Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2'-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2'-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2'-deoxycytidine to 2'-deoxyguanosine and was determined directly using 2'-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2'-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.

Several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. Recent studies of two long-recognized but unstudied stress proteins, heat shock protein (hsp) 110 and glucose-regulated protein (grp) 170, have shown them to be efficient peptide chain-binding proteins. The present investigation examines the vaccine potential of hsp110 and grp170. First, it is shown that prior vaccination with hsp110 or grp170 purified from methylcholanthrene-induced fibrosarcoma caused complete regression of the tumor. In a second tumor model, hsp110 or grp170 purified from Colon 26 tumors led to a significant growth inhibition of this tumor. In addition, hsp110 or grp170 immunization significantly extended the life span of Colon 26 tumor-bearing mice when applied after tumor transplantation. A tumor-specific cytotoxic T lymphocyte response developed in the mice immunized with tumor-derived hsp110 or grp170. Furthermore, treatments of the mice with bone marrow-derived dendritic cells pulsed with these two proteins from tumor also elicited a strong antitumor response. Last, we showed that mild, fever-like hyperthermic conditions enhance the vaccine efficiency of hsp110 as well as heat shock cognate 70, but not grp170. These studies indicate that hsp110 and grp170 can be used in hsp-based cancer immunotherapy, that Ag-presenting dendritic cells can be used to mediate this therapeutic approach, and that fever-level hyperthermia can significantly enhance the vaccine efficiency of hsps.

We have demonstrated previously that immunization with tumor-derived endoplasmic reticulum (ER) chaperone glucose-regulated protein 170 (grp170) elicits potent antitumor immunity. In the present study, we determine the impact of extracellular targeting grp170 by molecular engineering on tumor immunogenicity and potential use of grp170-secreting tumor cells as a cancer vaccine. grp170 depleted of ER retention sequence “KNDEL,” when secreted by B16 tumor cells, maintained its highly efficient chaperoning activities and was significantly superior to both hsp70 and gp96. The continued secretion of grp170 dramatically reduced the tumorigenicity of B16 tumor cells in vivo, although the modification did not alter its transformation phenotype and cell growth rate. C57BL/6 mice that rejected grp170-secreting B16 tumor cells (B16-sgrp170) developed a strong CTL response recognizing melanocyte differentiation Ag TRP2 and were resistant to subsequent tumor challenge. B16-sgrp170 cells also stimulated the production of proinflammatory cytokines by cocultured dendritic cells. Depletion studies in vivo indicate that NK cells play a primary role in elimination of viable B16-sgrp170 tumor cells inoculated into the animals, whereas both NK cells and CD8+ T cells are required for a long-term protection against wild-type B16 tumor challenge. Both the secreted and endogenous grp170, when purified from the B16 tumor, exhibited potent tumor-protective activities. However, the B16-sgrp170 cell appears to be more effective than tumor-derived grp170. Thus, molecular engineering of tumor cell to release the largest ER chaperone grp170 is capable of eliciting innate as well as adaptive immune responses, which may provide an effective cell-based vaccination approach for cancer immunotherapy.

bProteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1 gp-340 , mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen. Saliva plays a key role in host defense against invading pathogens (1-4). Among the more than 2,000 proteins and peptides found in saliva (5), many exhibit direct antimicrobial activity (6). Others can bind to bacteria to facilitate either their colonization on oral surfaces or their clearance from the oral cavity through agglutination (7,8). It has been suggested that systemic pathogens can be killed, inactivated, or agglutinated by salivary components and, thus, become cleared from the oral cavity through swallowing, thereby preventing them from colonizing the oral cavity of healthy individuals (2, 9). Thus, binding of salivary proteins to pathogens is thought to play an important role in preventing systemic infections. In hospitalized patients, the protective and antimicrobial functions of saliva, which play a crucial role in host defense against invading pathogens (1-3), are frequently impaired by reduction of salivary flow or lack of salivary secretion (9-11). Under such conditions of dry mouth and poor oral hygiene, the normal commensal oral microflora shifts to a community that harbors a higher number of pathogens (12, 13).Among the various systemic pathogens in the oral cavity, attention has been given to Staphylococcus aureus (14, 15), since both endocarditis and pneumonia have been related to oral colonization by this organism (16,17). Studies have shown the occurrence of S. aureus in oral biofilm and saliva of healthy individuals (18), bu...

The significant reduction of sRAGE in MS patients relative to healthy controls supports the potential role for RAGE axis in MS clinical pathology. Lower levels of sRAGE may be associated with enhanced inflammatory responses. Based on these observations, further investigations into the role of sRAGE in MS clinical pathology is warranted.