Novel H-2Db-restricted CD8 epitope derived from mouse MAGE-type antigen P1A mediates antitumor immunity in C57BL/6 mice.

BACKGROUND: Melanoma antigen gene (MAGE)-type antigens are promising targets for cancer immunotherapy as they are expressed in cancer cells but not in normal tissues, except for male germline cells. The mouse P1A antigen shares this MAGE-type expression pattern and has been used as a target antigen in preclinical tumor models aiming to induce antitumor CD8+ T-cell responses. However, so far only one MHC I-restricted P1A epitope has been identified. It is presented by H-2Ld in mice of the H-2d genetic background such as DBA/2 and BALB/c. Given the availability of multiple genetically altered strains of mice in the C57BL/6 background, it would be useful to define P1A T-cell epitopes presented by the H-2b haplotype, to facilitate more refined mechanistic studies. METHODS: We employed a heterologous prime-boost vaccination strategy based on a chimpanzee adenovirus (ChAdOx1) and a modified vaccinia Ankara (MVA) encoding P1A, to induce P1A-specific T-cell responses in C57BL/6 mice. Vaccine-induced responses were measured by intracellular cytokine staining and multiparameter flow cytometry. We mapped the immunogenic CD8 epitope and cloned the cognate T-cell receptor (TCR), which we used for adoptive cell therapy. RESULTS: ChAdOx1/MVA-P1A vaccination induces a strong P1A-specific CD8+ T-cell response in C57BL/6 mice. This response is directed against a single 9-amino acid peptide with sequence FAVVTTSFL, corresponding to P1A amino acids 43-51. It is presented by H-2Db. P1A vaccination, especially with ChAdOx1 administered intramuscularly and MVA delivered intravenously, protected mice against P1A-expressing EL4 (EL4.P1A) tumor cell challenge. We identified and cloned four TCRs that are specific for the H-2Db-restricted P1A43-51 peptide. T cells transduced with these TCRs recognized EL4.P1A but not MC38.P1A and B16F10.P1A tumor cells, likely due to differences in the proteasome subtypes present in these cells. Adoptive transfer of these T cells in mice bearing EL4.P1A tumors reduced tumor growth and increased survival. CONCLUSIONS: We identified the first CD8+ T-cell epitope of the MAGE-type P1A tumor antigen presented in the H-2b background. This opens new perspectives for mechanistic studies dissecting MAGE-type specific antitumor immunity, making use of the wealth of genetically altered mouse strains available in the C57BL/6 background. This should facilitate the advancement of specific cancer immunotherapies.

Preventing Candida albicans from subverting host plasminogen for invasive infection treatment.

Candida albicans is a common fungal pathogen in humans that colonizes the skin and mucosal surfaces of the majority healthy individuals. How C. albicans disseminates into the bloodstream and causes life-threatening systemic infections in immunocompromised patients remains unclear. Plasminogen system activation can degrade a variety of structural proteins in vivo and is involved in several homeostatic processes. Here, for the first time, we characterized that C. albicans could capture and "subvert" host plasminogen to invade host epithelial cell surface barriers through cell-wall localized Eno1 protein. We found that the "subverted" plasminogen system plays an important role in development of invasive infection caused by C. albicans in mice. Base on this finding, we discovered a mouse monoclonal antibody (mAb) 12D9 targeting C. albicans Eno1, with high affinity to the 254FYKDGKYDL262 motif in α-helices 6, ß-sheet 6 (H6S6) loop and direct blocking activity for C. albicans capture host plasminogen. mAb 12D9 could prevent C. albicans from invading human epithelial and endothelial cells, and displayed antifungal activity and synergistic effect with anidulafungin or fluconazole in proof-of-concept in vivo studies, suggesting that blocking the function of cell surface Eno1 was effective for controlling invasive infection caused by Candida spp. In summary, our study provides the evidence of C. albicans invading host by "subverting" plasminogen system, suggesting a potential novel treatment strategy for invasive fungal infections.

Dectin-1 plays an important role in host defense against systemic Candida glabrata infection.

Candida glabrata is the second most common pathogen of severe candidiasis in immunocompromised hosts, following C. albicans. Although C. glabrata and C. albicans belong to the same genus, they are phylogenetically distinct. C-type lectin receptors (CLRs), acting as pattern-recognition receptors (PRRs), play critical roles in host defense against C. albicans infections. However, our understanding of the specific roles of CLRs in host defense against C. glabrata is limited. Here, we explored the potential roles of the C-type lectins Dectin-1 and Dectin-2 in host defense against C. glabrata. We found that both Dectin-1-deficient mice (Dectin-1-/-) and Dectin-2-deficient mice (Dectin-2-/-) are more susceptible to C. glabrata infection. Dectin-1confers host higher sensitivity for sensing C. glabrata infections, while the effect of Dectin-2 in the host defense against C. glabrata is infection dose dependent. Dectin-1 is required for host myeloid cells recognition, killing of C. glabrata, and development of subsequent Th1 and Th17 cell-mediated adaptive immune response. Significantly impaired inflammatory responses such as inflammatory cells recruitment and cytokines release that were induced by C. glabrata were manifested in Dectin-1-deficient mice. Together, our study demonstrates that Dectin-1 plays an important role in host defense against systemic Candida glabrata infections, indicating a previous unknown control mechanism for this particular type of infection in host. Our study, therefore, provides new insights into the host defense against C. glabrata.

Up-regulation of TDAG51 is a dependent factor of LPS-induced RAW264.7 macrophages proliferation and cell cycle progression.

CONTEXT: As a component of the outer membrane in Gram-negative bacteria, lipopolysaccharide (LPS)-induced proliferation and cell cycle progression of monocytes/macrophages. It has been suggested that the proapoptotic T-cell death-associated gene 51 (TDAG51) might be associated with cell proliferation and cell cycle progression; however, its role in the interaction between LPS and macrophages remains unclear. OBJECTIVE: We attempted to elucidate the role(s) of TDAG51 played in the interaction between LPS and macrophages. MATERIALS AND METHODS: We investigated TDAG51 expression in RAW264.7 cells stimulated with LPS and examined the effects of RNA interference-mediated TDAG51 down-regulation. We used CCK-8 assay and flow cytometry analysis to evaluate the interaction between TDAG51 and LPS-induced proliferation and cell cycle progression in RAW264.7 cells. RESULTS: Our findings indicate that TDAG51 is up-regulated in LPS-stimulated RAW264.7 cells, the TDAG51 siRNA effectively reduced TDAG51 protein up-regulation following LPS stimulation in RAW264.7 cells, the significant changes of the proliferation and cell cycle progression of RAW264.7 cells in TDAG51 Knockdown RAW264.7 cells treated with LPS were observed. CONCLUSION: These findings suggested that TDAG51 up-regulation is a dependent event during LPS-mediated proliferation and cell cycle progression, and which increase our understanding of the interaction mechanism between LPS and macrophages.

PI3 kinase/Akt/HIF-1α pathway is associated with hypoxia-induced epithelial-mesenchymal transition in fibroblast-like synoviocytes of rheumatoid arthritis.

Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1α expression and activation of Akt. After knockdown or inhibition of HIF-1α in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1α could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1α pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.

Celastrus orbiculatus extract inhibits tumor angiogenesis by targeting vascular endothelial growth factor signaling pathway and shows potent antitumor activity in hepatocarcinomas in Vitro and in Vivo.

OBJECTIVE: Celastrus orbiculatus Thunb. has been used for thousands of years in China as a remedy against cancer and inflammatory diseases. This study aims to investigate whether C. orbiculatus extract (COE) could inhibit angiogenesis, which is the pivotal step in tumor growth, invasiveness, and metastasis. METHODS: In this study, the extract from the stem of C. orbiculatus was used. Mouse hepatic carcinoma cells (Hepa1-6) were treated with COE in different nontoxic concentrations (10, 20, 40, 80, and 160 µg/mL). The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively; the active fractions were further tested on C57BL/6 mice and human umbilical vein endothelial cells (HUVEC) for any antiangiogenic effects. RESULTS: COE significantly inhibited proliferation and induced apoptosis in Hepa1-6 cells and inhibited VEGF expression at both mRNA and protein levels. Furthermore, this agent inhibited the formation of the capillary-like structure in primary cultured HUVEC in a dose-dependent manner. In vivo, COE significantly reduced the volume and weight of solid tumors with low adverse effects and decreased tumor angiogenesis. CONCLUSIONS: In summary, COE could be used to treat hepatic carcinoma. The mechanisms of the antitumor activity of COE may be due to its effects against tumor angiogenesis by targeting the VEGF protein.

[A single administration of morphine suppresses the reduction of the systemic immune activity caused by acute inflammatory pain in rats].

BACKGROUND: Pain accompanying various diseases as well as invasion and postoperative pain reduce immune activities, and affect the prognosis of diseases and recovery after surgery (metastasis and relapse). While, some anesthetics and synthetic narcotics used to reduce pain are reported to suppress immune activities depending on the kind of medication and the dosing strategy. However, it is not clear how the single use of narcotics affects the immune activity at the acute stage of severe inflammatory pain. This study is undertaken to examine the effect of a single administration of morphine on the splenic NK cell activity in the acute inflammatory pain model rats. METHODS: Rats received a 50 microl s.c. injection of 4% formaldehyde into the plantar surface of the right hindpaw. The spleen was removed 2 hours later and the splenic NK-cell activity was measured by 51Cr release assay. RESULTS: Acute pain significantly reduced the splenic NK cell activity, but the single administration of morphine suppressed its reduction. CONCLUSIONS: It was indicated that the single administration of morphine could suppress the reduction of the systemic immune activity caused by acute inflammatory pain.

Losartan blocks the excitatory effect of peripheral hypertonic stimulation on vasopressinergic neurons in hypothalamic paraventricular nucleus in rats: electrophysiological and immunocytochemical evidence.

The effect of peripheral hypertonic stimulation on the neurons of hypothalamic paraventricular nucleus (PVN) was investigated in the present study with both electrophysiological and immunocytochemical methods. The discharge frequency of the neurons with phasic activity in PVN could be increased by intraperitoneal (i.p.) injection of hypertonic saline (HS, 1.5M NaCl) (from 2.8 +/- 0.5 Hz to 5.4 +/- 0.9 Hz, P<0.001). The Fos expression in PVN could be enhanced (from 21.2 +/- 12.9 to 217.3 +/- 38.5 Fos-positive neurons, P<0.001) by i.p. HS and the majority of AVP-positive neurons expressing Fos (91.7 +/- 3.6%) was in magnocellular subdivision of PVN. After intracerebroventricular (i.c.v.) injection of losartan, angiotensin II type 1 (AT1) receptor antagonist (5 microg/microl), the excitatory effect of peripheral hypertonic stimulation on PVN neurons with phasic activity was inhibited significantly, and the number of the neurons co-expressing Fos and AVP in PVN decreased significantly (P<0.001) as well. The result demonstrated that the vasopressinergic neurons in PVN could be excited by peripheral hypertonic stimulation, and this excitation might be mediated by angiotensin II fibers projecting from subfornical organ to PVN.

Sympathetic nervous system mediates cold stress-induced suppression of natural killer cytotoxicity in rats.

The aim of the present study is to investigate the mechanisms of suppression of splenic natural killer (NK) cytotoxicity caused by cold stress, using 6-hydroxydopamine (6-OHDA) as chemical sympathectomy. The NK activity was measured by (51)chromium release assay. Central sympathectomy with intracerebroventricular (i.c.v.) injection of 6-OHDA reduced significantly the elevation of plasma corticosterone level, the expression of Fos in hypothalamic paraventricular nucleus and in locus coeruleus, as well as the suppression of NK activity induced by cold stress at 4 degrees C for 4 h. Peripheral sympathectomy with intraperitoneal (i.p.) injection of 6-OHDA and blockade of beta-adrenergic receptor with i.p. injection of propranolol also reversed the cold stress-induced suppression of NK cytotoxicity, but without significant effect on Fos expression in brain. The results suggest that the activation of hypothalamic-pituitary-adrenal axis induced by cold stress might be mediated, at least partially, by central noradrenergic system, and that the cold stress-induced suppression of NK cytotoxicity might be mediated by the activation of peripheral sympathetic nerve.

Sympathetic nervous system mediates cold stress-induced suppression of natural killer cytotoxicity in rats.

The aim of the present study is to investigate the mechanisms of suppression of splenic natural killer (NK) cytotoxicity caused by cold stress, using 6-hydroxydopamine (6-OHDA) as chemical sympathectomy. The NK activity was measured by (51)chromium release assay. Central sympathectomy with intracerebroventricular injection of 6-OHDA significantly reduced the elevation of the plasma corticosterone level, the expression of Fos in hypothalamic paraventricular nucleus and in locus coeruleus, as well as the suppression of NK activity induced by cold stress at 4 degrees C for 4 h. Peripheral sympathectomy with intraperitoneal (i.p.) injection of 6-OHDA and blockade of beta-adrenergic receptor with i.p. injection of propranolol also reversed the cold stress-induced suppression of NK cytotoxicity, but without significant effect on Fos expression in the brain. The results suggest that the activation of the hypothalamic-pituitary-adrenal axis induced by cold stress might be mediated, at least partially, by the central noradrenergic system, and that the cold stress-induced suppression of NK cytotoxicity might be mediated by the activation of the peripheral sympathetic nerve.

[Cold stress induces the suppression of splenic NK cell activity and the c-fos expression in rat brain].

AIM: To observe the effect of cold stress on the splenic NK cell activity and the c-fos expression in rat brain. METHODS: Rats were maintained in cold chamber at 4 degrees for 4 h. The 51Cr release assay from YAC-1 cells was used to determine the splenic NK cell activity and the double staining of ABC method was employed to observe the immunoreactive expression of Fos, arginine-vasopressin and tyrosine hydroxylase. RESULTS: Cold stress could induce a marked suppression of splenic NK cell activity and a significant expression of Fos protein in hypothalamic paraventricular nucleus(PVN), as well as in the brainstem locus coeruleus (LC). Double staining showed c-expression of Fos and arginine-vasopressin in some of PVN neurons, and co-expression of Fos and tyrosine hydroxylase in the majority of LC neurons. CONCLUSION: The vasopressinergic neurons in PVN and the catecholaminergic neurons in LC might be probably involved in the suppression of splenic NK cell activity induced by cold stress.