Apoptosis inhibitor of macrophage as a biomarker for disease activity in Japanese children with IgA nephropathy and Henoch-Schönlein purpura nephritis.

BACKGROUND: To evaluate the apoptosis inhibitor of macrophage (AIM) deposition patterns on the kidneys of children with IgA nephropathy (IgAN) and Henoch-Schönlein purpura nephritis (HSPN) and to investigate the clinical usefulness of serum and/or urinary AIM levels as biomarkers for the disease activity. METHODS: Immunohistochemical study was performed in the kidneys of 37 patients with IgAN and 10 patients with HSPN. Serum and urinary AIM levels in the patients and 20 healthy controls (HCs) were quantified by enzyme-linked immunosorbent assay. The results were compared with clinical features. RESULTS: In patients with IgAN and HSPN, AIM expression was observed in various areas, including the glomerular mesangial and capillary areas, the proximal and distal tubular epithelial cells, and on infiltrating macrophages in the glomeruli and interstitial areas. Serum and urinary AIM levels were significantly elevated in these patients compared with the HCs. Urinary AIM levels were positively correlated with the histological severity and degree of proteinuria and hematuria as well as urinary ß2 microglobulin and urinary N-acetyl-ß-D-glucosaminidase levels. CONCLUSIONS: AIM plays an important role in the pathogenesis of IgAN and HSPN. Urinary AIM levels can potentially reflect active renal inflammation in these diseases and may represent a useful biomarker for disease activity. IMPACT: Urinary AIM levels may represent a useful biomarker for disease activity of IgAN and HSPN. AIM expression was observed in the glomeruli, tubular epithelial cells, and infiltrating macrophages in glomeruli and interstitial area. U-AIM/Cr were significantly correlated not only with proteinuria, hematuria, and u-ß2MG and u-NAG levels but also with the activity index of histological findings in kidney biopsy specimens. Our results can emphasize the important role of AIM in the pathogenesis of IgAN and HSPN.

Expression of apoptosis inhibitor of macrophages in tissue macrophages, leukocytes and vascular endothelial cells of dogs.

Apoptosis inhibitor of macrophages (AIM) is a protein which plays important roles in controlling the immune response and inflammation in human and mice. In dogs, AIM is reported to be expressed in cancerated macrophages and regulate the survival of these tumor cells. In this study, to elucidate the physiological expression pattern of AIM in dogs, systemic expression and distribution of AIM of dogs were investigated. Mature healthy Beagles were used. Various tissues, peripheral blood cells, and bone marrow cells of normal dogs were collected for in situ hybridization, real-time RT-PCR, and immunohistochemistry. AIM mRNA and protein were expressed in tissue macrophages of the spleen, liver, lungs, and lymph nodes, but not in the microglia of the cerebrum. Proximal tubules in the kidney also expressed AIM protein. Monocytes and B lymphocytes in circulating blood and a part of microvasculature endothelial cells showed AIM expression at both the mRNA and protein levels. In the bone marrow, early-stage monocyte progenitor-like cells expressed AIM mRNA and protein. These results clarified that AIM is expressed in more cell types than previously reported in human and mice. These data spread the possibility of AIM physiological functions and implies the relationship of AIM to the maturation of macrophage-strain cells in dogs and other species.

Morphological characteristics of fatal pediatric hand, foot and mouth disease: A clinicopathological study with related receptors of EV71.

OBJECTIVE: To investigate the pathological features of fatal pediatric hand foot and mouth disease (HFMD). METHODS: The histopathological features of HFMD were first summarized from literature, and then confirmed by in-house autopsies. Furthermore, immunohistochemistry was conducted to detect the distribution and expression level of two enterovirus 71 (EV71) receptors scavenger receptor class B, member 2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL1) in the samples of autopsies. RESULTS: The main symptoms of HFMD included hand and foot rashes, as well as oral herpes. The fatal HFMD patients had typical histopathological change in the central nervous system, such as encephaledema and encephalitis. As for respiratory system, the fatal HFMD patients suffered acute pulmonary edema and congestion. SCARB2 positive signaling was distributed equally in bronchial and bronchiolar epithelial cells, alveolar epithelial cells and inflammatory cells of all HFMD patients, healthy children and adults without significant difference. PSGL-1 dispersed in bronchial and bronchiolar epithelial cells of healthy adults, but no PSGL-1 expression was detected in HFMD patients and healthy children. CONCLUSIONS: Both of the central nervous and respiratory systems may be involved in the fatal HFMD patients. The EV71 receptor PSGL-1 might play essential parts in the pathogenesis of fatal HFMD, however, the hypothesis needs to be further investigated.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

BACKGROUND AIMS: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. METHODS: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. RESULTS: The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R2 = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. CONCLUSIONS: Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.

CXCL16 induces angiogenesis in autocrine signaling pathway involving hypoxia-inducible factor 1α in human umbilical vein endothelial cells.

Chemokine (C-X-C motif) ligand 16 (CXCL16) is a new angiogenic factor inducing angiogenesis via extracellular signal-regulated kinases pathway. To further understand the molecular mechanism underlying CXCL16­induced angiogenesis, we explored involvement of other relevant pathways in CXCL16-induced angiogenesis. In the present study, we investigated the mechanisms underlying CXCL16-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). CXCL16 promoted HUVEC proliferation, tube formation and migration. Enzyme-linked immunosorbent assay revealed that CXCL16 induced vascular endothelial growth factor secretion from HUVECs. Western blot analysis showed that CXCL16 increased the level of hypoxia­inducible factor 1α, p-extracellular signal-regulated kinases (ERK), p-p38 and p-Akt dose- and time-dependently. ERK-, p38- and Akt-selective inhibitors significantly suppressed HUVEC proliferation, migration, tube formation and hypoxia-inducible factor 1α (HIF-1α) expression induced by CXCL16. Furthermore, CXCL16 peptides induced CXCL16 secretion via ERK, p38 and Akt pathways, which was suppressed by HIF-1α-selective inhibitor PX12. Our data suggest that CXCL16 induces angiogenesis in autocrine manner via ERK, Akt, p38 pathways and HIF-1α modulation.

VISFATIN PROMOTES FOAM CELL FORMATION BY DYSREGULATING CD36, SRA, ABCA1, AND ABCG1 EXPRESSION IN RAW264.7 MACROPHAGES.

BACKGROUND: Visfatin is produced in and secreted from adipocytes. Increased circulating visfatin level is observed in obese subjects. Previous studies demonstrated that visfatin was involved in obesity-related cardiovascular diseases. AIMS: This study aims to explore the regulatory effects of adipokine visfatin on foam cell formation, a key step in the development of atherosclerosis. METHODS: Effect of visfatin on protein and mRNA expression of scavenger receptor and ATP binding cassette transporter in RAW264.7 macrophages were measured by western blotting and real-time RT-PCR. To confirm the influence of visfatin-regulated scavenger receptor and ATP binding cassette transporter to foam cell formation, the visfatin-caused changes of ox-LDL uptake, cholesterol efflux, and foam cell formation were determined. RESULTS: Visfatin significantly increased the expression of CD36 and scavenger receptor A (SRA), decreased the expression of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and had no effect on the expression of SR-B1. Visfatin increased oxidized-LDL (ox-LDL) uptake and decreased cholesterol efflux, which increased foam cell formation. The PI3K inhibitor LY294002 blocked the effect of visfatin on the protein and mRNA expression levels of CD36, SRA, and ABCG1 and ox-LDL uptake and cholesterol efflux. The ERK inhibitor PD98059 also prevented visfatin-induced ABCA1 instability and subsequently decreased cholesterol efflux. CONCLUSIONS: Visfatin upregulated CD36 and SRA expression and downregulated ABCA1 and ABCG1 expression, subsequently increased ox-LDL uptake and decreased cholesterol efflux, and finally promoted foam cell formation via the PI3K- and ERK-dependent pathways.

[Cloning and expression of scavenger receptor class B BmSCRB8 in silkworm Bombyx mori].

Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63.87 kDa and the isoelectric point (pI) is 6.06. The space-time expression profile of BmSCRB8 was detected by reverse transcription PCR (RT-PCR), which implicated that BmSCRB8 is extensively expressed in each tissue and at each stage of blood. In addition, BmSCRB8 is highest expressed in fat body of silkworm, and is highly expressed in metamorphosis periods. Anti-BmSCRB8 polyclonal antibody was generated through prokaryotic expression, protein purification and mice immunization. Simultaneously, we constructed BmSCRB8 eukaryotic vector and then transfected embryonic cell line of silkworm. Immunofluorescence and overexpression showed that BmSCRB8 expressed specifically in membrane. Western blotting demonstrated that BmSCRB8 protein can be specifically recognized by anti-serum generated after mice immunization.

CXCL16 in Vascular Pathology Research: from Macro Effects to microRNAs.

Chemokines and their receptors have become significant factors in atherosclerosis research. CXCL16 is a multifunctional agent located on a separate locus to all other known chemokines and binds only to its "unique" receptor named CXCR6. As a scavenger receptor, adhesion molecule, and chemokine, it quickly became an interesting target in atherosclerosis research as all its functions have a role in vascular pathology. The investigation of the role of CXCL16 in atherosclerosis, although shown in in vitro studies, animal knockout models, and CXCL16 gene polymorphisms, haplotypes, and circulating levels, still shows puzzling results. Genetic and epigenetic studies have just scratched the surface of research necessary for a better assessment of the significance and perspective of this marker in plaque development and progression. In this review, we will summarize current knowledge about CXCL16 in atherosclerosis. Additionally, we will point out the importance of bioinformatics tools for the detection of potentially new CXCL16 regulatory networks through microRNA activity. This review aims to provide a better understanding of the underlying mechanisms, define more specific biomarkers, and discover new therapeutic targets.

MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions.

Nasopharyngeal carcinoma (NPC) is a common malignancy in southern China and Southeast Asia. NPC frequently metastasizes to the bone in advanced patients resulting in high mortality. The molecular mechanisms for NPC development and cancer-induced bone lesions are unclear. In this study, we firstly determined chemokine receptor CCR2 and CXCR6 expressions in clinical specimens and CNE2, SUNE1, CNE1, and HK1 cell lines. Then, we measured chemokine CCL2 and CXCL16 production in these NPC cell lines by ELISA. Expression levels of these chemokines and their receptors were observed to positively correlate with tumor aggressiveness. Furthermore, U0126 (MEK inhibitor) was used to treat these NPC cell lines. CCL2 and CXCL16 expression levels and cell proliferation were significantly inhibited by U0126 in a dose- and time-dependent manner. Finally, we collected conditioned medium (CM) from NPC cell cultures in the presence of U0126 treatment. When mouse bone marrow non-adherent cells were treated with the CM, the numbers of multinucleated osteoclast formation were dramatically diminished. These results indicate that MEK inhibitor diminishes NPC cell proliferation and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions. This study provides novel therapeutic targets such as CCL2/CCR2 and CXCL16/CXCR6 for advanced NPC patients.

Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival.

BACKGROUND: The chemokine CXCL16 and its receptor CXCR6 are expressed by a variety of immune cells and have been shown to influence angiogenesis. The expression of CXCR6 and CXCL16 has been examined in numerous human cancers; however no studies have yet investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We aimed to explore their prognostic significance in NSCLC, in addition to examining associations with previously investigated markers. METHODS: Resected tumor tissue from 335 consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16 on tissue microarrays. In vitro, NSCLC cells (NCI-H460, A549 cells) were transfected with CXCL16 siRNA to examine effects on proliferation. RESULTS: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any prognostic impact. In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022). Knockdown of CXCL16 by siRNA resulted in accelerated proliferation of NSCLC cell lines. CONCLUSION: We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

CXCL16/CXCR6 chemokine signaling mediates breast cancer progression by pERK1/2-dependent mechanisms.

Our previous studies demonstrate that CXCL6/CXCR6 chemokine axis induces prostate cancer progression by the AKT/mTOR signaling pathway; however, its role and mechanisms underlying invasiveness and metastasis of breast cancer are yet to be elucidated. In this investigation, CXCR6 protein expression was examined using high-density tissue microarrays and immunohistochemistry. Expression of CXCR6 shows a higher epithelial staining in breast cancer nest site and metastatic lymph node than the normal breast tissue, suggesting that CXCR6 may be involved in breast cancer (BC) development. In vitro and in vivo experiments indicate that overexpression of CXCR6 in BC cells has a marked effect on increasing cell migration, invasion and metastasis. In contrast, reduction of CXCR6 expression by shRNAs in these cells greatly reduce its invasion and metastasis ability. Mechanistic analyses show that CXCL16/CXCR6 chemokine axis is capable of modulating activation of RhoA through activating ERK1/2 signaling pathway, which then inhibits the activity of cofilin, thereby enhancing the stability of F-actin, responsible for invasiveness and metastasis of BC. Taken together, our data shows for the first time that the CXCR6 / ERK1/2/ RhoA / cofilin /F-actin pathway plays a central role in the development of BC. Targeting the signaling pathway may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for BC.

Pharmacological inhibition of the chemokine CXCL16 diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of morbidity and mortality in developed countries, resulting in steatohepatitis (NASH), fibrosis and eventually cirrhosis. Modulating inflammatory mediators such as chemokines may represent a novel therapeutic strategy for NAFLD. We recently demonstrated that the chemokine receptor CXCR6 promotes hepatic NKT cell accumulation, thereby controlling inflammation in experimental NAFLD. In this study, we first investigated human biopsies (n = 20), confirming that accumulation of inflammatory cells such as macrophages is a hallmark of progressive NAFLD. Moreover, CXCR6 gene expression correlated with the inflammatory activity (ALT levels) in human NAFLD. We then tested the hypothesis that pharmacological inhibition of CXCL16 might hold therapeutic potential in NAFLD, using mouse models of acute carbon tetrachloride (CCl4)- and chronic methionine-choline-deficient (MCD) diet-induced hepatic injury. Neutralizing CXCL16 by i.p. injection of anti-CXCL16 antibody inhibited the early intrahepatic NKT cell accumulation upon acute toxic injury in vivo. Weekly therapeutic anti-CXCL16 administrations during the last 3 weeks of 6 weeks MCD diet significantly decreased the infiltration of inflammatory macrophages into the liver and intrahepatic levels of inflammatory cytokines like TNF or MCP-1. Importantly, anti-CXCL16 treatment significantly reduced fatty liver degeneration upon MCD diet, as assessed by hepatic triglyceride levels, histological steatosis scoring and quantification of lipid droplets. Moreover, injured hepatocytes up-regulated CXCL16 expression, indicating that scavenging functions of CXCL16 might be additionally involved in the pathogenesis of NAFLD. Targeting CXCL16 might therefore represent a promising novel therapeutic approach for liver inflammation and steatohepatitis.

CXCL16 is a novel diagnostic marker and predictor of mortality in inflammatory cardiomyopathy and heart failure.

BACKGROUND: Cardiac inflammation has been suggested to play a critical role in the pathogenesis of inflammatory cardiomyopathy as well as in progressive heart failure (HF). CXC motif ligand 16 (CXCL16) is a recently discovered chemokine produced by several inflammatory cells and representing an important pathogenic mediator in the development of HF. The present study evaluates the diagnostic and prognostic relevance of CXCL16 expression in endomyocardial biopsies of consecutive patients with congestive HF. METHODS AND RESULTS: 174 patients (age 54.4±14.6 years) with congestive HF undergoing endomyocardial biopsy for diagnostic reasons were prospectively enrolled. Biopsies were analyzed using established histopathological and immunohistological criteria together with CXCL16 staining. CXCL16 was significantly enhanced in patients with inflammatory cardiomyopathy (78/127, 61.4%) as compared to patients with non-inflammatory cardiomyopathy (17/47, 36.2%, p=0.003). During a mean follow-up of 27.5 months, 20 patients (11.5%) reached the primary endpoint (death of all causes). Of all clinical (age, gender, NYHA functional class, systolic pulmonary artery pressure, left ventricular ejection fraction), laboratory (brain natriuretic peptide) and immunohistological (CXCL16) parameters tested, CXCL16 was the only independent predictor of death (hazard ratio 5.4; 95% confidence interval 1.2 to 24.0; p=0.027). Subgroup analysis revealed CXCL16 as a predictor of death in both patients with inflammatory and with non-inflammatory cardiomyopathy. CONCLUSION: According to the present observations CXCL16 is enhanced in inflammatory cardiomyopathy and turned out as an independent predictor of death in patients with HF undergoing endomyocardial biopsy.

Detailed characterization of the endocannabinoid system in human macrophages and foam cells, and anti-inflammatory role of type-2 cannabinoid receptor.

OBJECTIVE: Cannabinoid receptors are activated in murine macrophages upon exposure to oxidized low-density lipoproteins (oxLDL), and type-1 cannabinoid receptor (CB1R) is considered as a risk factor in atherosclerosis, because it promotes cholesterol accumulation and release of inflammatory mediators. Conversely, accumulated evidence suggests a protective role for type-2 cannabinoid receptor (CB2R). Here, we sought to ascertain whether different elements of the endocannabinoid system (ECS) were activated in human lipid-laden macrophages, and whether CB2R played any role in atherogenesis and inflammation of these cells. METHODS AND RESULTS: Human macrophages were exposed to oxLDL in order to obtain lipid-laden foam cells. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the production of the endocannabinoids in both macrophages and foam cells, and radiometric assays were performed to measure cannabinoid receptor binding and activity of endocannabinoid metabolizing enzymes. OxLDL accumulation was investigated by confocal imaging, and cytokine production and release were measured by means of flow cytometry and ELISA. The results showed that human macrophages possess a fully functional ECS, which was modulated by oxLDL. Selective CB2R activation reduced cellular oxLDL accumulation, which was associated with decreased expression of CD36 scavenger receptor, and decreased production of TNFα, IL-12 and IL-10. These anti-atherogenic and anti-inflammatory effects were reverted by the selective CB2R antagonist SR144528. CONCLUSIONS: A fully active ECS is present in human macrophages and macrophage-derived foam cells. Selective activation of CB2R reduces CD36-dependent oxLDL accumulation and modulates production of inflammatory cytokines, thus representing a potential therapeutic strategy to combat atherosclerosis.

Elevated serum CXCL16 is an independent predictor of poor survival in ovarian cancer and may reflect pro-metastatic ADAM protease activity.

BACKGROUND: In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC). METHODS: CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays. RESULTS: sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29-4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells. CONCLUSIONS: High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

CXCL16 and CXCR6 in Ewing sarcoma family tumor.

Chemokines are a family of peptide mediators that play an essential role in cellular migration and intracellular communication in tumor cells as well as immune cells. We hypothesized that the CXCL16-CXCR6 ligand-receptor system plays an important role in Ewing sarcoma (ES) family tumor (ESFT) progression. Using real-time quantitative reverse transcription-polymerase chain reaction, we investigated the mRNA expression of CXCL16, CXCR6, and ADAM 10 in various cell lines. We also investigated the expression of CXCL16, CXCR6, ADAM 10, and ADAM 17 in tissue samples from 61 ESFT patients using immunohistochemistry. The mRNA expression levels of CXCL16 and CXCR6 in the ES cell line were higher than those in the other cell lines. Immunohistochemical staining revealed that CXCL16 and CXCR6 were highly expressed in tumor cells of ESFT and showed a positive correlation between them. The expression of CXCL16 and CXCR6 was associated with the occurrence of lung metastasis. Univariate analysis revealed that CXCL16 or CXCR6 expression was associated with worse prognosis of ESFT patients. In addition, CXCL16 and CXCR6 expression was associated with shorter overall survival irrespective of other prognostic factors. Our results suggest that the CXCL16/CXCR6 axis appears to be important in the progression of ESFT, resulting in more aggressive clinical behavior. Furthermore, there may be a decrease in the overall survival in ESFT patients who have tumors that stain strongly for CXCL16 and CXCR6.

Autophagy regulates phagocytosis by modulating the expression of scavenger receptors.

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.

Establishment of cell lines with increased susceptibility to EV71/CA16 by stable overexpression of SCARB2.

BACKGROUND: Human enterovirus type 71 (EV71) and Coxsackievirus A group type 16 (CA16) belong to human Enterovirus species A of the family Picornaviridae. These viruses are recognized as the major pathogens responsible for epidemics of hand-foot-mouth disease (HFMD), which presents with fever and vesicular eruptions of palms, soles of the feet or mouth. Human scavenger receptor class B, member 2 (SCARB2) has been identified as the receptor for both EV71 and CA16, as overexpression of SCARB2 in cells can enhance virus replication significantly. METHODS: In this study, we used a lentivirus packaging vector to transduce the SCARB2 gene into human embryonic kidney cells (293), human rhabdomyosarcoma cells (RD) and African green monkey kidney cells (Vero) to create stable expression lines. Expression of SCARB2 in the resulting three transgenic cell lines was confirmed by real-time RT-PCR, immunofluorescence and flow cytometry. RESULTS: Levels of SCARB2 mRNA determined by real-time RT-PCR in 293-SCARB2 (293S) or RD-SCARB2 (RDS) transgenic cell lines were approximately 2 × 10(2) times higher than those in 293 and RD cells, respectively, and three times higher in Vero-SCARB2 (VeroS) than in Vero cells. Furthermore, EV71 and CA16 virus titers in 293S and RDS cells were 10(2)-10(3)-fold higher (detected in RD cell) than those in the parental cells, and a 10-fold higher titer of EV71 was achieved in VeroS cells compared with that in Vero cells. CONCLUSIONS: We established for the first time three cell lines stably overexpressing SCARB2, which showed drastic increases in susceptibility to EV71/CA16 infection. These optimal cell lines may be utilized to develop inactivated vaccines for EV71/CA16 and facilitate rapid detection and isolation of HFMD pathogens or other Enterovirus serotypes. Furthermore, these stable cell lines also can serve as tools to facilitate drug screenings as well as molecular studies on virus-host interactions and pathogenesis of causative agents for HFMD.

A multilevel screening strategy defines a molecular fingerprint of proregenerative olfactory ensheathing cells and identifies SCARB2, a protein that improves regenerative sprouting of injured sensory spinal axons.

Olfactory ensheathing cells (OECs) have neuro-restorative properties in animal models for spinal cord injury, stroke, and amyotrophic lateral sclerosis. Here we used a multistep screening approach to discover genes specifically contributing to the regeneration-promoting properties of OECs. Microarray screening of the injured olfactory pathway and of cultured OECs identified 102 genes that were subsequently functionally characterized in cocultures of OECs and primary dorsal root ganglion (DRG) neurons. Selective siRNA-mediated knockdown of 16 genes in OECs (ADAMTS1, BM385941, FZD1, GFRA1, LEPRE1, NCAM1, NID2, NRP1, MSLN, RND1, S100A9, SCARB2, SERPINI1, SERPINF1, TGFB2, and VAV1) significantly reduced outgrowth of cocultured DRG neurons, indicating that endogenous expression of these genes in OECs supports neurite extension of DRG neurons. In a gain-of-function screen for 18 genes, six (CX3CL1, FZD1, LEPRE1, S100A9, SCARB2, and SERPINI1) enhanced and one (TIMP2) inhibited neurite growth. The most potent hit in both the loss- and gain-of-function screens was SCARB2, a protein that promotes cholesterol secretion. Transplants of fibroblasts that were genetically modified to overexpress SCARB2 significantly increased the number of regenerating DRG axons that grew toward the center of a spinal cord lesion in rats. We conclude that expression of SCARB2 enhances regenerative sprouting and that SCARB2 contributes to OEC-mediated neuronal repair.

CXC chemokine ligand 16, inversely correlated with CD99 expression in Hodgkin Reed-Sternberg cells, is widely expressed in diverse types of lymphomas.

The present study examined a correlation between CXC chemokine ligand 16 (CXCL16) and cell differentiation antigen 99 (CD99) expression and investigated the role of CXCL16 in human lymphoma cell lines and clinical samples. Cytokine antibody arrays were used to measure the differentially expressed cytokines in tumor tissues. The expression of CXCL16 and CD99 was analyzed by quantitative PCR (qPCR) and western blotting, while the pathways involved were assessed by western blotting and enzyme-linked immunosorbent assay (ELISA). The expression of CXCL16 was investigated in 9 lymphoma cell lines (L428, RPMI-8226, KM3, Jurkat, OCI-Ly1, OCI-Ly8, OCI-Ly10, Karpass299 and Raji) as well as in clinical lymphoma samples using qPCR, western blotting and immunochemistry. Soluble CXCL16 (sCXCL16) levels were measured by ELISA and proliferation was analyzed by CCK­8 proliferation assays. CXCL16 was one of the upregulated chemokines when lymphoma cells where transferred from in vitro to in vivo conditions. The increased expression and secretion of CXCL16 paralleled with a decrease of mCD99L2 and was accompanied by NF-κB pathway activation and vice versa. CXCL16 was expressed in all 9 lymphoma cell lines with the highest levels in the Hodgkin lymphoma (HL) cell line L428, the plasma cell-derived cell lines RPMI­8226 and KM3 and the T leukemia-derived cell line Jurkat. Higher levels of sCXCL16 were secreted by L428 cells, the diffuse large B-cell lymphoma (DLBCL)-derived cell lines (OCI-Ly1, OCI-Ly8 and OCI-Ly10) and Jurkat cells. CXCL16 was widely expressed in clinical samples of lymphoma patients with higher levels in HL compared to non-Hodgkin lymphoma. Human recombinant CXCL16 had no significant effect on L428 cell proliferation, but was able to stimulate CD4+ T lymphocytes to proliferate. CXCL16, inversely correlated with CD99 expression in Hodgkin Reed-Sternberg (H/RS) cells, is widely expressed in diverse types of lymphomas.