Human and Nonhuman Primate Lineage-Specific Footprints in the Salivary Proteome.

Proteins in saliva are needed for preprocessing food in the mouth, maintenance of tooth mineralization, and protection from microbial pathogens. Novel insights into human lineage-specific functions of salivary proteins and clues to their involvement in human disease can be gained through evolutionary studies, as recently shown for salivary amylase AMY1 and salivary agglutinin DMBT1/gp340. However, the entirety of proteins in saliva, the salivary proteome, has not yet been investigated from an evolutionary perspective. Here, we compared the proteomes of human saliva and the saliva of our closest extant evolutionary relatives, chimpanzees and gorillas, using macaques as an outgroup, with the aim to uncover features in saliva protein composition that are unique to each species. We found that humans produce a waterier saliva, containing less than half total protein than great apes and Old World monkeys. For all major salivary proteins in humans, we could identify counterparts in chimpanzee and gorilla saliva. However, we discovered unique protein profiles in saliva of humans that were distinct from those of nonhuman primates. These findings open up the possibility that dietary differences and pathogenic pressures may have shaped a distinct salivary proteome in the human lineage.

Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling.

Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.

Peripheral blood mononuclear cells of patients with breast cancer can be reprogrammed to enhance anti-HER-2/neu reactivity and overcome myeloid-derived suppressor cells.

Two major barriers in the immunotherapy of breast cancer include tumor-induced immune suppression and the establishment of long-lasting immune responses against the tumor. Recently, we demonstrated in an animal model of breast carcinoma that expanding and reprogramming tumor-sensitized lymphocytes, ex vivo, yielded T memory (Tm) cells as well as activated CD25+ NKT cells and NK cells. The presence of activated CD25+ NKT and NK cells rendered reprogrammed T cells resistant to MDSC-mediated suppression, and adoptive cellular therapy (ACT) of reprogrammed lymphocytes protected the host from tumor development and relapse. Here, we performed a pilot study to determine the clinical applicability of our protocol using peripheral blood mononuclear cells (PBMCs) of breast cancer patients, ex vivo. We show that bryostatin 1 and ionomycin combined with IL-2, IL-7, and IL-15 can expand and reprogram tumor-sensitized PBMCs. Reprogrammed lymphocytes contained activated CD25+ NKT and NK cells as well as Tm cells and displayed enhanced reactivity against HER-2/neu in the presence of MDSCs. The presence of activated NKT cells was highly correlated with the rescue of anti-HER-2/neu immune responses from MDSC suppression. Ex vivo blockade experiments suggest that the NKG2D pathway may play an important role in overcoming MDSC suppression. Our results show the feasibility of reprogramming tumor-sensitized immune cells, ex vivo, and provide rationale for ACT of breast cancer patients.

Allyl isothiocyanate arrests cancer cells in mitosis, and mitotic arrest in turn leads to apoptosis via Bcl-2 protein phosphorylation.

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and ß-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G(1) phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.

Characterization of a putative ovarian oncogene, elongation factor 1alpha, isolated by panning a synthetic phage display single-chain variable fragment library with cultured human ovarian cancer cells.

PURPOSE: In an effort to identify cell surface targets and single short-chain antibody (scFv) for ovarian cancer therapy, we used a phage display approach to isolate an antibody with high reactivity against ovarian cancer. EXPERIMENTAL DESIGN: A phage scFv library was subjected to panning against human SK-OV-3 ovarian cancer cells. A clone with high reactivity was selected and tested in immunoperoxidase staining on a panel of normal tissues and ovarian carcinoma. Using immunoprecipitation, a differentially expressed band was analyzed by mass spectrometry. The antigen subclass was characterized with reverse transcription-PCR on cDNA library of normal tissues, and 91 ovarian cancer specimens, and correlated with clinicohistopathologic characteristics. RESULTS: Ninety-six individual scFv clones were screened in ELISA following panning. scFv F7 revealed high reactivity with ovarian cancer cell lines and showed intense staining of 15 fresh ovarian cancer specimens and no staining of a panel of normal tissues. A 40-kDa protein was identified to be translation elongation factor 1alpha1 (EEF1A1; P < 0.05). The expression of EEF1A2, a highly homologous and functionally similar oncogene, was found to be restricted only to the normal tissues of the heart, brain, and skeletal muscle. Aberrant EEF1A2 mRNA expression was found in 21 of 91 (23%) of ovarian cancer specimens and significantly correlated with increased likelihood of recurrence (P = 0.021). CONCLUSIONS: scFv F7 may represent an ovarian cancer-specific antibody against translation EEF1A family of translational factors. We propose that EEF1A2 may be a useful target for therapy of human ovarian cancer.

Lipid and electroosmosis enhanced transdermal delivery of insulin by electroporation.

Transdermal transport of insulin and extraction of interstitial glucose under anodal iontophoresis (electroosmosis) following electroporation in the presence of 1,2-dimyristoylphophatidylserine (DMPS) was studied. An earlier study showed that DMPS increased the transport of insulin across porcine epidermis under electroporation by approximately fourfold. It was suggested that DMPS increased the lifetime of electropores in the epidermis resulting in an enhanced transport of permeants. When electroosmosis was applied across the epidermis following electroporation with DMPS, the enhancement of insulin transport was approximately 18-fold over electroporation alone. When the same strategy was applied to extract interstitial glucose, the enhancement was approximately 23-fold over electroporation alone. Real-time transdermal insulin transport kinetics was measured using FITC-labeled insulin and a custom-made vertical diffusion apparatus that had a fluorescence cuvette as the receiver compartment. Insulin transport by electroporation alone showed a nonlinear kinetics that is most likely due to the resealing of the electropores with time. The transport kinetics when electroporation was carried out in the presence of DMPS was more linear, confirming earlier studies that suggested the DMPS stabilizes transport paths formed by electroporation. The data suggests that in vivo, noninvasive insulin delivery to therapeutic levels and glucose extraction may be achieved by combining electroporation with anionic lipids and electroosmosis.

Distinct oligoclonal T cells are associated with graft versus host disease after stem-cell transplantation.

BACKGROUND: In patients with hematologic malignancies who receive stem-cell transplantation, donors' T cells can recognize minor histocompatibility antigens on recipient cells and generate an objective response against the tumor. However, a major side effect of such therapy is graft-versus-host disease (GVHD). The purpose of this study was to characterize distinct T-cell clones that were frequently and exclusively involved in GVHD or graft-versus-tumor (GVT) effects. METHODS: We hypothesized that distinct GVHD-associated T-cell clones can be identified during the disease progression. To test this, we conducted comparative analysis of T-cell receptor (TCR) Vßs in donor-recipient pairs of patients with GVHD versus those with GVHD-free and relapse-free survival using quantitative reverse-transcriptase polymerase chain reaction and spectratyping analyses. RESULTS: We identified three sets of T-cell clones that were either frequently involved in GVHD (TCR Vß4, 11, and 23) or GVT effect (TCR Vß9, 16, and 20), or were increased at the time of GVHD and GVT effects in a patient-specific manner (TCR Vß2, 3, 7, 12, 15, and 17). Spectratyping analysis showed restricted clonality of the identified TCR Vßs. Polymerase chain reaction analysis also confirmed the presence of GVHD-associated T-cell clones at the site of the disease. CONCLUSIONS: These data suggest that GVHD- and GVT-associated clones can be distinguished by molecular analysis of TCR Vß to develop targeted therapy for GVHD.

Emergence of immune escape variant of mammary tumors that has distinct proteomic profile and a reduced ability to induce "danger signals".

Breast tumors are shaped, in part, by a process termed immunoediting which selects for immunologically evasive phenotypes. In the present study we used the rat neu-transgenic mouse model of breast cancer and its congenic non-transgenic parental strain, FVB, to explore the phenotype of tumors that emerge in the presence of an immune response directed against the neu antigen. When inoculated into parental FVB mice, a neu-overexpressing mouse mammary carcinoma (MMC) cell line isolated from spontaneous breast tumors of the FVB neu (FVBN202) transgenic mouse, elicited a neu-specific immune response resulting in a tumor rejection because of the presence of the rat neu antigen. However, a neu negative variant (ANV) of MMC arose after a long latency in spite of the neu-specific immune response. We show that compared to MMC, ANV tumor cells have a significantly reduced ability to secrete pro-inflammatory cytokines and the CCL5 chemokine, to express immunostimulatory chaperones, and they have a distinct expression of proteins involved in cell motility, and metabolic and signal transduction pathways. These studies suggest that tumor escape through immunoediting can not be explained by the loss of a single tumor antigen, but rather by a selection process of a tumor variant that has a reduced ability to induce "danger signals" together with up-regulation of proteins involved in the tumor survival. Based on these findings, we propose to target novel antigens over-expressed in the escape variant of breast tumors to treat primary tumor and to prevent tumor relapse.

Cellular antitumor immune response to a branched lysine multiple antigenic peptide containing epitopes of a common tumor-specific antigen in a rat glioma model.

Human malignant gliomas contain epidermal growth factor receptor (EGFR) gene mutations that encode tumor-associated antigens (TAAs) that can be targeted using immunological techniques. One EGFR mutant gene (EGFRvIII) encodes a protein with an epitope that is not found in normal tissues. A number of studies have focused on this unique epitope as a potential target for tumor vaccines. In the present study, we examined the cellular immune effects of a peptide containing multiple copies of the unique EGFRvIII epitope linked together by way of a lysine bridge. Fischer rats were vaccinated with an EGFRvIII multiple antigenic peptide (MAP). While vaccination produced a humoral immune response, anti-MAP antibody production was not accompanied by expression of the Th2 response cytokine IL-4. In MAP/GM-CSF vaccinated animals, a cellular immune response was detected in association with the appearance of CD4+ and CD8+ T cells at the tumor site. Splenocytes and CD8+ T cells from vaccinated rats produced the Th1 cytokine IFN-gamma in vitro in response to stimulation by rat glioma cells expressing EGFRvIII, but not by those expressing wild-type EGFR. MAP vaccine also induced a specific lytic antitumor CTL immune response against F98 glioma cells expressing EGFRvIII, but not against F98 cells expressing either wild-type EGFR or no receptor. The in vivo growth of F98(EGFRvIII) cells was attenuated in vaccinated rats; whereas, growth of F98(EGFR) cells was not. The median survival of vaccinated rats was increased 72% over that of unvaccinated controls challenged with intracerebral F98(EGFRvIII) tumor implants. Therefore, MAP vaccination produced a predominantly cellular antitumor immune response directed against F98 gliomas expressing the EGFRvIII target antigen. The potent immunosuppressive effects of F98 glioma cells mimic the human disease and make this particular tumor model useful for studying immunotherapeutic approaches to malignant gliomas.