Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes T(H)1 cytokine expression while downregulating T(H)2 cytokine expression in PBMCs from human patients with ragweed allergy.

BACKGROUND: Recent studies have demonstrated that bacterially derived immunostimulatory sequences (ISSs) of DNA can activate the mammalian innate immune system and promote the development of T(H)1 cells. Promotion of T(H)1 immunity by means of immunotherapy in allergic patients has led to the alleviation of symptoms that result from allergen-specific T(H)2 responses. OBJECTIVE: Our purpose was to investigate whether the T(H)1-enhancing properties of ISSs could be used to alter the T(H)2-dominated immune response of allergic PBMCs in vitro. METHODS: Ragweed protein-linked ISS (PLI) was generated from a specific, highly active 22-base ISS and Amb a 1, the immunodominant allergen in ragweed pollen, to combine the T(H)1-enhancing properties of ISSs with allergen selectivity, and its activity was investigated in PBMC cultures from subjects with ragweed allergy. RESULTS: PLI was markedly successful at reversing the dominant allergen-induced T(H)2 profile while greatly enhancing IFN-gamma production. Delivering ISSs in a linked form proved to be much more effective at modulating the resulting cytokine profile than delivering free ISSs in a mixture with unlinked Amb a 1. PLI also demonstrated cytokine-modulating properties, even when used to stimulate cells that had already been primed for 6 days with Amb a 1. The antigen specificity of the action of PLI was confirmed by the observations that PLI enhances Amb a 1--specific T-cell proliferation. CONCLUSION: These data indicate that delivery of ISSs within an antigen-specific context exhibits potent cytokine-modulating activity and, combined with its reduced allergenicity, makes this molecule a strong candidate for use in improved immunotherapy applications.

Disulfide linkages in the in vitro refolded intermediates of recombinant human macrophage-colony-stimulating factor: analysis of the sulfhydryl alkylation of free cysteine residues by fast-atom bombardment mass spectrometry.

Fast-atom bombardment mass spectrometry was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophage-colony-stimulating factor. The content of iodoacetamide-alkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion high-performance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys157 and Cys159 and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.

Transformation of human mammary epithelial cells by oncogenic retroviruses.

We have introduced viral oncogenes into human mammary epithelial cells through the use of murine retroviruses. A continuous cell line (184A1N4) derived from benzo(a)pyrene treatment of normal breast epithelial cells was used as a recipient for the ras, mos, and T-antigen oncogenes. Each of these oncogenes enabled the 184A1N4 cells to grow in a selective medium, thus demonstrating the potential utility of these cells for oncogene detection and isolation. 184A1N4 cells transformed by T-antigen were nontumorigenic in athymic mice, but v-ras transformants were weakly tumorigenic. Transformants bearing both the T-antigen and ras oncogenes were strongly tumorigenic, however. The karyotype of these double transformants shows a high degree of stability. These results demonstrate the stepwise acquisition of the fully malignant phenotype by normal human epithelial cells in vitro.

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene.

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.

Biochemical and biological properties of the human N-ras p21 protein.

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.

Inhibition of cell surface ruffling and fluid-phase pinocytosis by microinjection of anti-ras antibodies into living cells.

Fibroblasts transformed by ras oncogenes display enhanced cell surface ruffling and fluid-phase pinocytotic activities. Microinjection of antibodies that specifically bind the ras proteins into these cells results in the inhibition of these two surface activities. The possible underlying biochemical basis of the influence of the ras proteins on membrane ruffling and pinocytosis and the potential relationship of these two biological activities to membrane signal transduction are discussed.

Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein.

The proteins encoded by the ras oncogene are thought to trigger expression of the transformed phenotype in some types of cancer cells. In human cells, the ras protein family consists of several members including normal (proto-oncogene) and mutant (oncogene) forms. In general, the proto-oncogene forms are thought to be involved in the normal growth control of cells, while the mutant forms (which apparently result from somatic mutation of the normal ras genes) appear to be responsible, in part, for the loss of normal growth control. On microinjection into living normal cells, the purified ras oncogene protein (p21) induces a characteristic loss of growth control in cells within several hours. The mutant forms of the different ras proteins typically contain a single amino-acid change, usually at position 12 or less frequently at position 61. Here we report that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype. The fact that this antibody inhibits binding of GTP to the v-Ki-ras protein supports the notion that GTP binding is essential to the transforming function of this oncogene product.