Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.     

Cellulose-binding domains: biotechnological applications

Many researchers have acknowledged the fact that there exists an immense potential for the application of the cellulose-binding domains (CBDs) in the field of biotechnology. This becomes apparent when the phrase "cellulose-binding domain" is used as the key word for a computerized patent search; more then 150 hits are retrieved. Cellulose is an ideal matrix for large-scale affinity purification procedures. This chemically inert matrix has excellent physical properties as well as low affinity for nonspecific protein binding. It is available in a diverse range of forms and sizes, is pharmaceutically safe, and relatively inexpensive. Present studies into the application of CBDs in industry have established that they can be applied in the modification of physical and chemical properties of composite materials and the development of modified materials with improved properties. In agro-biotechnology, CBDs can be used to modify polysaccharide materials both in vivo and in vitro. The CBDs exert nonhydrolytic fiber disruption on cellulose-containing materials. The potential applications of "CBD technology" range from modulating the architecture of individual cells to the modification of an entire organism. Expressing these genes under specific promoters and using appropriate trafficking signals, can be used to alter the nutritional value and texture of agricultural crops and their final products.     

Large-scale analysis of HLA peptides presented by HLA-Cw4

A large number of HLA-Cw4 (Cw *0402) peptides were purified, sequenced, and identified from breast and ovarian carcinoma cell lines. HLA-Cw4 molecules were expressed in these cells as soluble, secreted HLA (sHLA) and recovered from the growth medium. The peptides were separated by capillary reversed-phase HPLC and analyzed by tandem mass-spectrometry. The resulting peptides fit to some extent, but not completely, the known consensus of the Cw4 peptide-binding motif. Among the identified peptides, there are a few that originate from proteins of possible interest for cancer immunotherapy or diagnostics, including mucin-5B, ART-1, fatty acid synthase, putative prostate cancer tumor suppressor, DNA topoisomerase-1, and Rac1. This work demonstrates that large-scale identification of HLA peptides recovered from sHLA is an advantageous approach for establishing the HLA peptide consensus of different haplotypes and the identification of useful peptides for treatment of diseases such as cancer, viral, and autoimmune diseases.     

Evolutionary and dynamic stability in continuous population games

 Asymptotic stability under the replicator dynamics over a continuum of pure strategies is shown to crucially depend on the choice of topology over the space of mixed population strategies, namely probability measures over the real line. Thus, Strong Uninvadability, proved by Bomze (1990) to be a sufficient condition for asymptotic stability under the topology of variational distance between probability measures, implies convergence to fixation over a pure strategy x ^* only when starting from a population strategy which assigns to x ^* a probability sufficiently close to one. It does not imply convergence to x ^* when starting from a distribution of small deviations from x ^* , regardless of how small these deviations are. It is, therefore, suggested that when a metric space of pure strategies is involved, another topology, hence another stability condition, may prove more relevant to the process of natural selection. Concentrating on the case of a one dimensional continuous quantitative trait, we resort to the natural Maximum Shift Topology in which an ɛ-vicinity of the fixation on a pure strategy x ^* consists of all mixed population strategies with support which includes x ^* and is in the ɛ-neighborhood of x ^* . Under this topology, a relatively simple necessary and sufficient condition for replicator asymptotic stability, namely Continuous Replicator Stability (CRSS), is demonstrated. This condition is closely related to the static stability condition of Neighbor Invadability (Apaloo 1997), and slightly stronger than the condition of Continuous Stability (Eshel and Motro 1981). Received: 9 July 2002 / Revised version: 12 November 2002 / Published online: 19 March 2003 Key words or phrases: CSS – ESS – NIS – Strong uninvadability – Weak topology – Replicator dynamics – Long-term evolution – Continuous population games – Continuous replicator stability – Asymptotic stability     

Identification of an Escherichia coli operon required for formation of the O-antigen capsule

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.     

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.     

Sequence-based design of kinase inhibitors applicable for therapeutics and target identification

A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce, utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, three-dimensional structure, and involvement in protein-protein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.     

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.     

Unique molecular markers in human endometriosis: implications for diagnosis and therapy

Endometriosis originates in the uterine lining and affects ~20% of reproductive-age women. The disease often causes chronic pelvic pain, affects ovulatory function and influences fertility. Although laparoscopic diagnosis of uterine endometriosis is quite specific, direct visualisation can be difficult or inaccurate in some circumstances, and it is not useful for diagnosing extra-abdominal disease. Laparoscopy is an invasive procedure, has significant morbidity and cannot be carried out frequently to monitor efficacy of therapy and the possibility of recurrence. Thus, a specific, non-invasive diagnostic test is required. One intriguing area of research uses the technology of radioimmunotargeting, which has previously been used for cancer detection. This technique could have potential for the specific detection and eventual treatment of endometriosis. A marker, eosinophil peroxidase (EPO), has been identified that is expressed in ~90% of endometriosis specimens, and is not expressed or only weakly expressed in normal endometrium. The US Food and Drug Administration has approved a monoclonal antibody to EPO for investigation as an immunoimaging agent in cancers that exhibit eosinophilia. EPO targeting using this antibody could be useful for detecting and/or treating endometriosis.