Tumorigenicity,invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA

Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants,in vitro derivatives, andin vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examinedin vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was testedin vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasivein vitro and tumorigenicin vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passagein vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.     

Kalinin is abnormally expressed in epithelial basement membranes of Herlitz''s junctional epidermolysis bullosa patients

Kalinin is an extracellular adhesion molecule specific to epithelial basement membranes (BM) identified as a component of anchoring filaments of hemidesmosomes. This heterotrimeric protein is synthesized by cultured normal human keratinocytes and is involved in cell attachment. In indirect immunofluorescence studies, the epidermal BM of patients with junctional epidermolysis bullosa (JEB) of Herlitz's type were found not to be reactive with the anti-kalinin monoclonal antibodies ka146 and K140 and displayed a decreased immunoreactivity to two anti-kalinin antibodies cross-reacting with K-laminin, an anchoring filament component recently described. The intrinsic defect of JEB keratinocytes in the synthesis of kalinin was further documented by indirect immunofluorescence on in vitro cultures of these cells. In non-Herlitz JEB patients, staining of BM was constantly detected. Impairment of expression of kalinin correlated with the lack of reactivity to the monoclonal antibody GB3, which detects the BM component nicein/BM600. These results clearly demonstrate a defect of kalinin expression in epithelial basement membranes of Herlitz JEB patients and suggest that kalinin may play a role in the pathogenesis of the disease. Further studies are in progress to define possible relationships between kalinin and nicein.     

Vaccinia recombinants expressing early bovine papilloma virus (BPV1) proteins: retardation of BPV1 tumour development

Papillomaviruses are aetiological agents of epithelial proliferative diseases in animals and in man. It was previously demonstrated that animals inoculated with live vaccinia recombinants expressing early proteins of polyoma virus resist challenge with polyoma-tumour cells, and this approach has been extended to the development of a vaccine against papillomavirus-transformed cells. Bovine papillomavirus type 1 (BPV1), a virus responsible for dermal lesions in cattle, is a prototype virus of the papillomavirus group. Independent vaccinia recombinant viruses expressing the early E1, E2, E5, E6 or E7 open reading frames of BPV1 were tested for their ability to direct the expression of the corresponding protein in cultured cells. Recombinants were then assessed for their ability to elicit anti-tumour immunity in Fischer rats seeded with BPV1-transformed syngeneic FR3T3 cells. Retardation of tumour growth was observed in animals vaccinated with recombinants expressing E5, E6 or E7.     

State of viral DNA in BK virus-transformed rabbit cells

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.     

Identification of two rare and novel large deletions in ITGB4 gene causing epidermolysis bullosa with pyloric atresia

Epidermolysis bullosa with pyloric atresia (EB‐PA) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB‐PA is classified into Simplex form (EBS‐PA: OMIM #612138) and Junctional form (JEB‐PA: OMIM #226730), and it is caused by mutations in ITGA6, ITGB4 and PLEC genes. We report the analysis of six patients with EB‐PA, including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB4 gene. Two of the patients presented with non‐lethal EB‐PA associated with missense ITGB4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β4 integrin due to a variety of ITGB4 novel mutations (2 large deletions, 1 splice‐site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB4 gene. We have identified 6 novel mutations in the ITGB4 gene to be added to the mutation database. Our results reveal genotype–phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence‐based therapeutic options for EB management.     

Differential regulation of TNF receptors in maternal leukocytes is associated with severe preterm preeclampsia

We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n?=?15) with or without IUGR versus normotensive pregnant women (PREG, n?=?30), and non-pregnant controls (Con; n?=?20). Plasma levels of sTNFR1 were higher in PE (1675.0?±?227.1?pg/mL) compared with PREG (1035.0?±?101.1?pg/mL) and Con (589.3?±?82.67?pg/mL), with the highest values observed in PE with IUGR (2624.0?±?421.4?pg/mL; n?=?6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0?±?198.7?pg/mL; PREG: 1697.0?±?95.0?pg/mL) compared with Con (598.3?±?82.7?pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r?=??0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.