Variants of temporal defeasible logics for modelling norm modifications

This paper proposes some variants of Temporal Defeasible Logic (TDL) to reason about normative modifications. These variants make it possible to differentiate cases in which, for example, modifications at some time change legal rules but their conclusions persist afterwards from cases where also their conclusions are blocked.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the posttranslational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells, Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [H-3]mannose and [H-3]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [H-3]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

The role of vaccines in the control of STDs: HPV vaccines

Prophylactic vaccines for genital human papillomavirus (HPV) infection have been shown to be feasible in animal models, and suitable vaccine material based on virus-like particles can be produced in bulk at reasonable cost. Initiation of phase III clinical trials will follow definition of trial outcome measures through further epidemiological studies, and development-of assays of host protective immunity. Vaccines could in principle eliminate HPV-related disease, as the human race is the only natural host for the relevant papillomaviruses (PVs). Therapeutic vaccines for genital HPV infection are also possible, but have not yet been demonstrated as feasible in practice because the choice of vaccine antigens is difficult, the method of their optimal delivery is uncertain, and the nature of the relevant antiviral immunity is unknown. PV species specificity will require trials to be conducted in man, which will slow definition of an ideal vaccine.

Genomic organization of the CC chemokine MIP-3 alpha/CCL20/LARC/EXODUS/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3 alpha /CCL20 (HGMW-approved symbol SCYA20). The MIP-3 alpha /CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISK analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3 alpha /CCL20, Ala MLP-3 alpha /CCL20 and Ser MIP-3 alpha /CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ata MIP-3 alpha /CCL20 or Ser MIP-3 alpha /CCL20. Both forms of MIP-3cr/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-a-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3 alpha /CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3 alpha /CCL20 and Ala MIP-3 alpha /CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known. (C) 2001 Academic Press.

Papillomavirus research update: highlights of the Barcelona HPV 2000 international papillomavirus conference

The 18th international papillomavirus conference took place in Barcelona, Spain in July 2000. The HPV clinical workshop was jointly organised with the annual meeting of the Spanish Association of Cervical Pathology and Colposcopy. The conference included 615 abstracts describing ongoing research in epidemiology, diagnosis/screening, treatment/prognosis, immunology/human immunodeficiency virus, vaccine development/trials, transformation/progression, replication, transcription/translation, viral protein functions, and viral and host interactions. This leader summarises the highlights presented at the conference (the full text of the abstracts and lectures can be found at www.hpv2000.com). Relevant material in Spanish can be found at www.aepcc. org.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Specificity of 17 beta-oestradiol and benzo[a] pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg(48)Ala(119)Leu(432), Arg(48)Ala(119)Val(432), Gly(48)Ala(119)Leu(432), Gly(48)Ala(119)Val(432), Arg(48)Ser(119)Leu(432), Arg(48)Ser(119)Val(432), Gly(48)Ser(119)Leu(432) and Gly(48)Ser(119)Val(432) (all with Asn(453)), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17 beta -oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+. CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg(48) variants tended to have higher activities for 17 beta -oestradiol 4-hydroxylation than Gly(48) variants, although there were no significant variations in 17 beta -oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17 beta -oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val(432) forms than the corresponding Leu(432) forms. 4. In contrast, Leu(432) forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7, 8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val(432) forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17 beta -oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Novel MUCI splice variants are expressed in cervical carcinoma

Objectives. The MUC1 antigen can be used to identify epithelial cells from the background of hemopoietic cells. The present investigation describes patterns of overexpression of two novel MUC1 splice variants in human cervical carcinoma cell lines. Methods. RT-PCR was carried out to determine MUC1 splice variants in the cervical cancer cell lines C-4 II, C-33A, DoTc 2 4510, C-4 I, SiHa, HT3, Hs 636 T (C4-I), and HeLa. Results. The novel MUC1 splice variant D was expressed in all cell lines and the novel MUC1 splice variant C was expressed in all cell lines but C-33A. Variants A and B were expressed in all (variant A) and all but one (variant B) cell line. MUC1/REP was expressed in all cell lines and MUC1/SEC was positive in all but two cell lines (C-33 A, DoTc 2 4510). All but one cell line (C-33A) expressed MUC1/X and MUC1/Y, and two cell lines (C-33 A, DoTc 2 4510) did not express MUC1/Z, respectively. MUC1 variants A, D, and REP could be demonstrated consistently among all eight cervical carcinoma cell lines we have examined. Conclusions. The present study describes the feasibility of detecting a large number of MUC1 variants, including MUC1 variants C and D which are described for cervical carcinoma cells for the first time. Further studies will examine the presence of MUC1 splice variants' expression in human cervical carcinoma tissue.

Expression of MUC1 splice variants in benign and malignant ovarian tumours

MUC1 is expressed on the surface of ovarian cancer cells. Nine different splice variants of MUC1 have been described, but no study has reported on the expression of MUC1 iso-forms in human ovarian cancer. Our study compares patterns of expression of MUC1 splice variants of malignant and benign ovarian tumours. Ovarian tissue samples were taken from patients with benign ovarian tumours (n = 34) and from patients who had surgery for primary (n = 47) or recurrent (n = 8) ovarian cancer. RT-PCR for MUC1 splice variants A, B, C, D, X, Y, Z, REP and SEC was performed and their expression compared to clinical and histopathologic parameters. Variants A, D, X, Y and Z were more frequently expressed in malignant than in benign tumours. All primary ovarian cancer cases were positive for variant REP but negative for variant SEC. No significant association of the expression of MUC1 splice variants with the response to chemotherapy or patient survival could be demonstrated. Expression of MUC1 splice variants A, D, X, Y, Z and REP is associated with the presence of malignancy, whereas expression of MUC1/SEC is associated with the absence of malignancy. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens

Antibodies have been generated against two carboxyl-terminal splice variants of the glutamate transporter GLT1, namely, the originally described version of GLT1 and GLT1-B, and labelling has been examined in multiple species, including chickens and humans. Although strong specific labelling was observed in each species, divergent patterns of expression were noted. Moreover, each antibody was sensitive to the phosphorylation state of the appropriate protein, because chemical removal of phosphates using alkaline phosphatase revealed a broader range of labelled elements in most cases. In general, GLT1-B was present in cone photoreceptors and in rod and cone bipolar cells in the retinas of rabbits, rats, and cats. In the cone-dominated retinas of chickens and in marmosets, GLT1-B was associated only with cone photoreceptors, whereas, in macaque and human retinas, GLT1-B was associated with bipolar cells and terminals of photoreceptors. In some species, such as cats, GLT-B was also present in horizontal cells. By contrast, GLT1 distribution varied. GLT1 was associated with amacrine cells in chickens, rats, cats, and rabbits and with bipolar cells in marmosets and macaques. In the rat retina, rod photoreceptor terminals also contained GLT1, but this was evident only in enzymatically dephosphorylated tissues. We conclude that the two variants of GLT1 are present in all species examined but are differentially distributed in a species-specific manner. Moreover, each cell type generally expresses only one splice variant of GLT1. J. Comp. Neurol. 445:1-12, 2002. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT-1 (referred to hereafter as GLT-1alpha), and another against a C-terminal splice variant of this protein, GLT-1B. Both forms of GLT-1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT-1alpha was present in the intermediate lobe of the pituitary gland, whereas GLT-1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT-1B was only occasionally encountered. Conversely, GLT-1B was more extensively distributed, relative to GLT-1alpha, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT-1B was absent, whereas no such patches were evident for GLT-1alpha. At high resolution, other discrepancies were evident; double-labeling of areas such as hippocampus indicated that the. two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.