expressed in malignant cells and surrounding fibroblasts. These findings suggest that MMP-2 plays a more important role in invasion of NSCLC than MMP-9 and that TIMP-2 may have clinical relevance in NSCLC.
Sequencing Analysis of the Region Encoding the DNA Polymerase of Bovine Adenovirus Serotypes 2 and 3
Authors: Yagubi Abdelbaset , Ojkic Davor , Bautista Diosdado S. , Haj-Ahmad Yousef
Published: April 2003

Bovine adenoviruses (BAVs) are important pathogens causing significant economic losses to the cattle industry. We have been interested in the differences among serotypes of these viruses, particularly in their pathogenicity and host range. As part of our efforts to better understand these viruses, we have determined the nucleotide sequences for serotype 3 (BAV3) at map coordinates beween 11.7 and 23.7% and for serotype 2 (BAV2) between 13.1 and 24.0%. Analyses of these sequences revealed large open reading frames (ORFs) encoded within the leftward-reading strand of the viral DNA. The coding capacity of the ORF in BAV3 is 1,167 amino acid residues and 1,138 in BAV2. A search in the GenEMBL protein sequence databank for homology to the predicted polypeptide products of these ORFs established their identity as that for the adenovirus (Ad) DNA polymerase (DNA pol). The deduced polypeptide sequences were aligned with each other and with other known Ad DNA pols to reveal regions of homology and similarity. The comparison at the amino acid sequence level not only showed that the bovine Ad DNA pols from the two serotypes are quite distinct from each other, but also revealed that Ad DNA pols contain multiple domains that are highly conserved among human, canine and bovine Ads. These conserved domains are likely important for the multiple functions attributed to Ad DNA pol, which include catalysis of its own initiation complex, elongation of nascent DNA strand, as well as correction of DNA replication errors.
Interaction of Human Papillomavirus 8 Regulatory Proteins E2, E6 and E7 with Components of the TFIID Complex
Authors: Enzenauer Christina , Mengus Gabrielle , Lavigne Anne-Claire , Davidson Irwin , Pfister Herbert , May Michael
Published: April 2003

Human papillomavirus 8 (HPV8) is one of the oncogenic HPV types specifically associated with skin cancers of epidermodysplasia verruciformis patients. The early gene products of this virus exert functions in transformation (E2, E6, E7), replication (E1, E2) and in the control of viral transcription (E2, E7). Many viral and cellular transactivators of transcription have been shown to interact selectively and directly with a number of TATA-box-binding protein (TBP)-associated factors (TAFIIs), which then play a role as coactivators. Using glutathione-S-transferase (GST) pull-down experiments, we tested in vitro interactions between GST-HPV8-E1, -E2, -E6 and -E7 and 7 in-vitro-translated TAFIIs in the human (h) system (hTAFII18, hTAFII20, hTAFII28, hTAFII30, hTAFII55, hTAFII100, hTAFIIΔN135) or TBP. We could show that GST-HPV8-E2 interacts directly at least with hTAFII55 and TBP. Deletion analysis indicated that a domain overlapping with the C-terminal moiety of HPV8-E2 is required for binding to TBP, whereas determinants for interactions with hTAFII55 are in the central and C-terminal part of the E2 protein. In similar binding studies, GST-HPV8-E6 interacted with hTAFII28, hTAFIIΔN135 and TBP, and more weakly with hTAFII20, whereas GST- HPV8-E7 bound to hTAFII20, hTAFII28, hTAFII55, hTAFIIΔN135 and TBP. Deletion analysis revealed that the C-terminal part of HPV8-E7 is required for the interaction with these hTAFIIs. In contrast, no interactions were observed between GST-HPV8-E1 and in-vitro-translated hTAFIIs.
The US5 Open Reading Frame of Herpes simplex Virus Type 1 Does Encode a Glycoprotein (gJ)
Authors: Ghiasi Homayon , Nesburn Anthony B. , Cai Steve , Wechsler Steven L.
Published: April 2003

Based on sequence analysis, the protein encoded by the US5 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N-glycosylation site and was given the designation of glycoprotein J (gJ). However, the US5 gene product has not been identified and the identity of gJ as a glycoprotein has not been confirmed. We have cloned and expressed the DNA encoding the complete sequence of the US5 ORF, using a baculovirus expression system. Western blotting, using polyclonal antibody raised against synthetic US5 peptides, revealed two major baculovirus-US5-expressed protein bands with apparent molecular weights of 16–17 and 10 kD. The recombinant US5 was found on the membrane of Spodoptera frugiperda cells and was susceptible to tunicamycin, endoglycosidase H, glycosidase F and partially resistant to endoglycosidase F. Vaccination of mice with baculovirus-expressed US5 did not induce a neutralizing antibody to HSV-1 or provide protection against lethal HSV-1 challenge. However, serum from these vaccinated mice was able to recognize US5 in purified HSV-1 virions by Western blot analyses and on the surface of HSV-1-infected cells by immunofluorescence. These findings establish that US5 does encode a glycoprotein and confirm the appropriateness of naming the US5 gene product gJ.
Environmental Influence on Immune Inhibition of Release of Herpes simplex Virus from Cells
Authors: Benitez J. , Ahmad A. , Davies J. , Skinner G.R.B.
Published: April 2003

Immune inhibition of virus release (IVR) of herpes simplex virus type 1 (HSV-1) from baby hamster kidney cells (BHK-21) was mediated by antisera against BHK cells, HSV-1, human fibronectin and mouse heparan sulphate proteoglycan and was irreversible for at least 24 h following removal of antiserum. Enhancement of IVR by calf serum depletion of growth media was obtained in varying measure using each of these antisera and also by treatment of virus-infected cells by the lectin concanavalin A. Enhancement was reversible by replenishment of growth media with bovine serum components larger than 12 kD but this only occurred when replenishment was instituted prior to virus infection. There was also reversibility to varying degree following replenishment by ovine, equine and human serum which indicates that this phenomenon is not species specific. In addition to the presence of relevant antigens on the cell surface, IVR may also require an alteration in the cell membrane; this is evidenced by the absence of anticellular serum-mediated IVR when treatment was introduced less than 6 h after virus infection, suggesting that a certain level of alteration or possibly cell damage – in this case virus induced – is necessary. Enhancement of IVR by calf serum depletion would seem to operate through a specific alteration in the virus-infected cell membrane as serum-depleted cells did not show histological alteration and were able to replicate HSV-1 to usual titres; it is possible that this enhancement may represent an as yet unidentified host defence mechanism whereby extracellular release of virus will be reduced in ischaemic or necrotic tissue in the course of infectious inflammatory processes.
Lack of Detectable Interaction between HSV-1 and Integrins or Tachykinins
Authors: Israel Barbara A. , Schultz Kevin T. , Murphy Christopher J.
Published: April 2003

Several viruses are known to utilize cellular integrin molecules to gain entry into cells. Because of the ability of herpes simplex virus type 1 (HSV-1) to disrupt cellular adhesion, as seen particularly in ocular infections, we examined the ability of several peptides, containing known integrin recognition sequences, to interfere with plaque formation of HSV-1 in epithelial cells. We also examined the possible involvement of tachykinins in virus entry. We did not detect any decrease in plaque formation by HSV-1 in the presence of Arg-Gly-Asp, Asp-Gly-Glu-Ala, or EILDV peptides or in the presence of monoclonal antibodies to the human β1 or β4 integrin subunit. Substance P or inhibitors of the NK1 or NK2 tachykinin receptors also had no inhibitory effects on HSV-1 plaque formation.
Hepatitis B Maternal-Fetal Transmission in Southern Europe
Authors: Papaevangelou George , Farmaki Georgia , Kada Helen
Published: April 2003

Screening of pregnant women for hepatitis B surface antigen especially in countries with a high carrier rate as in Southern Europe is absolutely necessary for the control and elimination of the disease. In most Southern European countries screening of pregnant women is highly recommended. In Greece the carrier rate in pregnant women varies between 2.8 and 3.0%. However, only 63.1% of mothers at delivery had been screened before admission to the maternity hospital. To avoid legal problems arising from obligatory screening, educational campaigns, lectures, pamphlets and further measures to increase screening of pregnant women and administering hepatitis B immunoglobulin and vaccine should be taken.
Transmission of Cervical Cancer-Associated Human Papilloma Viruses from Mother to Child
Authors: Cason John , Rice Philip , Best Jennifer M.
Published: April 2003

There is now compelling evidence that persistent infection with certain types of human genital papillomaviruses (HPV) may, after many years, lead to cervical cancer. However, HPV have been detected in asymptomatic women, infants and children. Several studies have demonstrated that infants can acquire high-risk HPV infections from their mothers at birth. Thus, the traditional view that cervical-cancer associated HPV infections are primarily sexually transmitted needs to be re-assessed. Accordingly, the role of mother to child transmission of cancer-associated HPVs may need to be investigated further. These facts are pertinent to those developing prophylactic vaccines to prevent high-risk HPV infections and cervical carcinoma.
Antiviral Efficacy of Disinfectant Solution MRI-1
Authors: Skinner G.R.B. , Billstrom M. , Randall S. , Buchan A. , Davies J. , Ahmad A.
Published: April 2003

Disinfectant MRI-1 was prepared by dissolution of non-ionic and ionic detergent in ethanol. The disinfectant inactivated extracellular and intracellular enveloped and non-enveloped viruses including herpes viruses, influenza A and human immunodeficiency disease virus in suspension or on surfaces by pre-exposure or post-exposure to the disinfectant; in addition, cells were disabled as potential hosts for viral infection using concentrations of MRI-1 which were 50-fold less than the operative concentration for disinfection. There was no evidence of in vitro mutagenicity using Salmonella typhimurium or sensitization or other adverse effect in a guinea pig model or in human subjects.
Molecular Evolution of Hepatitis Viruses
Authors: Mizokami Masashi , Orito Etsuro
Published: April 2003

Methods and models for analytical and quantitative researches on the evolution of hepatitis viruses using nucleotide sequences and amino acids of hepatitis viruses are described. Methods for the calculation of evolutionary rates and the construction of molecular evolutionary trees, especially by estimating the number of nucleotide substitutions and the number of amino acids, are introduced hereinafter. In addition, the evolution of the hepatitis virus is discussed by applying these methods to hepatitis B virus and hepatitis C virus.
Problems in the Treatment of Hepatitis C with Interferon
Authors: Iino Shiro
Published: April 2003

A marked increase in the rate of eradication of hepatitis C virus (HCV) has been achieved by the combination of interferon-α2b and ribavirin when compared with interferon-α2b alone. However, even with combination therapy, hepatitis persists in more than half of the patients with chronic herpatitis C and progresses to liver cirrhosis and hepatocellular carcinoma with time. What needs to be kept in mind is that, whether by its natural course or by therapy to suppress liver inflammation or by interferon therapy, the rate of development of hepatocellular carcinoma is reduced if ALT is maintained at low levels. Attention should therefore be focused on the development of drugs which enhance the effect of interferon as well as drugs which suppress liver inflammation.
Hepatitis E Virus
Authors: Yarbough Patrice O.
Published: April 2003

Hepatitis E, previously known as enterically transmitted, enteric, or epidemic hepatitis, is a worldwide public health problem. The causative agent, the hepatitis E virus, is involved in epidemic, sporadic, and fulminant hepatitis cases worldwide. This review describes the advances in the biology of the hepatitis E virus and the progress made to develop simple and robust serologic assays for the diagnosis of HEV infection. Genomic sequence comparisons with a recently identified US isolate now suggests three genetic groups of HEV viruses. A highly conserved animal isolate found in pigs suggest the coexistence of animal and human isolates of HEV. The use of recombinant technology to develop an effective subunit vaccine capable of providing cross-protection for the most divergent HEV strains has been established and is reviewed.
Effects of Varying Concentrations of Bleach on in vitro HIV-1 Replication and the Relevance to Injection Drug Use
Authors: Contoreggi Carlo , Jones Susan , Simpson Pippa , Lange W. Robert , Meyer III William A.
Published: April 2003

The use of bleach (hypochlorite) as a disinfectant for drug injection equipment in the intravenous-drug-using population was recommended early in the HIV-1/AIDS epidemic. Epidemiological studies have challenged the use of bleach as an effective measure to prevent HIV-1 transmission. However, in vitro HIV-1 coculture studies have shown that a high concentration of bleach is an effective cytotoxic and potentially virucidal agent. In this study, we demonstrate that HIV-1 peripheral blood mononuclear cell cocultures containing low concentrations of hypochlorite in the media showed earlier conversion to HIV-1 positivity, as measured by the presence of p24 antigen. HIV-1 cocultures with high concentrations of hypochlorite in the culture media, which appeared to be highly cytotoxic, and HIV-1 cocultures without bleach in the media did not exhibit this early p24 antigen positivity. Hypochlorite chemically disinfects by releasing free chlorine that is a potent oxidant. In injection drug equipment, a low residual concentration of bleach is likely to remain in cleaned equipment despite rinsing with water. Low concentrations of oxidants have been shown to enhance tissue inflammation, in vivo, as well as HIV-1 replication in vitro. Previous studies have shown that despite vigorous cleaning of blood-contaminated injection syringes with bleach followed by water, microaggregates of residual blood remained in bleach-cleaned blood-contaminated syringes. Hypothetically, oxidant effects of the residual bleach in the bleach-cleaned syringes could enhance the possibility of infection by remaining HIV-1 contained in a contaminated syringe. We suggest that the likelihood of an injection drug user contracting HIV-1 through the sharing of a bleach-cleaned blood-contaminated syringe may be increased by the cotransmission of residual bleach and its localized tissue-inflammatory effects; however, this has not been statistically proven in epidemiological studies.
Temperature-Dependent Phosphorylation State of the H5R Protein Synthesised at the Early Stage of Infection in Cells Infected with Vaccinia Virus ts Mutants of the B1R and F10L Protein Kinases
Authors: Beaud Georges , Beaud Ruth
Published: April 2003

The phosphorylation state of H5R protein was investigated by two-dimensional gel electrophoresis of proteins of BSC-40 cells infected at 32° or 39.5° with vaccinia virus ts mutants of the viral B1R or F10L protein kinase genes. A temperature-dependent increase of underphosphorylated H5R protein (pI 6.8) was demonstrated in the case of the B1R, but not of the F10L gene. The temperature-dependent cytoplasmic location of underphosphorylated H5R protein after infection with the ts mutants of the B1R gene was the consequence of the associated viral DNA replication block. These results show that the B1R protein kinase controls the phosphorylation state of the H5R protein synthesised at the early stage in vaccinia-virus-infected cells.
Propagation of Human Papillomavirus Type 59 in the Athymic Mouse Xenograft System
Authors: Bryan Janine T. , Tekchandani Jenifer , Schroeder Jill M. , Brown Darron R.
Published: April 2003

Studies of human papillomavirus (HPV) infection are hampered by the lack of an adequate culture system. The athymic mouse xenograft system permits propagation of HPV, but only a few HPV types have been grown in this manner. To produce an oncogenic type for studies of HPV pathogenesis, a condylomata acuminata lesion from an immunosuppressed patient was used to prepare an infectious extract. The patient’s lesion was shown by PCR analysis to contain abundant HPV 59 (an oncogenic type) and a lesser amount of HPV 11, a nononcogenic type. The extract was used to infect human foreskin tissue which was subsequently implanted into athymic mice. Characterization of implants recovered after 3–4 months of growth revealed the presence of HPV 59 exclusively. A second extract was prepared from one of these implants and used in an additional experiment to demonstrate the passage of HPV 59. Compared to the histopathologic changes induced by the prototypic nononcogenic HPV 11, infection with HPV 59 caused a higher degree of basal cell crowding, less acanthosis, minimal papillomatosis and less pronounced koilocytosis.
Glomerular Endothelial Cell Injury Mediated by Shiga-Like Toxin-1
Authors: Adler Stephen , Bollu Ravindra
Published: April 2003

The hemolytic uremic syndrome is an important cause of acute renal failure and is often associated with prior infection with enterotoxigenic strains of Escherichia coli. Significant pathologic changes in the glomerulus include endothelial cell swelling, detachment, and intravascular coagulation. We investigated the potential pathogenetic mechanisms of this disease by exposing cultured rat glomerular endothelial cells to shiga-like toxin 1 (SLT-1). Glomerular endothelial cell viability and protein synthesis were not affected by 10–9 M SLT-1. Synthesis and release of thromboxane (Tx) A2, measured as the stable metabolite TxB2, and of 12-(S)-HETE were each increased 1.6 ± 0.1 fold (p < 0.05). No change was observed in the production of PGE2 or 6-keto-PGF1α, the active metabolite of PGI2. SLT-1 (10–9 M) significantly increased cell retraction and the formation of gaps in the glomerular endothelial cell monolayer for up to 6 h following exposure. A similar effect was seen with 0.1 μM 12-(S)-HETE. SLT-1 and 0.01 μM 12-(S)-HETE also significantly decreased adhesion of glomerular endothelial cells to fibronectin (75 ± 4 and 65 ± 2% of control, respectively; p < 0.001) and laminin (81 ± 5 and 67 ± 4% of control, respectively; p < 0.01). These results support a role for SLT-1 in the production of the pathologic changes seen in the hemolytic uremic syndrome through stimulation of production of procoagulant, vasoconstrictor arachidonic acid metabolites with potential to produce endothelial injury and through alterations in glomerular endothelial cell adhesion to components of the glomerular basement membrane.
Effects of Neutral Endopeptidase Inhibition in the Rat Remnant Kidney Model
Authors: Jandeleit-Dahm Karin , Burrell Louise M. , Kanazawa Masayuki , Casley David , Jackson Bruce , Johnston Colin I.
Published: April 2003

The orally active neutral metalloendopeptidase (NEP) inhibitor SCH34826 was given by oral gavage in a dose of 90 mg/kg twice daily for 3 days to rats with subtotal nephrectomy (n = 7) and effects were compared to a placebo group receiving phosphate buffer (n = 5). Inhibition of neutral endopeptidase in the remnant kidney was measured by in vitro autoradiography using the specific radioligand [125I]–SCH 47896. Treatment with the NEP inhibitor SCH34826 caused a 60% reduction in the neutral endopeptidase radioligand–binding site density in the kidneys of the SCH34826–treated animals compared to the placebo group (81.6±3.7 versus 214.5±4.2 dpm/mm2, p<0.01). This was associated with a marked increase in urinary atrial natriuretic peptide (ANP) from 3,930±295 to 9,094±1,089 pg/24 h in the SCH34826–treated group (p<0.01). Concomitantly there was a transient increase in natriuresis in the SCH34826–treated group [baseline 2.03±0.55 to 3.77±0.58 mmol/24 h on treatment day 1 (p = 0.02) and 2.58±0.19 mmol/24 h on treatment day 3 (p = 0.09)] which was not observed in the placebo group. Urinary protein excretion, glomerular filtration rate (determined by 99mTc–DTPA clearance), systemic blood pressure, plasma ANP concentration and urinary cyclic GMP excretion were not changed by SCH34826 treatment. These results suggest that oral administration of the NEP inhibitor SCH34826 inhibits renal neutral endopeptidase, increases urinary ANP and modulates natriuresis without alteration of systemic blood pressure, plasma ANP and renin level, glomerular filtration or protein excretion.
Leukotriene D4 Inhibits Na+ Uptake through cAMP and PLC Pathways in Primary Cultured Renal Proximal Tubular Cells
Authors: Han Ho-Jae , Park Soo-Hyun , Lee Jae-Cheon , Lee Hwanghee-Blasie , Park Haeng-Soon
Published: April 2003

Leukotriene D4 (LTD4) is one of the slow–reacting substances of anaphylaxis and is reported to have a diverse response including the mediation of glomerular nephritis. However, little is known about the functions of LTD4 and its mechanisms of action in primary cultured rabbit renal proximal tubular cells (PTCs). The purpose of this study is to investigate the effect of LTD4 on Na+ uptake and its related signal transduction pathways in PTCs. LTD4 (>10–9 M) significantly inhibited the Na+ uptake after 15 min (in nmol/mg protein: controls 431.7±11.4 vs. LTD4 (10–9 M) 355.0±23.6; p<0.05); and its effect was blocked by MK–571 (10–6 M), a leukotriene receptor antagonist, in PTCs. Preincubation with cilastatin, a renal dipeptidase inhibitor, and polyclonal antibody against renal dipeptidase potentiated the inhibitory effect of LTD4 on Na+ uptake. SQ 22536 (10–6 M), an adenylate cyclase inhibitor, and the myristoylated protein kinase A inhibitor amide 14–22 (PKI; 10–5 M) blocked the effect of LTD4 on Na+ uptake (in nmol/mg protein: LTD4 349.9±18.5 vs. SQ 22536+LTD4 476.5±22.0 and PKI+LTD4 440.3±19.3; p<0.05), and LTD4 induced an increase in cyclic adenosine monophosphate (cAMP), suggesting the involvement of cAMP in the inhibition of Na+ uptake. In addition, U 73122 (10–6 M) and neomycin (10–4 M), phospholipase C (PLC) inhibitors, W–7 (10–4 M), a calmodulin antagonist, and bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, blocked the LTD4–induced inhibition of Na+ uptake, strongly suggesting involvement of the PLC–PKC signal pathways in the effect of LTD4. LTD4 significantly increased [Ca2]i by 49±7% as compared with baseline. TMB–8 (10–5 M) and BAPTA/AM (10–5 M), intracellular calcium mobilization blockers, completely blocked the LTD4–induced inhibition of Na+ uptake (in nmol/mg protein: LTD4 347.6±19.0 vs. TMB–8+LTD4 436.4±22.3 and BAPTA/AM+LTD4 419.9±14.3; p<0.05); however, EGTA (1 mM), a calcium chelator, partially blocked the LTD4–induced inhibition of Na+ uptake. In conclusion, LTD4–induced inhibition of Na+ uptake may be involved in both cAMP and PLC–PKC signal pathways in PTCs. In addition, Ca2+, which comes from the intracellular Ca2+ mobilization, is primarily responsible for the LTD4–induced inhibition of Na+ uptake.