Purpose: Tumor-derived exosomes are proposed as a new type of cancer vaccine. Heat shock proteins are potentTh1adjuvant, and heat stress can induce heat shock protein and MHC-I expression in tumor cells, leading to the increased immunogenicity of tumor cells. To improve the immunogenicity of exosomes as cancer vaccine, we prepared exosomes from heat-stressed carcinoembryonic antigen (CEA)^positive tumor cells (CEA +

IntroductionThe first recorded outbreak of severe acute respiratory syndrome (SARS) in late February 2003 has led to thousands of infected patients and hundreds of deaths. The etiologic agent of the syndrome, a novel coronavirus termed SARS-associated coronavirus (SARS-CoV), has since been identified and isolated, 1-4 and its genome sequenced. 5,6 SARS is characterized by high fever, rigor, headache, nonproductive cough, or dyspnea and may progress to generalized, interstitial infiltrates in the lung, requiring intubation and mechanical ventilation. 1,2,7-9 A common observation in patients with SARS is pronounced lymphopenia. 1,10,11 A notable drop in CD4 ϩ and CD8 ϩ T lymphocyte counts occurs early in the course of the disease and is associated with adverse outcomes. 12 To date, studies on SARS have generally been retrospective or limited to the description of initial clinical, hematologic, radiologic, and microbiologic findings. Further studies to evaluate the mechanisms of these manifestations may help us to better understand this disease.Evidence suggests that CD8 ϩ cytotoxic T lymphocytes (CTLs) play a pivotal role in both virus elimination and induction of immunopathology in respiratory syncytial virus (RSV) infection, following recognition of epitopes presented on target cells in the context of major histocompatibility complex (MHC) class I. [13][14][15][16][17] Similarly, CTLs may participate in the clearance of virus in recovered SARS patients but also contribute to immunopathology in early stages of the disease; the molecular mechanisms underlying these CD8 ϩ CTL-mediated effects remain poorly defined. HLA-A2 is the most common HLA-A allele in Asian populations, particularly in the Chinese, with an estimated frequency of more than 50%. 18 As SARS affected many parts of Asia, and a reservoir of infection may persist in these regions, the identification of HLA-A*0201-restricted CTL epitopes of SARS-CoV is an important contribution to future studies concerning the role of CTLs in SARS-CoV pathogenesis and protection in at-risk populations.Here we have studied a panel of SARS-CoV spike protein (SARS/S)-derived peptides to identify those with binding motifs for HLA-A*0201 molecules. We evaluated the ability of HLA-A*0201 binding peptides to provoke in vivo-and in vitro-specific CTL responses in HLA-A2.1/K b transgenic (Tg) mice and peripheral blood lymphocyte (PBL) preparations from MHC-matched healthy donors, using dendritic cells (DCs) prepulsed with the peptides. Our findings show that anti-SARS-CoV CTLs generated from Tg mice and PBLs of healthy donors can elicit an antigenspecific, HLA-A2.1-restricted response, effectively killing peptidepulsed T2 cells or HLA-A2.1 ϩ tumor cell lines endogenously For personal use only. on May 10, 2018. by guest www.bloodjournal.org From expressing S protein. To the best of our knowledge, our study is the first successful prospective identification of a novel HLA-A*0201-restricted CD8 ϩ T-cell epitope from the SARS-CoV spike protein. Materials and methods PeptidesTo ide...

Objective. To study the osteogenic differentiation capacity of bone marrow-derived mesenchymal stem cells (BM-MSCs) from patients with ankylosing spondylitis (AS) and to investigate the mechanisms of abnormal osteogenic differentiation of BM-MSCs in AS.Methods. BM-MSCs from healthy donors (HDMSCs) and patients with AS (AS-MSCs) were cultured in osteogenic differentiation medium for 0-21 days, after which their osteogenic differentiation capacity was determined using alizarin red S and alkaline phosphatase assays. Gene expression levels of osteoblastic markers and related cytokines were detected by high-throughput quantitative reverse transcription-polymerase chain reaction. Enzyme-linked immunosorbent assay was performed to detect protein levels of bone morphogenetic protein 2 (BMP-2) and Noggin in the cell culture supernatant. The activation of Smad1/5/8 and MAPK signaling pathways was measured by Western blotting. The balance between BMP-2 and Noggin expression was regulated using lentiviruses encoding short hairpin RNA and exogenous Noggin, respectively, which enabled evaluation of how this balance affected osteogenic differentiation of AS-MSCs.Results. AS-MSCs outperformed HD-MSCs in osteogenic differentiation capacity. During osteogenic differentiation, AS-MSCs secreted more BMP-2 but less Noggin, accompanied by an overactivation of Smad1/5/ 8 and ERK-1/2. When the Noggin concentration was increased or BMP-2 expression was inhibited, the abnormal osteogenic differentiation of AS-MSCs was rectified. In addition, the balance between BMP-2 and Noggin secretion was restored.Conclusion. The results of this study demonstrate that an imbalance between BMP-2 and Noggin secretion induces abnormal osteogenic differentiation of AS-MSCs. These findings reveal a mechanism of pathologic osteogenesis in AS and provide a new perspective on inhibiting pathologic osteogenesis by regulating the balance between BMP-2 and Noggin.

The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns (DAMPs) can activate immune cells, including dendritic cells (DCs), via Toll-like receptors (TLRs); however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer (CRC) cells treated with oxaliplatin (OXA) and/or 5-fluorouracil (5-Fu) released high levels of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-1b, TNF-a, MIP-1a, MIP-1b, RANTES and IP-10 production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-c-producing Th1 response both in vitro and in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-c-producing Th1 response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs via TLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.

Background Rehabilitation is crucial for postoperative patients with low back pain (LBP). However, the implementation of traditional clinic-based programs is limited in developing countries, such as China, because of the maldistribution of medical resources. Mobile phone–based programs may be a potential substitute for those who have no access to traditional rehabilitation. Objective The aim of this study was to examine the efficacy of mobile phone–based rehabilitation systems in patients who underwent lumbar spinal surgery. Methods Patients who accepted spinal surgeries were recruited and randomized into 2 groups of rehabilitation treatments: (1) a mobile phone–based eHealth (electronic health) program (EH) or (2) usual care treatment (UC). The primary outcomes were (1) function and pain status assessed by the Oswestry Disability Index (ODI) and (2) the visual analog scale (VAS). Secondary outcomes were (1) general mental health and (2) quality of life (Likert scales, EuroQol-5 Dimension health questionnaire, and 36-item Short-Form Health Survey). All the patients were assessed preoperatively and then at 3, 6, 12, and 24 months postoperatively. Results A total of 168 of the 863 eligible patients were included and randomized in this study. Our analysis showed that the improvement of primary outcomes in the EH group was superior to the UC group at 24 months postoperatively (ODI mean 7.02, SD 3.10, P <.05; VAS mean 7.59, SD 3.42, P <.05). No significant difference of primary outcomes was found at other time points. A subgroup analysis showed that the improvements of the primary outcomes were more significant in those who completed 6 or more training sessions each week throughout the trial (the highest compliance group) compared with the UC group at 6 months (ODI mean 17.94, SD 5.24, P <.05; VAS mean 19.56, SD 5.27, P <.05), 12 months (ODI mean 13.39, SD 5.32, P <.05; VAS mean 14.35, SD 5.23, P <.05), and 24 months (ODI mean 18.80, SD 5.22, P <.05; VAS mean 21.56, SD 5.28, P <.05). Conclusions This research demonstrated that a mobile phone–based telerehabilitation system is effective in self-managed rehabilitation for postoperative patients with LBP. The effectiveness of eHealth was more evident in participants with higher compliance. Future research should focus on improving patients’ compliance. Trial Registration Chinese Clinical Trial Registry ChiCTR-TRC-13003314; http://www.chictr.org.cn/showproj.aspx?proj=6245 (Archived by WebCite at http://www.webcitation.org/766RAIDNc)

IntroductionAnkylosing spondylitis (AS) is a chronic autoimmune disease, and the precise pathogenesis is largely unknown at present. Bone marrow-derived mesenchymal stem cells (BMSCs) with immunosuppressive and anti-inflammatory potential and Th17/Treg cells with a reciprocal relationship regulated by BMSCs have been reported to be involved in some autoimmune disorders. Here we studied the biological and immunological characteristics of BMSCs, the frequency and phenotype of CCR4+CCR6+ Th/Treg cells and their interaction in vitro in AS.MethodsThe biological and immunomodulation characteristics of BMSCs were examined by induced multiple-differentiation and two-way mixed peripheral blood mononuclear cell (PBMC) reactions or after stimulation with phytohemagglutinin, respectively. The interactions of BMSCs and PBMCs were detected with a direct-contact co-culturing system. CCR4+CCR6+ Th/Treg cells and surface markers of BMSCs were assayed using flow cytometry.ResultsThe AS-BMSCs at active stage showed normal proliferation, cell viability, surface markers and multiple differentiation characteristics, but significantly reduced immunomodulation potential (decreased 68 ± 14%); the frequencies of Treg and Fox-P3+ cells in AS-PBMCs decreased, while CCR4+CCR6+ Th cells increased, compared with healthy donors. Moreover, the AS-BMSCs induced imbalance in the ratio of CCR4+CCR6+ Th/Treg cells by reducing Treg/PBMCs and increasing CCR4+CCR6+ Th/PBMCs, and also reduced Fox-P3+ cells when co-cultured with PBMCs. Correlation analysis showed that the immunomodulation potential of BMSCs has significant negative correlations with the ratio of CCR4+CCR6+ Th to Treg cells in peripheral blood.ConclusionsThe immunomodulation potential of BMSCs is reduced and the ratio of CCR4+CCR6+ Th/Treg cells is imbalanced in AS. The BMSCs with reduced immunomodulation potential may play a novel role in AS pathogenesis by inducing CCR4+CCR6+ Th/Treg cell imbalance.

IntroductionThe Th1 cellular immune response is crucial for antitumor and antimicrobial immunity, 1,2 and powerful immunomodulatory adjuvants that induce Th1 polarization are an important facet of vaccination strategies. 3,4 While traditional adjuvants, such as aluminum salts and oil emulsions, mainly evoke Th2 responses, characterized by production of antibodies specific for conformational antigenic determinants, 5 new generation adjuvants, including CpG-rich motifs, monophosphoryl lipid A (MPA), and quil a saponin (QS21) promote Th1 polarization. 6 These effects are thought to result from the activation of antigen-presenting cells (APCs), in particular, dendritic cells (DCs). 7,8 In recent years the molecular chaperone heat shock protein (HSP) has been revealed to interact with APCs, and its considerable capacity to induce antigen-specific CD8 ϩ cytotoxic T lymphocyte (CTL) and Th1 responses has attracted much attention. 9,10 Extracellular HSPs can interact with APCs, exhibiting potent adjuvant functions in stimulating the host immune response. 10,11 This interaction triggers a cascade of events, including re-presentation of the chaperoned peptides by the major histocompatibility complex (MHC), secretion of proinflammatory cytokines, and maturation of DCs. [12][13][14] These properties combine to make HSPs a potent adjuvant, eliciting significant immune responses against associated antigens.The Hsp70 subfamily is one of the most important HSP subfamilies. Hsp70 prepared from tumor cells or virus-infected cells are capable of eliciting potent antigen-specific CD8 ϩ CTL responses 10,15 ; these responses are CD4 ϩ T-cell-independent and have been attributed to antigenic peptides bound to the HSP. 16 It also has been shown that Hsp70 complexed with ovalbumin (OVA) antigen-specific epitopes can activate DCs and induce OVAspecific CTL responses. 17 Hsp70 can bind to CD91, CD14, and TLR2/4 receptors on the surface of APCs, activating APCs and facilitating the re-presentation of HSP-associated antigen via a TAP-dependent or TAP-independent route. 13,14,18,19 These findings demonstrate the adjuvant effects of Hsp70 and encourage the potential application of Hsp70 in vaccination.We report here a new member of the Hsp70 subfamily cloned from a human DC cDNA library, Hsp70-like protein 1 (Hsp70L1). Recombinant Hsp70L1 can bind DCs, resulting in DC maturation and activation. While Hsp70L1 shares common receptors (CD91, TLR2, TLR4) on DCs with Hsp70, there are functional differences between Hsp70L1 and Hsp70. Hsp70L1 can induce DCs to secrete IP-10, a cytokine vital for Th1 polarization, but Hsp70 cannot. Moreover, Hsp70L1 is more potent than Hsp70 in the stimulation of IL-12p70, MIP-1␣, MIP-1␤, RANTES, CCR7, and CXCR4 expression by DCs. Hsp70L1 may therefore polarize Th1 responses more effectively than Hsp70. We used OVA as a model antigen to evaluate the adjuvant effects of Hsp70L1 in vivo, finding that Hsp70L1-OVA 257-264 hybrid peptide immunization could strongly Materials and methods Cell linesE.G7-OVA, an OVA-express...

PD-1 blockade has demonstrated impressive clinical outcomes in colorectal cancers that have high microsatellite instability. However, the therapeutic efficacy for patients with tumors with low microsatellite instability or stable microsatellites needs further improvement. Here, we have demonstrated that low-dose decitabine could increase the expression of immune-related genes such as major histocompatibility complex genes and cytokine-related genes as well as the number of lymphocytes at the tumor site in CT26 colorectal cancer-bearing mice. A more significant inhibition of tumor growth and a prolongation of survival were observed in the CT26 mouse model after treatment with a combination of PD-1 blockade and decitabine than in mice treated with decitabine or PD-1 blockade alone. The anti-tumor effect of the PD-1 blockade was enhanced by low-dose decitabine. The results of RNA sequencing and whole-genome bisulfite sequencing of decitabine-treated CT26 cells and tumor samples with microsatellite stability from the patient tumor-derived xenograft model have shown that many immune-related genes, including antigen-processing and antigen-presenting genes, were upregulated, whereas the promoter demethylation was downregulated after decitabine exposure. Therefore, decitabine-based tumor microenvironment re-modulation could improve the effect of the PD-1 blockade. The application of decitabine in PD-1 blockade-based immunotherapy may elicit more potent immune responses, which can provide clinical benefits to the colorectal cancer patients with low microsatellite instability or stable microsatellites.