Nucleic acids in mummified plant seeds : biochemistry and molecular genetics,of pre-Columbian maize

SummaryNucleic acids fractions were isolated from pre-Columbian maize seeds and characterized using different approaches such as polyacrylamide gel electrophoresis, anti-DNA antibody binding, HPLC fractionation, molecular hybridization with cloned genes, and DNA amplification by the polymerase chain reaction. The nucleic acids were found to be very depolymerized (≤140 base pairs in length) and composed mainly of ribosomal RNA. Despite the very low amount and degree of polymerization of seed DNA, specific maize nuclear Mul, Mu4, Mu8 and, possibly, Mu5 element components could be detected, thanks to the use of amplification systems as short as 90 bp. The results suggest that evaluation of the relative proportions of Mu-type element components and, possibly, other maize genomic components in single mummified kernels, may offer a new key to the study of ancient maize populations

Administration of DNA Plasmid Coding Protein Aggregating Domain Induces Inflammatory Bone Loss.

Background: Plasmids coding protein aggregation polypeptides from different sources have beenproposed as genetic adjuvants for DNA vaccines. We reported that a plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates and subsequent cell death. Aims: In the present study we addressed the question of whether there is any substantial immunotoxicity associated with the use of self-aggregating proteins as genetic adjuvants. Results: Here we report, by mean of histology, X-ray and molecular examinations of bone specimens, the unexpected finding that intramuscular injection of pATRex in mice triggers, per se, severe bone loss (osteoporosis) independently from the sex and genotype of the treated animals. Conclusion: Even though the study suggests that proteinaceous “sticky “ adjuvants are unlikely to find their way into practical vaccination, the information gained is of value as ATRex injections could provide an additional, simplified, mouse model of osteoporosis. Moreover, our results provide an experimental support to the hypothesis that proteotoxic aggregates chronically activate the innate immune system in amyloid and aggregosome disorders

Methods and compositions relating to p62 for the treatment and prophylaxis of cancer

Provided herein are novel p62 compositions for the prophylaxis and treatment of cancer and related methods. The invention also provides modified p62 compositions that increase the anti-cancer activity of p62

HER-2/neu oncogene sequence revisited

No abstract availabl

Co-Expression of Flt-3 Ligand Gene Ablates Tumor Immunity Elicited by HER-2/neu DNA Vaccine in Transgenic Mice

Fms-like tyrosine-kinase 3 ligand (Flt-3L), is a powerful hematopoyetic growth factor, known to modulate the immune response against delivered antigens by acting either as an adjuvant or tolerogenic stimulus. In this study we evaluated the use of murine Flt-3 ligand plasmid (pFl) in combination with a DNA vaccine encoding rat-p185 oncoprotein extra cellular domain (pECD) in the prevention of mammary carcinogenesis in rat-neu HER-2 mutated (neuT) transgenic mice. We demonstrate that intramuscular (i.m.) co-immunization of pFl inhibits the production of anti-HER-2 antibody elicited by pECD vaccine, resulting in the development of spontaneous carcinomas in all co-immunized mice. The inhibitory effect on antibody production by mFlt3 gene appeared to be: dose-dependent, linked to the injection site and timing, and transient in nature. Additionally, we show that co-administration of pFI and pECD plasmids was unable to trigger cytotoxic T-cell immune response in neuT mice. On the other hand, we found that the combination of pFl with pECD had no impact on the ability of pECD to reject HER-2+ transplantable tumors in parental mice. In summary our results demonstrate that, depending on tumor model, co-administration of pFl gene can produce untoward effects to immune response, and thus its application as a vaccine adjuvant should be carefully evaluated

Recombinant DNA Technology for Melanoma Immunotherapy: Anti-Id DNA Vaccines Targeting High Molecular Weight Melanoma-Associated Antigen

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (VH) and light (VL) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format VH/VL, and VL/VH were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy

Molecular stability of DNA typing short tandem repeats in the mammary tree of breast cancer patients.

Archival pathologic specimens are a rich source for the studies of hereditary diseases, cancer genetics, and identification cases in forensic science. In this study, the intraindividual consistency of eight identifying microsatellite polymorphisms (i.e., HMTH01, vWFA31, F13A, MITMH26, FES-FPS, CD4, TPOX, CSF1PO)in a cohort of 40 patients with invasive breast carcinoma were analyzed. Nests of cancer and adjacent morphologically normal ductal-lobular structures (TDLUs) were microdissected as discrete regions from hematoxylin-eosin-stained slides. As controls for each case, DNA templates were prepared from TDLUs located in nontumor quadrants and from unaffected breast skin. Over 1,400 carefully controlled PCR reactions were reviewed, and no evidence was found for microsatellite mismatches among intraindividual cancer and control DNAs. The negative results, supported by validation experiments, strongly argue that alterations of simple repeats are rare somatic events during the onset and progression of breast cancer. This study suggests that PCR artifacts may be a relevant cause of misdiagnosis of microsatellite instability in human sporadic cancer

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immunization against differentiation Ag

BACKGROUND: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target

p62-DNA-encoding plasmid reverts tumor grade, changes tumor stroma, and enhances anticancer immunity

Previously, we reported that the administration of a p62/SQSTM1-encoding plasmid demonstrates high safety and signs of clinical benefits for human cancer patients. The treatment also suppressed tumor growth and metastasis in dogs and mouse models. Here we investigated some mechanistic aspects of these effects. In mammary tumors bearing-dogs, i.m. injections of p62 plasmid reduced tumor sizes and their aggressive potential in 5 out of 6 animals, with one carcinoma switching to adenoma. The treatment increased levels of smooth muscle actin in stroma cells and type III collagen in the extracellular matrix, which correlate with a good clinical prognosis. The p62 treatment also increased the abundance of intratumoral T-cells. Because of the role of adaptive immunity cannot be tested in dogs, we compared the protective effects of the p62 plasmid against B16 melanoma in wild type C57BL/6J mice versus their SCID counterpart lacking lymphocytes. The plasmid was only protective in the wild type strain. Also, p62 plasmid amplified the anti-tumor effect of T-cell transfer from tumor-bearing animals to animals challenged with the same tumors. We conclude that the plasmid acts via re- modeling of the tumor microenvironment, making it more favorable for increased anti-cancer immunity. Thus, the p62-encoding plasmid might be a new adjuvant for cancer treatments