Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Dpc4 is expressed in virtually all primary and metastatic pancreatic endocrine carcinomas

DPC4/Smad4 is inactivated in about 50% of pancreatic ductal cancers. It has been recently reported that this gene is also inactivated in neoplasms arising from pancreatic islet cells, a phenomenon suggested to be related to similar progressions of malignancy found in common ductal cancers. To evaluate this possibility, we analysed 20 metastases of pancreatic endocrine carcinomas and their corresponding primary lesion for inactivation of DPC4 using immunohistochemical staining. In fact, immunohistochemical labelling has been shown to correlate with DPC4 gene status with high sensitivity and specificity. The cancers included 18 nonfunctioning tumours, one gastrinoma and one ViPoma all with liver, nodal and/or adrenal metastases. Seventeen were well-differentiated and three poorly differentiated endocrine carcinomas. Dpc4 expression was absent in only one primary well-differentiated endocrine cancer and its liver metastasis, while all the remaining 19 primary tumours and their metastases stained positive for the protein. All positively staining cases showed diffuse cytoplasmic and nuclear staining in virtually all neoplastic cells. Our data suggest that DPC4 is only rarely involved in pancreatic endocrine tumourigenesis and give further weight to the hypothesis that tumours arising from pancreatic exocrine and endocrine epithelia are genetically distinct.     

Characterization of integrin chains in normal and neoplastic human pancreas.

Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1-6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening and genetic counselling for relatives of patients with breast cancer in a family cancer clinic.

Family history is the major risk factor in the aetiology of breast cancer. Breast screening is currently available to women from the age of 50 to 64 through the National Breast Screening Programme. There is, however, an equivalent risk of developing breast cancer below 50 for first degree relatives of women diagnosed with breast cancer premenopausally. We have estimated the risk of breast cancer for relatives of women affected at different ages and used these to establish a family cancer clinic offering breast screening based on individual risk. In three years we have seen 851 patients. Compliance for annual radiology was in excess of 83% over this period and of five cancers detected one had a lump at presentation, two developed interval breast lumps, and two were asymptomatic.     

The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission

Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increased in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60) repeats for FRAXA, 31-60 for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.      

Methodology of post-mortem research. Apropos of setting-up a data bank for studying the biology of depression

Investigation of post mortem neurobiochemical changes is an important field in biological psychiatry which allows analyses of the analogies between peripherical markers and the central neurochemical processes they represent. Our study discusses the pathological neuroanatomy (collection and conservation of the anatomical specimens) and clinical criteria (selection of an evaluation and diagnostic scale, analysis of results of retrospective clinical interviews: "the psychological autopsy"). We also discuss the many methodological issues which arise in interpretations of these correlations. We wish to encourage others to apply a standardized methodology to these scientific studies of post mortem neurochemical parameters and retrospective clinical diagnosis.     

Localization of expression of fibroblast growth-factors and their receptors in pancreatic adenocarcinoma by in-situ hybridization

Growth factors may play a role in autocrine or paracrine growth control of tumour cells. We have now examined the expression pattern in vivo by in situ hybridization (ISH) on a series of 13 ductal adenocarcinomas of the pancreas using the non-radioactive digoxigenin system to generate specific antisense orientated riboprobes for FGF-1 and FGF-2, and the four FGFRs (FGFR-1, -2, -3 and -4). We confirmed the expression of both FGF/FGFR by tumour cells, with the potential of a potential autocrine loops in 46% of the cases studied. FGF-2 and FGFR-3 were the most commonly expressed ligand and receptor (46% and 76% respectively). Endothelial cells lining vessels within an around invasive tumours were frequently positive for expression of FGFR-1 and/or FGFR-3. In the normal pancreas remote from the tumour, the acinar cells were found to have a heterogeneous expression pattern for FGFRs while duct cells, islet cells and stromal components including nerves and vascular endothelium were negative. The data suggest a role for FGFs and their high affinity receptors in the control of growth of human pancreatic adenocarcinoma and its supporting stroma.