CCL13 is upregulated in alopecia areata lesions and is correlated with disease severity.

Alopecia areata (AA) is a multi-factors disease characterized by non-scarring hair loss. AA could be classified into three main clinical phenotypes including patchy type AA (AAP), alopecia totalis (AT) and alopecia universalis (AU) based on the severity and areas of hair loss. Recent studies suggested immunological factor was critical in AA, but the precise aetiology and pathogenesis of AA still need exploration. In the work, we screened two gene expression profiles (GSE45512 and GSE68801) from Gene Expression Omnibus (GEO). Based on the two data sets, 10 upregulated genes and 107 downregulated genes in AA skin biopsies were identified. CCL13, as one of the remarkably upregulated genes, was found to have potential biological functions in aberrant immune response of AA according to the GO and KEGG analyses. The PPI network showed CCL13 was associated with multiple immune-related genes. The expression of CCL13 was increased depending on the severity of disease in AA patients. Cytotoxic lymphocytes, T cells and myeloid dendritic cells accumulated remarkably in scalp tissue depending on the severity of AA, and CCL13 was significantly correlated to cytotoxic lymphocytes, T cells and myeloid dendritic cells in AA patients. Our RT-PCR and ELISA results found CCL13 was upregulated in skin biopsy and serum of AA patients, and the immunohistochemistry (IHC) detection showed CCL13 was expressed by both the hair follicle epithelium and infiltrating immune cells. In conclusion, the upregulated of CCL13 and subsequent immune cell infiltration was related to AA, which could be a promising target for diagnosis and therapy in AA patients.

Osteoarthritis-associated basic calcium phosphate crystals alter immune cell metabolism and promote M1 macrophage polarization.

OBJECTIVE: A number of studies have demonstrated that molecules called 'alarmins' or danger-associated molecular patterns (DAMPs), contribute to inflammatory processes in the OA joint. Metabolic reprogramming of immune cells, including macrophages, is emerging as a prominent player in determining immune cell phenotype and function. The aim of this study was to investigate if basic calcium phosphate (BCP) crystals which are OA-associated DAMPs, impact on macrophage phenotype and metabolism. METHODS: Human monocyte derived macrophages were treated with BCP crystals and expression of M1 (CXCL9, CXCL10) and M2 (MRC1, CCL13)-associated markers was assessed by real-time PCR while surface maturation marker (CD40, CD80 & CD86) expression was assessed by flow cytometry. BCP induced metabolic changes were assessed by Seahorse analysis and glycolytic marker expression (hexokinase 2(HK2), Glut1 and HIF1α) was examined using real-time PCR and immunoblotting. RESULTS: Treatment with BCP crystals upregulated mRNA levels of CXCL9 and CXCL10 while concomitantly downregulating expression of CCL13 and MRC1. Furthermore, BCP-treated macrophages enhanced surface expression of the maturation makers, CD40, CD80 and CD86. BCP-treated cells also exhibited a shift towards glycolysis as evidenced by an increased ECAR/OCR ratio and enhanced expression of the glycolytic markers, HK2, Glut1 and HIF1α. Finally, BCP-induced macrophage activation and alarmin expression was reduced in the presence of the glycolytic inhibitor, 2-DG. CONCLUSIONS: This study not only provides further insight into how OA-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for calcium crystal-related arthropathies.

The CC chemokines CCL8, CCL13 and CCL20 are local inflammatory biomarkers of HLA-B27-associated uveitis.

PURPOSE: To determine the concentrations of the CC chemokines CCL2, CCL7, CCL8, CCL11, CCL13, CCL20, CCL24 and CCL26 in aqueous humour (AH) samples from patients with specific uveitic entities. METHODS: Aqueous humour samples from patients with active uveitis associated with Behçet's disease (BD) (n = 13), sarcoidosis (n = 8), HLA-B27-related inflammation (n = 12), Vogt-Koyanagi-Harada (VKH) disease (n = 12) and control patients (n = 9) were assayed with the use of a multiplex assay. RESULTS: When considering all uveitis patients as one group, all chemokine levels except CCL2 were significantly increased compared to controls. CCL8, CCL13 and CCL20 were the most strongly upregulated, 48-fold, 118-fold and 173-fold, respectively, above control AH levels. CCL8 and CCL13 levels were significantly higher in HLA-B27-associated uveitis than in sarcoidosis and VKH disease. CCL20 levels were significantly higher in HLA-B27-associated uveitis than in BD, sarcoidosis and VKH disease. In addition, CCL20 levels were significantly higher in BD than in VKH disease. In HLA-B27-associated uveitis, CCL8, CCL13 and CCL20 were upregulated 111-fold, 255-fold and 465-fold, respectively, compared with controls. CCL8, CCL13 and CCL20 levels were significantly higher in nongranulomatous uveitis (BD and HLA-B27-associated uveitis) than in granulomatous uveitis (sarcoidosis and VKH disease). CONCLUSION: Immune responses mediated by CCL8, CCL13 and CCL20 appear to be more potent in nongranulomatous uveitis, particularly in HLA-B27-associated uveitis.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

Galectin-8 activates dendritic cells and stimulates antigen-specific immune response elicitation.

Galectin-8 (Gal-8) is a mammalian ß-galactoside-binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen-specific immune responses in vivo, we investigated whether Gal-8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow-derived DCs (BMDCs) treated with exogenous Gal-8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal-8-treated BMDCs (Gal-8-BMDCs) stimulated antigen-specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL-3, IL-2, IL-6, TNF, MCP-1, and MCP-5, as well as growth factor G-CSF, were augmented in Gal-8-BMDC conditioned media, with IL-6 as the most prominent. Remarkably, BMDCs from Gal-8-deficient mice (Lgals8-/- BMDC) displayed reduced CD86 and IL-6 expression and an impaired ability to promote antigen-specific CD4 T cell activation. To test if Gal-8-induced activation correlates with the elicitation of an effective immune response, soluble Gal-8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot-and-mouth disease virus (FMDV) model. When a single dose of Gal-8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL-6 and IFN-γ, as well as lymphoproliferative responses, were also incremented in Gal-8/antigen-immunized animals only at 48 h after immunization, suggesting that Gal-8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal-8 as an immune-stimulator molecule to enhance the adaptive immune response.

MDR1A deficiency restrains tumor growth in murine colitis-associated carcinogenesis.

Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.

Duodenal Mucosa of Patients With Type 1 Diabetes Shows Distinctive Inflammatory Profile and Microbiota.

Context: Increasing evidences suggest a correlation between gut and type 1 diabetes (T1D). Objective: The objective of this study is to evaluate the gut inflammatory profile and microbiota in patients with T1D compared with healthy control (CTRL) subjects and patients with celiac disease (CD) as gut inflammatory disease controls. Design/Setting/Participants: The inflammatory status and microbiome composition were evaluated in biopsies of the duodenal mucosa of patients with T1D (n = 19), in patients with CD (n = 19), and CTRL subjects (n = 16) recruited at San Raffaele Scientific Institute, in Milan, Italy, between 2009 and 2015. Main Outcome Measures: Inflammation was evaluated by gene expression study and immunohistochemistry. Microbiome composition was analyzed by 16S ribosomal RNA gene sequencing. Results: An increased expression of CCL13, CCL19, CCL22, CCR2, COX2, IL4R, CD68, PTX3, TNFα, and VEGFA was observed in patients with T1D compared with CTRL subjects and patients with CD. Immunohistochemical analysis confirmed T1D-specific inflammatory status compared with healthy and CD control tissues, mainly characterized by the increase of the monocyte/macrophage lineage infiltration. The T1D duodenal mucosal microbiome results were different from the other groups, with an increase in Firmicutes and Firmicutes/Bacteroidetes ratio and a reduction in Proteobacteria and Bacteroidetes. The expression of genes specific for T1D inflammation was associated with the abundance of specific bacteria in the duodenum. Conclusions: This study shows that duodenal mucosa in T1D presents disease-specific abnormalities in the inflammatory profile and microbiota. Understanding the mechanisms underlying these features is critical to disentangle the complex pathogenesis of T1D and to gain new perspectives for future therapies targeting the intestine.

Beneficial impact of CCL2 and CCL12 neutralization on experimental malignant pleural effusion.

Using genetic interventions, we previously determined that C-C motif chemokine ligand 2 (CCL2) promotes malignant pleural effusion (MPE) formation in mice. Here we conducted preclinical studies aimed at assessing the specific therapeutic potential of antibody-mediated CCL2 blockade against MPE. For this, murine MPEs or skin tumors were generated in C57BL/6 mice by intrapleural or subcutaneous delivery of lung (LLC) or colon (MC38) adenocarcinoma cells. Human lung adenocarcinoma cells (A549) were used to induce MPEs in severe combined immunodeficient mice. Intraperitoneal antibodies neutralizing mouse CCL2 and/or CCL12, a murine CCL2 ortholog, were administered at 10 or 50 mg/kg every three days. We found that high doses of CCL2/12 neutralizing antibody treatment (50 mg/kg) were required to limit MPE formation by LLC cells. CCL2 and CCL12 blockade were equally potent inhibitors of MPE development by LLC cells. Combined CCL2 and CCL12 neutralization was also effective against MC38-induced MPE and prolonged the survival of mice in both syngeneic models. Mouse-specific CCL2-blockade limited A549-caused xenogeneic MPE, indicating that host-derived CCL2 also contributes to MPE precipitation in mice. The impact of CCL2/12 antagonism was associated with inhibition of immune and vascular MPE-related phenomena, such as inflammation, new blood vessel assembly and plasma extravasation into the pleural space. We conclude that CCL2 and CCL12 blockade are effective against experimental MPE induced by murine and human adenocarcinoma in mice. These results suggest that CCL2-targeted therapies may hold promise for future use against human MPE.

Early induction of CCL7 downstream of TLR9 signaling promotes the development of robust immunity to cryptococcal infection.

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.

Antitumor activity mediated by CpG: the route of administration is critical.

Unmethylated CpG oligodeoxynucleotides (CpG) are synthetic toll-like receptor 9 agonists that activate innate immune cells and which have been tested as an immune therapy in a number of cancer clinical trials. Although some antitumor immune responses have been reported, so far the majority of studies have failed to show significant clinical responses to CpG. Here we showed that the route of administration is critical to the antitumor activity of CpG. Although intravenous (i.v.) injection of CpG was capable of inducing the activation and expansion of tumor antigen-specific T cells, most of these activated T cells failed to migrate to tumor sites. By contrast, intratumoral (i.t.) injection of CpG led to extensive tumor infiltration of antigen-specific T cells and subsequent tumor suppression. We further showed that very high levels of inflammatory chemokines [regulated upon activation, normal T-cell expressed, and secreted (RANTES), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, monocyte chemotactic protein (MCP5), macrophage inflammatory proteins (MIP1α, and MIP1ß)] were induced in the tumor microenvironment after i.t. CpG injection, compared with administration by the i.v. route. It is interesting to note that, in vivo depletion of plasmacytoid dendritic cells greatly reduced the levels of chemokines induced; also, T-cell accumulation and antitumor effect were impaired. We also showed that i.t. but not i.v. CpG injection induced a broad antigen-specific T-cell response against tumor-derived antigens. Collectively, our data provides evidence that the route of CpG administration is a critical factor in mediating antitumor activity. By inducing localized inflammatory signals at tumor sites, i.t. CpG effectively promotes the migration, activation and function of immune cells, ultimately leading to improved tumor control.

Combining epitope-distinct antibodies to HER2: cooperative inhibitory effects on invasive growth.

Monoclonal antibodies (mAbs) to HER2 are currently used to treat breast cancer, but low clinical efficacy, along with primary and acquired resistance to therapy, commonly limit clinical applications. We previously reported that combinations of antibodies directed at non-overlapping epitopes of HER2 are endowed with enhanced antitumor effects, probably due to accelerated receptor degradation. Here, we extend these observations to three-dimensional mammary cell models, and compare the effects of single mAbs with the effects of antibody combinations. Collectively, our in vitro assays and computational image analyses indicate that combining mAbs against different epitopes of HER2 better inhibits invasive growth. Importantly, while growth factors are able to reduce intraluminal apoptosis and induce an invasive phenotype, combinations of mAbs better than single mAbs can reverse the growth factor-induced phenotypes of HER2-overexpressing spheroids. In conclusion, our studies propose that mAb combinations negate the biological effects of growth factors on invasive growth of HER2-overexpressing cells. Hence, combining mAbs offers a therapeutic strategy, potentially able to enhance clinical efficacy of existing antireceptor immunotherapeutics.

CCL2 blockade augments cancer immunotherapy.

Altering the immunosuppressive microenvironment that exists within a tumor will likely be necessary for cancer vaccines to trigger an effective antitumor response. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immunoinhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the human papillomavirus E7 antigen expressed by a non-small cell lung cancer (NSCLC) line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus-expressing IFN-alpha to treat a nonimmunogenic NSCLC line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intratumoral CD8+ T cells that were more activated and more antitumor antigen-specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T regulatory cells. CCL2 seems to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T-cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.

CDIP-2, a synthetic peptide derived from chemokine (C-C motif) ligand 13 (CCL13), ameliorates allergic airway inflammation.

Airway inflammation is characterized by selective recruitment of mononuclear and granulocytic cells. This recruitment is mediated by the action of chemotactic cytokines, such as chemokines. A number of chemokines and their receptors have been identified and proposed as potential therapeutic agents in allergic airway inflammation. One of these chemokines is chemokine (C-C motif) ligand 13 (CCL13), a CC chemokine that has been associated with allergic inflammatory diseases such as asthma and allergic rhinitis. To investigate alternative therapeutic agents to alleviate allergic inflammatory diseases, a number of chemokine-derived synthetic peptides were designed and tested for their ability to modulate in vitro and in vivo chemokine-mediated functions. Our results show that one of these peptides, CDIP-2, displayed antagonist functions in in vitro chemotaxis assays using monocytic cell lines. In addition, we found that CDIP-2 significantly reduced peribronchial, perivascular infiltrate and mucus overproduction in an ovalbumin-induced allergic lung inflammation murine model. Thus, CDIP-2 may be considered as part of a novel group of anti-inflammatory agents based on chemokine-derived synthetic peptides.

Chemokine-mediated inflammation: Identification of a possible regulatory role for CCR2.

The chemokine receptor CCR2 binds four pro-inflammatory monocyte chemoattractant proteins, designated MCP1/CCL2, MCP2/CCL8, MCP3/CCL7 and MCP4/CCL13. This study demonstrates the important biology of this receptor during the response to the chemokine milieu. Competitive chemotaxis and calcium flux assays were performed utilising mixtures of chemokines to assess a hierarchal arrangement of chemokine prepotency; these demonstrated that the MCP2-CCR2 interaction is able to supersede signals generated by RANTES, another pro-inflammatory chemokine, or the homeostatic chemokine SDF1. These observations were validated using three physiologically relevant monocytic cell lines. Having identified the importance of CCR2, experiments were then performed to examine the signal transduction processes coupled to this receptor. G protein coupling was initially examined; Cholera toxin reduced the chemotactic response to MCP2 (p<0.001), whilst the response to the other MCP chemokines remained normal. The response to MCP2 was uniquely inhibited by elevated concentrations of cAMP and, unlike MCP1, 3 and 4 (p<0.05), MCP2 failed to inhibit adenylate cyclase. Expression of dominant negative H-ras demonstrated that each MCP chemokine required active ras in order to elicit ERK activation and a chemotactic response. Unlike MCP1, MCP2 failed to induce nuclear translocation of activated ERK1 or subsequent induction of c-Myc expression. Akt activation also showed ligand-specific differences, with MCP2 producing a delayed response compared to the other MCP chemokines. Together these data highlight the importance of CCR2 and suggest that it is a powerful tool for fine tuning the immune response.

Generation, characterization and biological activity of CCL2 (MCP-1/JE) and CCL12 (MCP-5) specific antibodies.

The human CCL2 chemokine is implicated in many chronic inflammatory conditions. In the mouse, there are two CCL2 homologues, CCL2 (MCP-1/JE) and CCL12 (MCP-5). Both are potent monocyte chemoattractants and bind to and activate the same receptor, CCR2. The overlapping activities of these chemokines complicate the design of mouse model studies that are intended to mimic human disease. To study the roles of CCL2 and CCL12, we generated neutralizing antibodies specific to each chemokine. Consistent with binding and affinity analyses, the antibodies specifically inhibited CCL2- or CCL12- mediated Ca(2+) mobilization in THP-1 cells. When tested in nude mice bearing human PANC-1 pancreatic tumor cells in Matrigel plugs, CCL2 and CCL12 antibodies potently inhibited tumor angiogenesis, indicating that both CCL2 and CCL12 may contribute to tumor angiogenesis.

Inflammatory markers and sleep disturbance in major depression.

OBJECTIVE: This study was conducted to determine whether immune activation occurs in major depression, and to evaluate the associations between disordered sleep and markers of inflammation in patients with major depressive disorder. METHODS: All-night polysomnography was obtained in patients with acute Diagnostic and Statistical Manual of Mental Disorders, 4th edition major depressive disorder (n = 22) and age-, gender-, and body weight-matched comparison controls (n = 18). After the onset of sleep, nocturnal serum levels of interleukin-6 (IL-6), soluble intercellular adhesion molecule (sICAM), monocyte chemotactic protein (MCP-1), and IL-6 soluble receptor (IL-6sR) were sampled. RESULTS: As compared with matched controls, depressed patients showed significant (p <.05) nocturnal elevations of circulating levels of IL-6 and sICAM. Both sleep latency and rapid eye movement (REM) density had moderate correlations with IL-6 and sICAM (r's > or = 0.30). Backward regression analyses indicated that sleep latency (beta = 0.34, p <.05) and REM density (beta = 0.27, p = .09) were better predictors of IL-6 than depressive status. Similarly, sleep latency (beta = 0.27, p = .06) and REM density (beta = 0.32, p = .02) were also better predictors of sICAM. CONCLUSION: These findings support the hypothesis that sleep disturbance is associated with elevated levels of the inflammatory markers IL-6 and sICAM. This relationship was not accounted for by other confounding factors such as age and body weight. These findings suggest that the elevations in inflammatory markers found in depressive subjects may be partially the result of disturbances of sleep initiation found in this population.

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells.

The stromal compartments of hematopoietic organs (eg, spleen) are known to influence the viability and growth of diseased hematopoietic progenitors. Here we have used Friend murine leukemia virus (F-MuLV)-induced erythroleukemia to investigate factors of the splenic microenvironment that may make it fertile for the expansion and survival of malignant erythroblasts. We found that splenectomized, erythroleukemic mice exhibited extended survival compared with age-matched sham controls. In vitro, the proliferation of primary erythroleukemic cells cocultured with leukemic-derived splenic adherent cells or their conditioned media was found to be significantly higher than that observed in cocultures with healthy-derived adherent splenic cells. Cytokine protein arrays revealed that F-MuLV-infected splenocytes secreted elevated levels of interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), macrophage chemoattractant protein-5 (MCP-5), soluble tumor necrosis factor receptor-1 (sTNFR1), IL-12p70, tumor necrosis factor-alpha (TNF-alpha), and IL-2 over normal splenocytes. Medium supplemented with both VEGF-A and MCP-5 could sustain proliferation of primary erythroleukemic cells in vitro, and significant proliferative suppression was observed upon addition of neutralizing antibodies to either of these factors. Furthermore, in vivo administration of a neutralizing antibody to VEGF-A extended survival times of erythroleukemic mice in comparison with controls. These findings suggest that VEGF-A and MCP-5 are potentially pivotal paracrine mediators occurring within the diseased splenic microenvironment capable of promoting disease acceleration and expansion of erythroleukemic blasts.

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination.

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-gamma production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8(+) T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

Alterations in eotaxin, monocyte chemoattractant protein-4, interleukin-5, and interleukin-13 after systemic steroid treatment for nasal polyps.

OBJECTIVE: To determine alterations in Th2 chemokines eotaxin and monocyte chemoattractant protein-4 (MCP-4), and cytokines interleukin-5 (IL-5) and interleukin-13 (IL-13), in nasal polyps (NP) after steroid treatment. STUDY DESIGN: Cytokine/chemokine levels were measured in NP before and after steroid therapy and compared to control sinus mucosa. RESULTS: Twenty-one patients (control = 7, NP = 14) were enrolled. Eotaxin and MCP-4 were significantly higher than control tissue (P = 0.004 and 0.003). All four mediators decreased after steroid treatment (P < 0.03). IL-5 and IL-13 in untreated polyps were not significantly different from controls. Patients showed clinical improvement according to SNOT-20 scores (average presteroid score 19, poststeroid score 13) and endoscopic grading (1.75 each side presteroid, 1.13 poststeroid). CONCLUSIONS: Steroids significantly decreased all cytokine/chemokine levels, but the impact on Th2 chemokines was of a much greater magnitude. SIGNIFICANCE: Novel approaches to block inflammatory mediators, particularly Th2 chemokines, may lead to better control of nasal polyposis in the future.

[Construction of a DNA vaccine against human B cell lymphoma].

AIM: To construct a DNA vaccine based on surface Ig V(H) gene of tumor cells in the human B-cell lymphoma biopsy tissue. METHODS: The V(H) gene fragment was amplified by RT-PCR using Ig superfamily primers. Also, the murine monocyte chemotactic protein 3(MCP-3) cDNA was cloned. The fusion gene fragment of MCP-3 gene with V(H) gene was constructed by recombinant PCR and then cloned into the eukaryotic expression vector pcDNA3.1 to construct the DNA vaccine plasmid pcDNA3.1/MCP-V(H). The vaccine plasmid was transiently expressed in the eukaryotic cell line COS-7. RESULTS: The DNA vaccine plasmid was successfully constructed and expressed in COS-7 cells in the form of fusion protein MCP-V(H). CONCLUSION: The DNA vaccine plasmid pcDNA3.1/MCP-V(H) is constructed and expressed successfully, which plays the foundation for further experimental research in animal model.