Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Effects of progestins on the proliferation of estrogen-dependent human breast cancer cells under growth factor-defined conditions.

The effect was studied of four different synthetic progestins (Org 30659, gestodene, 3-ketodesogestrel and levonorgestrel) on the proliferation of the 17 beta estradiol (E2)-dependent human breast cancer cell line MCF7. All progestins were found to stimulate proliferation, but only at high pharmacological dosages. Moreover, like estrogens the progestins at high concentrations synergistically stimulated MCF7 cell proliferation together with low concentrations of insulin. This stimulatory effect could be blocked by antiestrogens, but not by antiglucocorticoids and antiprogestins. This suggests that growth stimulation by these progestins (or their metabolites) occurs through crossreaction with the E2 receptor (ER). This is confirmed by the observation that the strong synthetic progestin Org 2058 does not stimulate proliferation. The absence of a progesterone receptor (PR)-mediated growth response seems not to be due to aberrant PR expression in these cells; 27,000 receptors (Kd 1.7 x 10(-10)M) per cell were present under growth-assay conditions. Growth stimulation by E2 in the absence or presence of insulin, is slightly inhibited or unaffected by the progestins, respectively. Our data do not support a role for the recently identified gestodene binding sites [Colletta et al., J. Steroid Biochem. 33 (1989) 1055-1061] in mediating gestodene effects on breast cancer cells: gestodene and 3-ketodesogestrel, a compound that does not bind to these gestodene binding sites, showed a similar biological activity. The effects of the progestins on the MCF7 breast cancer cell line, indicate that the use of these compounds at very high concentrations may be unfavourable, but do not support a role for them in directly stimulating breast tumor proliferation at the low progestin concentration which are reached in the serum in oral contraceptive users.     

Identification and characterization of (GA/CT) n - microsatellite loci from Quercus petraea

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)_n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.     

Why do reed beds decline and fail to re‐establish? A case study of Dutch peat lakes

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