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T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DCOVA)-activated CD4+ Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DCOVA and acted as CD4+ Th-APC capable of stimulating OVA-specific CD8+ CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC’s stimulatory effect. We demonstrated that DCOVA-stimulated OT II CD4+ Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DCOVA activation. CD4+ Th-APC with acquired pMHC II and I were capable of stimulating CD4+ Th1 and central memory CD8+44+CD62LhighIL-7R+ T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8+ CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8+ T cells in vivo via acquired pMHC I. In addition, CD4+ Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DCOVA and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4+ Th-APC can modulate immune responses by stimulating CD4+ Th1 and central memory CD8+ T cell responses and eliminating DCOVA and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.

Core fucosylation (CF) patterns of some glycoproteins are more sensitive and specific than evaluation of their total respective protein levels for diagnosis of many diseases, such as cancers. Global profiling and quantitative characterization of CF glycoproteins may reveal potent biomarkers for clinical applications. However, current techniques are unable to reveal CF glycoproteins precisely on a large scale. Here we developed a robust strategy that integrates molecular weight cutoff, neutral loss-dependent MS 3 , database-independent candidate spectrum filtering, and optimization to effectively identify CF glycoproteins. The rationale for spectrum treatment was innovatively based on computation of the mass distribution in spectra of CF glycopeptides. The efficacy of this strategy was demonstrated by implementation for plasma from healthy subjects and subjects with hepatocellular carcinoma. Over 100 CF glycoproteins and CF sites were identified, and over 10,000 mass spectra of CF glycopeptide were found. The scale of identification results indicates great progress for finding biomarkers with a particular and attractive prospect, and the candidate spectra will be a useful resource for the improvement of database searching methods for glycopeptides. Molecular & Cellular Proteomics 8:913-923, 2009.

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up-regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS after overnight incubation of immature bone-marrow-derived DCs (5 × 106 cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A-specific T-cell responses after immunization. We demonstrate that EXOHSP are able to stimulate type 1 CD4+ helper T (Th1) cell responses, and more efficient P1A-specific CD8+ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXOHS. In addition, we further elucidate that EXOHSP-stimulated antitumour immunity is mediated by both P1A-specific CD8+ CTL and non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

Six new 5,6-dihydro-α-pyrone derivatives (1-6), namely, brevipolides A-F, together with seven known compounds, including a 5,6-dihydro-α-pyrone derivative (7), three flavonoids, a steroid glycoside, and two triterpenoids, were isolated from the entire plant of Hyptis brevipes. Compounds 1-7 were assigned with the absolute configuration, 5R, 6S, 7S, and 9S, as elucidated by analysis of data obtained from their CD spectra and by Mosher ester reactions. Compounds 2, 6, and 7 exhibited ED 50 values of 6.1, 6.7 and 3.6 μM against MCF-7 cells, and compounds 1, 2, 6, and 8 (the known 5,6,3′-trihydroxy-3,7,4′-trimethoxyflavone) gave ED 50 values of 5.8, 6.1 7.5, and 3.6 μM against HT-29 cells, respectively. However, no significant cytotoxicity was found against Lu1 cells for any of the compounds isolated. When these compounds were subjected to evaluation in a panel of mechanism-based in vitro assays, compound 7 were found to be active in an enzyme-based ELISA NF-κB assay, with an ED 50 value of 15.3 μM. In a mitochondrial transmembrane potential assay, compounds 3, 7, and 8 showed ED 50 values of 8.5, 75, and 310 nM, respectively. However, no potent activity was found in a proteasome inhibition assay for any of the isolated compounds.The genus Hyptis (Lamiaceae) is composed of approximately 350-400 species in the form of small herbs to large bushes, which are distributed in the tropics and warmer temperate regions all over the world. Previous studies on the constituents of species of the genus Hyptis have revealed diterpenoids, 1,2 flavonoids, 3,4 lignans, 4,5 and α-pyrone derivatives. 6,7 Hyptis brevipes Piot. originated from tropical America but is now widely distributed in other tropical regions of the world as an alien invasive species. Biological studies on crude extracts of this plant have shown inhibitory activities against bacterial and fungal growth, as well as DNA intercalation activity. 8,9 There has been no previous report on the phytochemical evaluation of this plant to date.* To whom correspondence should be addressed. Tel: .edu. † Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University. ‡ University of Illinois at Chicago. § Division of Pharmacy Practice and Administration, The Ohio State University. ⊥ Research Center for Indonesian Medicinal Plants, Tawangmangu, Indonesia. || Indonesian Institute of Science, Tangerang, Indonesia. ∇ Present address: College of Pharmacy, University of Hawaii-Hilo, Hilo, Hawaii 96720. Supporting Information Available: 1 H and 13 C NMR spectra of compounds 1 -6, DEPT and 2D NMR spectra including 1 H-1 H COSY, HSQC, HMBC, and NOESY of compound 1, as well as 1 H NMR spectra of R-and S-TPMA esters of compound 3. This material is available free-of-charge via the Internet at http://pubs.acs.org. As part of a collaborative, multi-disciplinary approach to the discovery of new naturally occurring anticancer drugs, 10,11 the entire plant of H. brevipes was selected for further investigation, following initial cytotoxicity screening using the MCF-7 human b...

Simulated microgravity (SMG) was reported to affect tumor cell proliferation and metastasis. However, the underlying mechanism is elusive. In this study, we demonstrate that clinostat-modelled SMG reduces BL6-10 melanoma cell proliferation, adhesion and invasiveness in vitro and decreases tumor lung metastasis in vivo. It down-regulates metastasis-related integrin α6β4, MMP9 and Met72 molecules. SMG significantly reduces formation of focal adhesions and activation of focal adhesion kinase (FAK) and Rho family proteins (RhoA, Rac1 and Cdc42) and of mTORC1 kinase, but activates AMPK and ULK1 kinases. We demonstrate that SMG inhibits NADH induction and glycolysis, but induces mitochondrial biogenesis. Interestingly, administration of a RhoA activator, the cytotoxic necrotizing factor-1 (CNF1) effectively converts SMG-triggered alterations and effects on mitochondria biogenesis or glycolysis. CNF1 also converts the SMG-altered cell proliferation and tumor metastasis. In contrast, mTORC inhibitor, rapamycin, produces opposite responses and mimics SMG-induced effects in cells at normal gravity. Taken together, our observations indicate that SMG inhibits focal adhesions, leading to inhibition of signaling FAK and RhoA, and the mTORC1 pathway, which results in activation of the AMPK pathway and reduced melanoma cell proliferation and metastasis. Overall, our findings shed a new light on effects of microgravity on cell biology and human health.

Active T cells release bioactive exosomes (EXOs). However, its potential modulation in immune responses is elusive. In this study, we in vitro generated active OVA-specific CD8+ T cells by cultivation of OVA-pulsed dendritic cells (DCOVA) with naive CD8+ T cells derived from OVA-specific TCR transgenic OTI mice and purified EXOs from CD8+ T cell culture supernatant by differential ultracentrifugation. We then investigated the suppressive effect of T cell EXOs on DCOVA-mediated CD8+ CTL responses and antitumor immunity. We found that DCOVA uptake OTI T cell EXOs expressing OVA-specific TCRs and Fas ligand via peptide/MHC Ag I–TCR and CD54–LFA-1 interactions leading to downregulation of peptide/MHC Ag I expression and induction of apoptosis of DCOVA via Fas/Fas ligand pathway. We demonstrated that OVA-specific OTI T cell EXOs, but not lymphocytic choriomeningitis virus-specific TCR transgenic mouse CD8+ T cell EXOs, can inhibit DCOVA-stimulated CD8+ CTL responses and antitumor immunity against OVA-expressing B16 melanoma. In addition, these T cell EXOs can also inhibit DCOVA-mediated CD8+ CTL-induced diabetes in transgenic rat insulin promoter-mOVA mice. Interestingly, the anti–LFA-1 Ab treatment significantly reduces T cell EXO-induced inhibition of CD8+ CTL responses in both antitumor immunity and autoimmunity. EXOs released from T cell hybridoma RF3370 cells expressing OTI CD8+ TCRs have a similar inhibitory effect as T cell EXOs in DCOVA-stimulated CTL responses and antitumor immunity. Therefore, our data indicate that Ag-specific CD8+ T cells can modulate immune responses via T cell-released EXOs, and T cell EXOs may be useful for treatment of autoimmune diseases.

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