Description: More than 350 million people are chronically infected with hepatitis B virus, and dysfunctional T cell responses contribute to persistent viral infection and immunopathogenesis in chronic hepatitis B (CHB). However, the underlying mechanisms of T cell hyporesponsiveness remain largely undefined. Given the important role of microRNA-146a (miR-146a) in diverse aspects of lymphocyte function, we investigated the potential role and mechanism of miR-146a in regulating T cell immune responses in CHB. We found that miR-146a expression in T cells is significantly upregulated in CHB compared with healthy controls, and miR-146a levels were correlated with serum alanine aminotransaminase levels. Both inflammatory cytokines and viral factors led to miR-146a upregulation in T cells. Stat1 was identified as a miR-146a target that is involved in antiviral cytokine production and the cytotoxicity of CD4 + and CD8 + T cells. In vitro blockage of miR-146a in T cells in CHB greatly enhanced virus-specific T cell activity. Therefore, our work demonstrates that miR-146a upregulation in CHB causes impaired T cell function, which may contribute to immune defects and immunopathogenesis during chronic viral infection.

Description: Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene–induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell–dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Description: Anti-dsDNA Ab is reported to be the central pathogenic autoantibody involved in systemic lupus erythematosus (SLE) pathogenesis. However, the mechanisms involved in anti-dsDNA Ab production remain unclear. Recent evidence indicated that DNA-containing immune complexes (ICs) in circulation (termed "circulating DNA-containing ICs"), which are one of the hallmarks of SLE, might be involved in autoantibody production. In this study, we explored their potential role in anti-dsDNA Ab production and the underlying mechanisms in patients with SLE. We demonstrated that circulating DNA-containing ICs were able to induce anti-dsDNA Ab. Of note, HMGB1 in circulating DNA-containing ICs was crucial for anti-dsDNA Ab induction. The HMGB1 content of circulating DNA-containing ICs also correlated positively with anti-dsDNA Ab production in patients with SLE. Further, we revealed that the TLR2/MyD88/microRNA-155 (miR-155) pathway was pivotal for HMGB1 to confer anti-dsDNA Ab induction, and Ets-1 was a functional target of miR-155 in the induction of anti-dsDNA Ab by circulating DNA-containing ICs. Finally, we validated the expression of miR-155 and Ets-1 and their correlation with anti-dsDNA Ab production in patients with SLE. To our knowledge, this is the first report of the crucial role of HMGB1 in autoantibody production mediated by the TLR2/MyD88/miR-155/Ets-1 pathway. These findings identify a novel mechanism to account for the persistent production of anti-dsDNA Ab in SLE and a clue for developing a novel therapeutic strategy against SLE.

Description: 18 F-FPPRGD2, which was approved for clinical study recently, has favorable properties for integrin targeting and showed potential for antiangiogenic therapy and early response monitoring. However, the time-consuming multiple-step synthesis may limit its widespread applications in the clinic. In this study, we developed a simple lyophilized kit for labeling PRGD2 peptide ( 18 F-AlF-NOTA-PRGD2, denoted as 18 F-alfatide) using a fluoride–aluminum complex that significantly simplified the labeling procedure. Methods: Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18 F-alfatide, and 1 tuberculosis patient was investigated using both 18 F-alfatide and 18 F-FDG imaging. Standardized uptake values were measured in tumors and other main organs at 30 min and 1 h after injection. Kinetic parameters were calculated by Logan graphical analysis. Immunohistochemistry and staining intensity quantification were performed to confirm the expression of integrin α v β 3 . Results: Under the optimal conditions, the whole radiosynthesis including purification was accomplished within 20 min with a decay-corrected yield of 42.1% ± 2.0% and radiochemical purity of more than 95%. 18 F-alfatide PET imaging identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. Conclusion: 18 F-alfatide can be produced with excellent radiochemical yield and purity via a simple, 1-step, lyophilized kit. PET scanning with 18 F-alfatide allows specific imaging of α v β 3 expression with good contrast in lung cancer patients. This technique might be used for the assessment of angiogenesis and for planning and response evaluation of cancer therapies that would affect angiogenesis status and integrin expression levels.

Description: G protein–coupled receptor kinases (GRKs) phosphorylate the activated form of G protein–coupled receptors leading to receptor desensitization and downregulation. We have recently shown that the chemokine receptor, CXCR2, couples to GRK6 to regulate cellular responses including chemotaxis, angiogenesis, and wound healing. In this study, we investigate the role of GRK6 in tumorigenesis using murine models of human lung cancer. Mice deficient in GRK6 (GRK6 –/– ) exhibited a significant increase in Lewis lung cancer growth and metastasis relative to control littermates (GRK6 +/+ ). GRK6 deletion had no effect on the expression of proangiogenic chemokine or vascular endothelial growth factor, but upregulated matrix metalloproteinase (MMP)-2 and MMP-9 release, tumor-infiltrating PMNs, and microvessel density. Because β-arrestin-2–deficient (βarr2 –/– ) mice exhibited increased Lewis lung cancer growth and metastasis similar to that of GRK6 –/– , we developed a double GRK6 –/– /βarr2 –/– mouse model. Surprisingly, GRK6 –/– /βarr2 –/– mice exhibited faster tumor growth relative to GRK6 –/– or βarr2 –/– mice. Treatment of the mice with anti-CXCR2 Ab inhibited tumor growth in both GRK6 –/– and GRK6 –/– /βarr2 –/– animals. Altogether, the results indicate that CXCR2 couples to GRK6 to regulate angiogenesis, tumor progression, and metastasis. Deletion of GRK6 increases the activity of the host CXCR2, resulting in greater PMN infiltration and MMP release in the tumor microenvironment, thereby promoting angiogenesis and metastasis. Because GRK6 –/– /βarr2 –/– showed greater tumor growth relative to GRK6 –/– or βarr2 –/– mice, the data further suggest that CXCR2 couples to different mechanisms to mediate tumor progression and metastasis.

Description: Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti–non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti–non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.

Description: A single dynamic PET acquisition using multiple tracers administered closely in time could provide valuable complementary information about a tumor’s status under quasiconstant conditions. This study aimed to investigate the utility of dual-tracer dynamic PET imaging with 18 F-alfatide II ( 18 F-AlF-NOTA-E[PEG 4 -c(RGDfk)] 2 ) and 18 F-FDG for parametric monitoring of tumor responses to therapy. Methods: We administered doxorubicin to one group of athymic nude mice with U87MG tumors and paclitaxel protein-bound particles to another group of mice with MDA-MB-435 tumors. To monitor therapeutic responses, we performed dual-tracer dynamic imaging, in sessions that lasted 90 min, starting with injection via the tail vein catheters with 18 F-alfatide II, followed 40 min later by 18 F-FDG. To achieve signal separation of the 2 tracers, we fit a 3-compartment reversible model to the time–activity curve of 18 F-alfatide II for the 40 min before 18 F-FDG injection and then extrapolated to 90 min. The 18 F-FDG tumor time–activity curve was isolated from the 90-min dual-tracer tumor time–activity curve by subtracting the fitted 18 F-alfatide II tumor time–activity curve. With separated tumor time–activity curves, the 18 F-alfatide II binding potential (Bp = k 3 / k 4 ) and volume of distribution (V D ) and 18 F-FDG influx rate (( K 1 x k 3 )/( k 2 + k 3 )) based on the Patlak method were calculated to validate the signal recovery in a comparison with 60-min single-tracer imaging and to monitor therapeutic response. Results: The transport and binding rate parameters K 1 – k 3 of 18 F-alfatide II, calculated from the first 40 min of the dual-tracer dynamic scan, as well as Bp and V D correlated well with the parameters from the 60-min single-tracer scan ( R 2 〉 0.95). Compared with the results of single-tracer PET imaging, 18 F-FDG tumor uptake and influx were recovered well from dual-tracer imaging. On doxorubicin treatment, whereas no significant changes in static tracer uptake values of 18 F-alfatide II or 18 F-FDG were observed, both 18 F-alfatide II Bp and 18 F-FDG influx from kinetic analysis in tumors showed significant decreases. For therapy of MDA-MB-435 tumors with paclitaxel protein-bound particles, a significant decrease was observed only with 18 F-alfatide II Bp value from kinetic analysis but not 18 F-FDG influx. Conclusion: The parameters fitted with compartmental modeling from the dual-tracer dynamic imaging are consistent with those from single-tracer imaging, substantiating the feasibility of this methodology. Even though no significant differences in tumor size were found until 5 d after doxorubicin treatment started, at day 3 there were already substantial differences in 18 F-alfatide II Bp and 18 F-FDG influx rate. Dual-tracer imaging can measure 18 F-alfatide II Bp value and 18 F-FDG influx simultaneously to evaluate tumor angiogenesis and metabolism. Such changes are known to precede anatomic changes, and thus parametric imaging may offer the promise of early prediction of therapy response.

Description: The advancement of breast cancer therapy is limited by the biologic behaviors of cancer cells, such as metastasis and recurrence. β-adrenoceptors (ADRB) are reported to be associated with the biologic behaviors of breast cancer and may influence glucose metabolism. Here, we sought to investigate the relationship between the activation of ADRB and the expression of glucose transporter (GLUT)-1 and hexokinase (HK)-2 and to clarify the impact of ADRB on 18 F-FDG PET imaging in breast cancer. Methods: ADRB1/2 expression in 4T1, MDA-MB-231, and MCF-7 breast cancer cell lines was detected by Western blotting and immunofluorescence. ADRB-dependent regulation of GLUT-1 and HK-2 was determined by in vitro pharmacologic intervention. 4T1 breast cancer cells were treated with phosphate-buffered saline, isoproterenol, or propranolol, and the transcription and expression of GLUT-1 and HK-2 were measured by quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. ADRB1/2 was, respectively, blocked by small-interfering RNA to investigate the direct relationship between ADRB1/2 and HK-2. To evaluate the impact of ADRB on 18 F-FDG PET imaging, BALB/c mice bearing 4T1 tumors were injected with phosphate-buffered saline, isoproterenol, or propranolol, and 18 F-FDG PET imaging was performed. The tumor-to-nontumor (T/NT) values of tumors and brown adipose tissue were calculated by defining the liver as a reference. The in vivo expression of GLUT-1 and HK-2 was observed by immunohistochemical analysis and Western blotting. Results: MDA-MB-231, MCF-7, and 4T1 breast cancer cells were positive for ADRB1/2 expression. The protein expression and posttranscriptional level of HK-2 were significantly decreased by treatment with propranolol in vitro, whereas GLUT-1 expression was not significantly altered by pharmacologic intervention. The expression of HK-2 could be reduced in ADRB2-blocked 4T1 cells. Mice in the propranolol-treated group exhibited lower T/NT values for the tumors and brown adipose tissue than the control group. Immunohistochemical analysis and Western blotting revealed reduced HK-2 expression in the tumors of propranolol-treated mice. Conclusion: The expression of HK-2 was regulated by the activation of ADRB2 in 4T1 breast cancer cells primarily at the posttranscriptional level. Additionally, propranolol prevented glucose metabolism and 18 F-FDG PET imaging of 4T1 breast cancer tumors.

Description: Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. In this study, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow–derived mast cells enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null bone marrow–derived mast cells showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1 deficiency led to specific attenuation of IB–NF-B pathway and IKK–SNAP23 pathway, whereas calcium flux, MAPK, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.

Description: HLA-DO/H2-O is a highly conserved, nonpolymorphic MHC class II-like molecule expressed in association with H2-M in thymic epithelial cells, B lymphocytes, and primary dendritic cells. The physiological function of DO remains unknown. The finding of cell maturation-dependent DO expression in B lymphocytes and dendritic cells suggests the possibility that H2-O functions to promote the presentation of exogenous Ag by attenuating presentation of endogenous self-peptides. In the current study, we report that H2-O –/– mice spontaneously develop high titers of IgG2a/c antinuclear Abs (ANAs) with specificity for dsDNA, ssDNA, and histones. Reconstitution of RAG1 – / – mice with T and B cells from H2-O – / – or wild-type mice demonstrated that production of ANAs requires participation of CD4 + T cells from H2-O – / – mice. Bone marrow chimeras demonstrated that loss of H2-O expression in thymic epithelial cells did not induce ANAs, and that lack of H2-O expression in bone marrow-derived cells was sufficient to induce the autoimmune phenotype. Despite production of high titers of autoantibodies, H2-O –/– mice exhibit a delayed generation of humoral immunity to model Ags (OVA and keyhole limpet hemocyanin), affecting all major T-dependent Ig classes, including IgG2a/c. Ag presentation experiments demonstrated that presentation of exogenous Ag by H2-O –/– APC was inefficient as compared with wild-type APC. Thus, H2-O promotes immunity toward exogenous Ags while inhibiting autoimmunity. We suggest that H2-O, through spatially or temporally inhibiting H2-M, may enhance presentation of exogenous Ag by limiting newly generated MHC class II molecules from forming stable complexes with endogenous self-peptides.