Human papillomavirus 16-specific T cell responses in classic HPV-related vulvar intra-epithelial neoplasia. Determination of strongly immunogenic regions from E6 and E7 proteins.

Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in 16 patients affected with classic vulvar intra-epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14-34) and/or E6/4 (45-68) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospot-interferon (IFN)-gamma assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14-34) and E6/4 (45-68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA-DR class II molecules. In order to build a therapeutic anti-HPV-16 vaccine, E6/2 (14-34) and E6/4 (45-68) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions.

Overexpression of the V3 vasopressin receptor in transgenic mice corticotropes leads to increased basal corticosterone.

The vasopressin V3 receptor (V3) is specifically expressed in pituitary corticotropes and mediates the stimulatory effect of vasopressin on adrenocorticotropic hormone (ACTH) release. The V3 gene is overexpressed in corticotrope pituitary tumours compared to normal pituitaries. We hypothesized that V3 overexpression might induce changes in corticotrope function and alter the regulation of the hypothalamic-pituitary-adrenal axis. Thus, we generated transgenic mice (POMV3) expressing the human V3 receptor in the pituitary under the control of rat pro-opiomelanocortin (POMC) promoter sequences. The transgene was efficiently transcribed and vasopressin binding was increased in both corticotropes and melanotropes. In-vitro ACTH release and inositol phosphate formation were unchanged in POMV3 pituitaries, but the responses to vasopressin were significatively increased. In vivo, basal circulating concentrations of ACTH in POMV3 mice were similar to those of controls but corticosterone concentrations were moderately increased. In addition, the levels of POMC mRNA in the transgenic pituitaries were comparable to those of control mice. Finally, POMV3 mice responded with a similar maximal increase of ACTH and corticosterone to a 20-min acute restraint stress. Together, these results show that hypophyseal V3 overexpression led to increased basal concentrations of corticosterone and suggest that the negative glucocorticoid feedback may be altered at the pituitary level.

Role of the PKA-regulated transcription factor CREB in development and tumorigenesis of endocrine tissues.

The cAMP pathway plays a major role in the development of endocrine tissues and various molecular defects of key components of this pathway (G protein, receptors, PKA, etc.) have been observed in endocrine tumors. The ubiquitous transcription factor CREB (cAMP-response element binding protein) binds to the cAMP response element (CRE) and stimulates transcription after phosphorylation on Ser(133) by PKA. The CREB family of transcription factors contains three members: CREB, CREM, and ATF-1. Targeted expression of dominant-negative mutants of CREB in transgenic mice leads to somatotrophs or thyroid hypoplasia. GH-secreting adenomas are benign secreting tumors expressing an activated mutant G alpha s protein (Gsp) in about 40% of cases. In GH-secreting adenomas CREB is always expressed and often highly phosphorylated. The CREM isoform ICER is stimulated by cAMP, and its expression is increased in Gsp-harboring tumors. After transfection in pituitary somatotroph cells, activating mutations of Gs protein (Gsp) and overexpression of wild-type G alpha S stimulate transcription of various CRE-containing promoters via CREB in a Ser(133)-specific-dependent manner. Activation of the cAMP pathway by ACTH is required for adrenal cortex (AdCx) maintenance and steroidogenesis. CREB is expressed in normal AdCx. Alterations of CRE binding proteins with loss of CREB expression and compensatory overexpression of CREMtau is observed in the human adrenocortical cancer cell line H295R. Similar alterations are found at the protein level in human malignant adrenocortical tumors. In conclusion, the CREB family of transcription factors plays an important role in the development, differentiation, and proliferation of endocrine tissues. Various alterations of the CREB family of transcription factors can be observed in endocrine tumors.

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Human skeletal myosin heavy chain genes are tightly linked in the order embryonic-IIa-IId/x-ILb-perinatal-extraocular.

Myosin heavy chain (MyHC) is the major contractile protein of muscle. We report the first complete cosmid cloning and definitive physical map of the tandemly linked human skeletal MyHC genes at 17p13.1. The map provides new information on the order, size, and relative spacing of the genes. and it resolves uncertainties about the two fastest twitch isoforms. The physical order of the genes is demonstrated to contrast with the temporal order of their developmental expression. Furthermore, nucleotide sequence comparisons allow an approximation of the relative timing of five ancestral duplications that created distinct genes for the six isoforms. A firm foundation is provided for molecular analysis in patients with suspected primary skeletal myosinopathies and for detailed modelling of the hypervariable surface loops which dictate myosin's kinetic properties.

EAAT1 is involved in transport of L-glutamate during differentiation of the Caco-2 cell line.

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism of L-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity, D-aspartate-sensitive L-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na(+) dependent, with a Hill coefficient of 2. 9 +/- 0.3, suggesting a 3 Na(+)-to-1 glutamate stoichiometry and corresponding to the well-characterized X(A,G)(-) system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B(0) and y(+), which have previously been reported to be downregulated when Caco-2 cells stop proliferating, L-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.

Absence of human papillomavirus DNA detected by polymerase chain reaction in French patients with esophageal carcinoma.

BACKGROUND & AIMS: Recent studies have suggested that esophageal human papillomavirus infection could be a risk factor for esophageal squamous cell carcinoma. The aim of this study was to evaluate the prevalence of human papillomavirus DNA sequences in the esophagus of French patients with esophageal squamous cell carcinoma. METHODS: Multiplex polymerase chain reactions with consensus primers directed to the L1 gene or specific primers for human papillomavirus types 6, 11, 16, 18, 31, and 33 directed to E6 gene (40 cycles followed by restriction mapping of the amplified products) were used to determine the presence of human papillomavirus DNA sequences in esophageal squamous cell carcinoma (n = 75), normal adjacent mucosa (n = 49), and metastatic lymphadenopathies (n = 5). As an internal control, a target located in the embryonic myosin heavy-chain gene was used in each reaction. RESULTS: Human papillomavirus DNA sequences could not be detected in any of the tumoral samples, the normal adjacent mucosa, or the metastatic lymphadenopathies. CONCLUSIONS: Human papillomavirus seems not to be implicated in esophageal carcinogenesis, at least in French patients, because the viral genomes are not associated with esophageal squamous cell carcinomas.

Characterization of a human perinatal myosin heavy-chain transcript.

Using a monoclonal antibody specific to the neonatal myosin heavy chain, we have cloned the full-length heavy chain cDNA from an 18-week human fetal cDNA library. Ribonuclease protection assays were used to survey a human muscle collection ranging from 11 weeks gestation to 16 years. Expression of the RNA encoded by this cDNA was observed at 20 and 21 weeks gestation and at 2 days after birth. No expression was observed at 13.5 weeks, before 2 years, at 2 years, or after 2 years gestation. Due to the timing of its expression, this cDNA appears to represent of the human fetal myosin heavy chain. Sequencing of the entire 6010 bases showed high similarity to the rat perinatal myosin heavy chain [Periasamy, M., Wieczorek, D. F. & Nadal-Ginard, B. (1984) J. Biol. Chem. 21, 13,573-13,578]. However, moderate divergence was observed when compared to a previously described human perinatal myosin heavy chain [Karsch-Mizrachi, I., Feghali, R., Shows, T. B. & Leinwand, L. A. (1990) Gene 89, 289-294; Feghali, R. & Leinwand, L. A. (1989) J. Cell Biol. 108, 1791-1797]. Restriction fragment-length polymorphism analyses of sites in both the S1 and rod domains showed the presence of this fetal myosin heavy chain sequence in all 27 genomic samples examined. Restriction fragment-length polymorphism analysis failed to find the previously described perinatal isoform in any sample.

Excretion of platelet activating factor-acetylhydrolase and phospholipase A2 into nasal fluids after allergenic challenge: possible role in the regulation of platelet activating factor release.

Platelet activating factor (PAF), a proinflammatory mediator synthesized through a phospholipase A2 (PLA2)-dependent reaction, is hydrolyzed into its inactive metabolite, lyso-PAF, by a specific acetylhydrolase. Previous studies have shown that allergen challenge of patients with allergic rhinitis leads to an increase of the concentrations of lyso-PAF in nasal lavage fluid (NLF), whereas PAF is detected only marginally. PAF-hydrolyzing enzymes are expected to be released on allergenic challenge, to account for the reduced concentrations of PAF in NLF. Here, we show that allergen challenge of patients with allergic rhinitis induces an increase of acetylhydrolase-like activity in NLF, which peaks within 10 minutes and returns to basal values 1 hour later. Acetylhydrolase hydrolyzed exogenous PAF with a complete loss of its ability to induce platelet aggregation. Allergen challenge also led to a parallel release of a PLA2 in nasal fluids. This enzyme preferentially hydrolyzes negatively charged phospholipids (phosphatidic acid monomethyl ester and phosphatidylgylcerol) versus phosphatidylcholine. More interestingly, the hydrolysis of phosphatidic acid monomethyl ester and phosphatidylglycerol by NLF was completely abolished by the addition of ethylenediaminetetraacetic acid which had no effect on the hydrolysis of PAF, indicating that the PLA2 secreted in nasal fluids is not involved in the degradation of PAF. Finally, our results show that allergen-induced increase in the concentrations of lyso-PAF and prostaglandin D2 occurred with a kinetic similar to that of tosyl-L-arginine-methyl-ester esterase, suggesting that mast cells are implicated in this process. Although no direct relationship was demonstrated between the absence of PAF and the increase of acetylhydrolase levels in NLF, we suggest a potential role for this enzyme in the inactivation of PAF if the latter is released in the nasal lumen.

Improved detection of human papillomavirus types 16 and 18 in cervical scrapes by a multiplex polymerase chain reaction: a 4% prevalence among 120 French women with normal cytology.

BACKGROUND: Cancer of the cervix is still a deadly disease. Since the finding of an association between human papillomavirus (HPV) and cervical carcinoma, the development of a reliable means of detecting viral DNA in cervical scrapes has become a priority. We have used the polymerase chain reaction to detect DNA from HPV types 16 and 18 in cervical scrapes. EXPERIMENTAL DESIGN: We designed a protocol that minimizes manipulation steps and improves control of the reaction. Our technique involves elaboration of a unique reaction mixture (core reagent) containing all reagents except Taq polymerase. Each cervical sample from controls and patients treated during the same experiment, received an aliquot of this core reagent, with the DNA polymerase added just before dispensing. The results of the amplification are visualized on a polyacrylamide gel stained with ethidium bromide. Positive results for viral DNA are confirmed by restriction mapping of the amplified products. We used HeLa cells as the positive control for HPV 18 and negative control for HPV 16 and SiHa cells for the reciprocal controls. As an internal control, we used a target in the exon 3 of the human embryonic myosin heavy chain gene. RESULTS: The polymerase chain reaction in our experiments assure a sensitivity at least equal to two copies of target per cell. We analyzed 120 cervical smears with normal cytology; only 4% gave a positive result for HPV 16. We did not detect any HPV 18 DNA. CONCLUSIONS: This prevalence, which is among the lowest reported in the literature to date, supports the concept that HPV detection may have value in aiding the prevention of cervical cancer.

C-fos and c-myc modulation, mitogenic effect and Ia expression in the P388D1 murine macrophage line treated by immunomodifiers.

The aim of the present study was to investigate whether the early modulation of the c-fos and c-myc oncogenes could give some orientation to the impact of immunomodulators on the monocyte-macrophage lineage. In order to work in a homogeneous system we used the P388D1 mouse macrophage cell-line which is considered as an almost mature macrophage. When P388D1 cells were stimulated by LPS, interferon-gamma or the association of both compounds, no direct correlation could be found between the modulation of DNA synthesis and the early expression of the c-fos and c-myc oncogenes. The positive regulation of Ia antigen expression seemed to correlate with the absence of induction of c-fos oncogene.

Expression of c-fos and c-myc oncogenes in the P388D1 murine macrophage line treated by immunomodulators: absence of direct correlation with DNA synthesis.

Complex patterns of metabolic and functional characteristics are induced in macrophages by biological response modifiers. The study of the early events resulting from the transduction of immunomodulatory signals could be an approach for a better understanding of this activation process. The transcription of c-fos and c-myc genes has been shown to be rapidly modified in many cells responding to various signals. Since murine peritoneal macrophages are a rather heterogeneous population we chose to investigate the c-fos and c-myc modulation in the P388D1 murine macrophage line. Owing to the frequent implication of the c-myc gene in the tumorigenicity of hematopoietic cells we first demonstrated the normal c-myc status in this cell line by Southern analysis. The modulation of the c-fos and c-myc expression has been studied by Northern analysis, 15, 30 and 60 minutes after treatment of the P388D1 cells by the phorbol ester (TPA), the Calcium ionophore A 23187 (Ca2+I), the N-acetyl muramyldipeptide (MDP) or the macrophage Colony Stimulating Factor (CSF-1). The mitogenic activity of these compounds, as evaluated by [3H] thymidine incorporation, has been measured either after a 30 minute or a 24 hour treatment. An early increase in c-fos expression always preceded a c-myc augmentation. The highest modulation of c-fos and c-myc was observed with TPA. Ca2+I and TPA presented a low mitogenic effect if compared to CSF-1. MDP did not change DNA synthesis even after 24 hours. Therefore, in the present study on the P388D1 macrophage cell line, no direct correlation could be evidenced between the mitogenic effect and the modulation of c-fos and c-myc induced by these immunomodifiers. Investigations are in progress in order to evaluate the role of these proto-oncogenes on terminal differentiation induced by immunomodulators in this cell line.

The human embryonic myosin heavy chain. Complete primary structure reveals evolutionary relationships with other developmental isoforms.

We have isolated a single 6021-nucleotide cDNA fragment encoding the full length of the myosin heavy chain (MHC) isoform initially expressed in developing human limb muscle. The corresponding transcript is expressed in fetal, but not adult, human muscle, and the corresponding gene maps to human chromosome 17. Comparison of the full length nucleotide sequence with that of the orthologous rat gene transcript reveals 74, 90, and 80% similarities in the 5'-untranslated, coding, and 3'-untranslated regions, respectively. To precisely quantitate the degree of nucleotide sequence divergence between the human embryonic and other developmentally regulated MHC gene transcripts, we utilize the algorithm of Perler et al. (Perler, F., Efstratiadis, A., Lomedico, P., Gilbert, W., Kolodner, R. & Dodgson, J. (1980) Cell 20, 555-566) and make use of the codon-for-codon register attainable in alignments of the MHC rod encoding cDNA fragments. The results allow reconstruction of the order and relative timing of certain gene duplication events involved in the evolution of the multimembered mammalian MHC loci. By this analysis, the principal sarcomeric MHC gene expressed in the 14-day chick embryo is shown to be more distantly related to the mammalian embryonic MHC genes than to those expressed peri- and postnatally. Attention is focused on regional patterns of MHC sequence conservation, ordered with reference to the topology of our phylogenetic tree. We present a composite map depicting the deduced evolutionary age of various primary structural subdomains of the human embryonic MHC.