BACKGROUND

Cia5a is a locus on rat chromosome 10 that regulates disease severity and joint damage in two models of rheumatoid arthritis, collagen- and pristane-induced arthritis (PIA). In this study, we aimed to identify cellular and molecular processes regulated by Cia5a using microarray-based gene expression analysis of synovial tissues from MHC identical DA (severe erosive disease) and DA.F344(Cia5a) congenics (mild non-erosive disease) rats.

RESULTS

Synovial tissues from six DA and eight DA.F344(Cia5a) rats were analyzed 21 days after the induction of PIA using the Illumina RatRef-12 BeadChip (21,922 genes) and selected data confirmed with qPCR. There was a significantly increased expression of pro-inflammatory mediators such as Il1b (5-fold), Il18 (3.9-fold), Cxcl1 (10-fold), Cxcl13 (7.5-fold) and Ccl7 (7.9-fold), and proteases like Mmp3 (23-fold), Mmp9 (32-fold), Mmp14 (4.4-fold) and cathepsins in synovial tissues from DA, with reciprocally reduced levels in congenics. mRNA levels of 47 members of the Spleen Tyrosine Kinase (Syk) pathway were significantly increased in DA synovial tissues compared with DA.F344(Cia5a), and included Syk (5.4-fold), Syk-activating receptors and interacting proteins, and genes regulated by Syk such as NFkB, and NAPDH oxidase complex genes. Nuclear receptors (NR) such as Rxrg, Pparg and Rev-erba were increased in the protected congenics, and so was the anti-inflammatory NR-target gene Scd1 (54-fold increase). Tnn (72-fold decrease) was the gene most significantly increased in DA.

CONCLUSIONS

Analyses of gene expression in synovial tissues revealed that the arthritis severity locus Cia5a regulates the expression of key mediators of inflammation and joint damage, as well as the expression of members of the Syk pathway. This expression pattern correlates with disease severity and joint damage and along with the gene accounting for Cia5a could become a useful biomarker to identify patients at increased risk for severe and erosive disease. The identification of the gene accounting for Cia5a has the potential to generate a new and important target for therapy and prognosis.

Dietary influences may affect microbiome composition and host immune responses, thereby modulating propensity toward inflammatory bowel diseases (IBDs): Crohn disease (CD) and ulcerative colitis (UC). Dietary n-6 fatty acids have been associated with UC in prospective studies. However, the critical developmental period when (n-6) consumption may induce UC is not known. We examined the effects of transiently increased n-6 consumption during pediatric development on subsequent dextran-sulfate-sodium (DSS)-induced acute murine colitis. The animals transiently became obese then rapidly lost this phenotype. Interestingly, mice were protected against DSS colitis 40 days after n-6 consumption. The transient high n-6-induced protection against colitis was fat type- and dietary reversal-dependent and could be transferred to germ-free mice by fecal microbiota transplantation. We also detected decreased numbers of chemokine receptor (Cxcr)5(+) CD4(+) T cells in the mesenteric lymph nodes (MLNs) of transiently n-6-fed mice. Further experiments revealed that anti-chemokine ligand (Cxcl)13 (the ligand of Cxcr5) antibody treatment decreased DSS colitis severity, implicating the importance of the Cxcr5-Cxcl13 pathway in mammalian colitis. Consecutively, we found elevated CXCL13 concentrations (CD: 1.8-fold, P = 0.0077; UC: 1.9-fold, P = 0.056) in the serum of untreated pediatric IBD patients. The human serologic observations supported the translational relevance of our findings.

Interacting with T cells, cytokine-producing B cells play a critical protective role in autoimmune diseases. However, the interaction between malignant B and T cells remains to be fully elucidated. In a previous study, we have reported that ligation of CCL19-CCR7 and CXCL13-CXCR5 activates paternally expressed gene 10 (PEG10), resulting in an enhancement of apoptotic resistance in B-cell acute lymphocytic leukemia (B-ALL) CD23+CD5+ B cells. Here, we report that B-ALL CD23+CD5+ B cells produce IL-10 at high level, which can be further elevated by costimulation with CCL19 and CXCL13. CCL19/CXCL13-activated B-ALL CD23+CD5+ B cells, in turn, increase IL-10 expression in syngeneic CD8+ T cells in a B cell-derived IL-10-dependent manner and requiring a cell-cell contact. IL-10 secreted from B-ALL CD23+CD5+ B cells in vitro impairs tumor-specific CTL responses of syngeneic CD8+ T cells. The impairment of cytotoxicity of syngeneic CD8+ T cells is escalated by means of CCL19/CXCL13-induced up-regulation of IL-10 from B-ALL CD23+CD5+ B cells. Moreover, using a short hairpin RNA to knockdown PEG10, we provide direct evidence that increased expression of PEG10 in B-ALL CD23+CD5+ B cells is involved in malignant B-T cell interaction, contributing to the up-regulation of IL-10 expression, as well as to the impairment of cytotoxicity of syngeneic CD8+ T cells. Thus, malignant B-ALL CD23+CD5+ B cells play an immunoregulatory role in controlling different inflammatory cytokine expressions. IL-10 may be one of the critical cellular factors conferring B-ALL CD23+CD5+ B cells to escape from host immune surveillance.

BACKGROUND

Peripheral nodal follicular T-cell lymphomas expressing follicular helper T-cell (T(FH)) markers have recently been identified. Such lymphomas are characterized by a nodal neoplastic T-cell proliferation accompanied by numerous reactive B cells and demonstrate some overlap with nodal angioimmunoblastic T-cell lymphoma (AITL). We identified 5 cases of cutaneous T-cell lymphoma with a peculiar pathologic aspect and expression of T(FH) markers.

METHODS

The mean age of the patients was 61 years (range, 33-78 years). Four patients had multiple papules, plaques, and nodules predominating on the trunk and the head. One had a nodular plaque on the face. Lesional T-cell clonality was found in all 5 patients, and blood T-cell clonality in 4 of the 5. Nodal involvement was never found. Patients had no systemic symptoms and no biological signs of AITL. In 3 cases, findings from skin biopsy specimens were initially misdiagnosed as primary cutaneous follicle B-cell lymphoma due to major B-cell infiltrate and CD10 positivity. Rituximab-containing therapies were ineffective in these cases, and biopsy specimens after treatment with rituximab showed medium- to large-sized atypical T-cell skin infiltrate expressing T(FH) markers (CD10, Bcl-6, PD-1, CXCL13, and ICOS). The final diagnosis proposed for all patients was cutaneous T(FH) lymphoma. The patient with localized disease was successfully treated with radiotherapy. Patients with diffuse disease showed marked resistance to treatments, with only 1 case of complete remission after allogeneic hematopoietic stem cell transplantation followed by bortezomib and donor-lymphocyte infusion. Bexarotene, methotrexate, thalidomide, interferon alfa, gemcitabine, liposomal doxorubicin, or multiagent chemotherapy with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) were either ineffective or induced transitory partial remission.

CONCLUSIONS

We describe an original clinicopathologic series of primary cutaneous lymphomas with T(FH) phenotype, suggesting the existence of a new entity among cutaneous T-cell lymphomas. Relations of these lymphomas with the provisional entity of primary cutaneous small to medium CD4 pleomorphic T-cell lymphoma need to be further addressed.

Cholesterol-enriched lipid microdomains regulate L-selectin signaling, but the role of membrane cholesterol in L-selectin adhesion is unclear. Arrest chemokines are a subset of endothelial chemokines that rapidly activate leukocyte integrin adhesiveness under shear flow. In the absence of integrin ligands, these chemokines destabilize L-selectin-mediated leukocyte rolling. In the present study, we investigated how cholesterol extraction from the plasma membrane of peripheral blood T or B cells affects L-selectin adhesions and their destabilization by arrest chemokines. Unlike the Jurkat T cell line, whose L-selectin-mediated adhesion is cholesterol dependent, in primary human PBLs and in murine B cells and B cell lines, cholesterol depletion did not impair any intrinsic adhesiveness of L-selectin, consistent with low selectin partitioning into lipid rafts in these cells. However, cholesterol raft disruption impaired the ability of two arrest chemokines, CXCL12 and CXCL13, but not of a third arrest chemokine, CCL21, to destabilize L-selectin-mediated rolling of T lymphocytes. Actin capping by brief incubation with cytochalasin D impaired the ability of all three chemokines to destabilize L-selectin rolling. Blocking of the actin regulatory phosphatidylinositol lipid, phosphatidylinositol 4,5-bisphosphate, did not affect chemokine-mediated destabilization of L-selectin adhesions. Collectively, our results suggest that L-selectin adhesions are inhibited by actin-associated, cholesterol-stabilized assemblies of CXCL12- and CXCL13-binding receptors on both T and B lymphocytes. Thus, the regulation of L-selectin by cholesterol-enriched microdomains varies with the cell type as well as with the identity of the destabilizing chemokine.

Chemokines contribute to the maintenance of cartilage homeostasis. To evaluate the role of CXC chemokines CXCL8 (interleukin-8), CXCL10 (interferon-gamma-inducible protein-10), CXCL12 (stroma-derived factor-1) and CXCL13 (B-cell attracting chemokine-1) and their receptors, respectively CXCR1-2, CXCR3, CXCR4, and CXCR5, during chondrogenic differentiation of human mesenchymal stromal cells (h-MSCs), we used a well-defined in vitro model. Chondrogenic differentiation was analyzed on h-MSCs grown on hyaluronic acid-based biomaterial in the presence or absence of transforming growth factor-beta, and the expression and modulation of CXC chemokines and receptors were evaluated at different time points. Real-time polymerase chain reaction was performed to analyze their expression at the messenger ribonucleic acid (mRNA) level, and immunohistochemistry and enzyme-linked immunosorbent assay were used to evaluate their expression at the protein level. Human articular cartilage biopsies were used to evaluate chemokine and receptor expression in normal tissue. We found no expression of CXCR1, CXCR2, CXCR3, or CXCL10 at the mRNA level. CXCL8 mRNA was down-modulated, whereas at the protein level, we found greater release of this chemokine. CXCR4 and its ligand CXCL12 were down-modulated during chondrogenesis. By contrast, CXCR5 was up-regulated, whereas its ligand CXCL13 was lower. These data were also confirmed on human articular cartilage. These findings show that, during in vitro h-MSC chondrogenic differentiation, chemokine and receptor expression was specifically induced or repressed. This was in line with what the authors also found in normal articular cartilage, suggesting a role in differentiation and maturation of a cartilage-like structure in vitro and consequently the regulation of cartilage homeostasis.

OBJECTIVE

To investigate the association of posterior vitreous detachment (PVD) with aqueous levels of vascular endothelial growth factor (VEGF) and other inflammatory cytokines.

METHODS

These are prospective comparative studies. Subjects comprised 98 eyes for VEGF concentration and 80 eyes for other cytokines, which are normal except for cataract. PVD was examined by B-mode ultrasonography, and the subjects were divided into complete PVD group (PVD group) or the other group (without PVD group). At the beginning of cataract surgery, aqueous humour was collected and the concentrations of VEGF and other inflammatory cytokines were determined using ELISA and a multiplex cytokine assay, respectively. The concentrations of these cytokines were compared between the two groups.

RESULTS

Complete PVD was observed in 56 (57%) eyes for VEGF concentration analysis, and 51 (64%) eyes for the other cytokines analysis. The concentrations of VEGF, adjusted for the average age, axial length and gender distribution, was 47 pg/mL in the PVD group and 72 pg/mL in the without PVD group. The concentrations of IP-10, MCP-1, CXCL13 and CCL11 were 53, 450, 3.8 and 6.0 pg/mL in the PVD group, and 100, 560, 7.0 and 8.4 pg/mL in the without PVD group, respectively. Multiple regression analysis revealed that the logarithmic concentration of VEGF, IP-10, MCP-1, CXCL13 and CCL11 were significantly lower in the eyes with PVD than in those without PVD independently of age, sex and axial length (p=0.01, p=0.002, p=0.009, 0.006 and 0.03, respectively).

CONCLUSIONS

PVD is related to the change in the multiple intraocular inflammatory cytokines.

Surface properties affect the biological properties of cells modulating the expression of different factors. Osteoblasts contribute both to new bone formation and controlling haematopoiesis through cytokines and growth factors. We analyzed the effect of bone (calcium-phosphate bone slides), cartilaginous (hyaluronan-based scaffold), and plastic substrate culture on human osteoblast proliferation, bone matrix molecule, and inflammatory factor expression. Osteoblast proliferation increased to a greater extent when the cells were grown for 14 days on plastic and bone slides, whereas hyaluronan-based scaffold (HA-scaffold) induced only a minimal increase. Collagen type I, osteonectin, alkaline phosphatase and osteocalcin were expressed on osteoblasts grown on plastic and bone slides and down-modulated at mRNA and protein level by HA-scaffold. Bone slides showed the ability to increase osteopontin mRNA expression. The expression of CXCR4 and CXCL13 was upregulated by bone slides and HA-scaffold, while CXCL12 and CXCR5 expression was down-modulated. These data suggest a substrate-dependent modulation of human osteoblast proliferation, bone matrix and inflammatory factor expression, which might help to understand the active role played by osteoblasts in bone microenvironment by coupling bone extracellular matrix, chemokines and the haematopoietic system.

Angioimmunoblastic T-cell lymphoma (AITL) is considered to originate from follicular helper T (TFH) cells. Currently, neoplastic cells in AITL are considered to express CXCL13 as a tumor marker. However, the identification of CXCL13(+) cells remains unclear in terms of whether they are neoplastic cells (or TFH cells) or follicular dendritic cells (FDCs) in both AITL and normal germinal centers. Therefore, the exact identification of CXCL13(+) cells was performed using 33 cases of AITL and normal germinal centers. Single-labeling immunohistochemistry and double-labeling immunofluorescent microscopy first confirmed that CXCL13 was expressed mainly in FDCs in the normal germinal centers. In 28 of 33 AITL cases, CXCL13 was expressed mainly in FDCs as a meshwork pattern, which was associated with CXCL13(+) neoplastic cells. In the other five cases, CXCL13 was expressed mainly in neoplastic cells, which were densely distributed in and around the FDC meshwork. These findings indicate the abundance of CXCL13(+) cells in the FDC meshwork irrespective of the cell type. Triple-labeling immunofluorescent microscopy showed that the CXCL13(+) FDC meshwork in AITL harbored both neoplastic cells and B cells. CXCR5, the cognate receptor of CXCL13, was expressed in neoplastic cells in AITL. The present study suggests that neoplastic cells in AITL preserve a certain level of TFH-cell function since neoplastic cells and B cells are closely enmeshed in the CXCL13(+) cell-rich FDC meshwork in a similar way as in normal germinal centers.

Androgen receptor (AR) is a key transcription factor playing a critical role in prostate cancer (PCa) initiation and progression. However, the molecular mechanisms of AR action in prostate cancer are not very clear. CXCL13, known as B cell attracting chemokine1 (BCA-1), is a member of CXC chemokine family and relevant to cancer metastasis. This study shows that CXCL13 is an androgen-responsive gene and involved in AR-induced PCa cell migration and invasion. In clinical specimens, expression of CXCL13 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of CXCL13 is up-regulated by androgen-AR axis at both mRNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen responsive elements (ARE) at CXCL13 enhancer and dual-luciferase reporter assays reveals that the ARE is highly responsive to androgen while mutations of the ARE abolish the reporter activity. Additional chromatin immunoprecipitation (ChIP) assays also identify that the ARE presents androgen responsiveness. In addition, CXCL13 promotes G2/M phase transition by increasing Cyclin B1 levels in PCa cells. Functional studies demonstrate that reducing endogenous CXCL13 expression in LNCaP cells largely weakens androgen-AR axis induced cell migration and invasion. Taken together, our study implicates for the first time that CXCL13 is an AR target gene and involved in AR-mediated cell migration and invasion in primary PCa.

Oral symptoms are among the most distressing manifestations for patients with Sjögren's syndrome (SS). The feeling of dry mouth is unpleasant, and hyposalivation may contribute to difficulty in speaking, chewing and swallowing and reduced quality of life. Reduced salivary flow increases the risk for dental caries and problems with prosthetic replacement. It seems that SS is not as frequently occurring as previously anticipated. Population-based prevalence studies on primary SS in Europe, conducted on large background populations and in accordance with the AECG criteria, reported of a prevalence of 1-9 cases per 10,000 people. This gives a combined prevalence of nearly 39/100,000 (~0.04 %). The cause of Sjögren's syndrome is even now not fully understood, and the treatment of oral symptoms is still mostly palliative. Hopefully, useful information will appear from the new methods that are now available for genome wide association studies, epigenetics, DNA methylation studies, and proteomics. Similarly, this is anticipated for the immunological side of the story. The interferon signature, the interferon γ/interferon α mRNA ratio, and CXCL13 are among the proposed biomarkers of active disease. In this review, we provide an update on oral aspects of Sjögren's syndrome with emphasis on the latest publications on these topics.

OBJECTIVE

Mesenchymal stromal cells (MSC) are pluripotent adult stem cells capable of osteogenesis and chondrogenesis to form bone and cartilage. This characteristic gives them the potential for bone and cartilage regeneration. Synthetic polymers have been studied to examine whether they could be used as a scaffold for tissue engineering. In the current study a two-dimensional (2-D) poly(l-lactic acid) (PLLA) scaffold was treated with chemokine, adhesion and extracellular matrix molecules with the aim of using biologic molecules to improve the attachment of human MSC.

METHODS

MSC were isolated from human bone marrow and applied to a 2-D PLLA scaffold. Chemokines ligand (CXCL12 and CXCL13), adhesion molecules [P-selectin, vascular cell adhesion molecule (VCAM)-1 and heparin] and extracellular matrix molecules (fibronectin and type IV collagen) were coated on the scaffold and their effects on the number of MSC that adhered were recorded.

RESULTS

When used alone CXCL12 and CXCL13 enhanced MSC adhesion, as did VCAM-1, P-selectin, fibronectin and collagen, but not heparin. The effects of VCAM-1, P-selectin and heparin were enhanced by the addition of CXCL12. Incubation of MSC with antibodies to integrins α4 and α5β1 inhibited their adhesion to VCAM-1 and fibronectin-treated PLLA respectively, suggesting that these integrins were involved in the MSC interactions.

CONCLUSIONS

The use of certain chemokines and adhesion and extracellular matrix molecules, alone or in combination, is beneficial for the attachment of MSC to PLLA, and may be helpful as natural molecules in scaffolds for regenerative medicine.

The interleukin-17 (IL-17) cytokine family plays a central role in the coordination of inflammatory responses. In fish species, three genes that have a similar homology to both IL-17A and IL-17F were designated IL-17A/F1, 2, and 3. In this study, we identified three IL-17A/F homologues (LycIL-17A/F1, 2, and 3) from large yellow croaker (Larimichthys crocea). The deduced LycIL-17A/F1 and 3 had four cysteine residues conserved in teleost IL-17A/F1 and 3 homologues and shared a domain similar to the B chain of human IL-17F. The deduced LycIL-17A/F2 possessed the unique arrangement of six cysteine residues as teleost IL-17A/F2 (except Fugu IL-17A/F2) and higher vertebrate IL-17A and F, and shared a domain similar to the D/E chain of human IL-17A. Phylogenetic analysis showed that teleost IL-17A/F1 and 3 fall into a major clade, whereas IL-17A/F2 forms a separated clade and is clustered with IL-17N. Based on structural and phylogenetic analyses, we suggest that teleost IL-17A/Fs may be classified into two subgroups: one consisting of IL-17A/F1 and 3, and the other composed of IL-17A/F2. The three LycIL-17A/Fs were constitutively expressed in all tissues examined although at a different level. Following challenge with Aeromonas hydrophila, expression of these three LycIL-17A/Fs was rapidly increased in head kidney and gills. The in vivo assays showed that recombinant LycIL-17A/F1, 2, and 3 all were able to enhance the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α2), chemokines (CXCL8 and CXCL13), and antimicrobial peptide hepcidin in head kidney. Furthermore, LycIL-17A/Fs appeared to mediate pro-inflammatory responses via NF-κB signalling. These results therefore reveal similar functions between the two subgroup members，LycIL-17A/F1 and 3 and LycIL-17A/F2, in promoting inflammation and host defences.

The overlap of morphology and immunophenotype between angioimmunoblastic T-cell lymphoma (AITL) and other nodal peripheral T-cell lymphomas (n-PTCLs) is a matter of current interest whose clinical relevance and pathogenic background have not been fully established. We studied a series of 98 n-PTCL samples (comprising 57 AITL and 41 PTCL-NOS) with five TFH antibodies (CD10, BCL-6, PD-1, CXCL13, ICOS), looked for mutations in five of the genes most frequently mutated in AITL (TET2, DNMT3A, IDH2, RHOA and PLCG1) using the Next-Generation-Sequencing Ion Torrent platform, and measured the correlations of these characteristics with morphology and clinical features. The percentage of mutations in the RHOA and TET2 genes was similar (23.5% of cases). PLCG1 was mutated in 14.3%, IDH2 in 11.2% and DNMT3A in 7.1% of cases, respectively. In the complete series, mutations in RHOA gene were associated with the presence of mutations in IDH2, TET2 and DNMT3A (p < 0.001, p = 0.043, and p = 0.029, respectively). Fourteen cases featured RHOA mutations without TET2 mutations. A close relationship was found between the presence of these mutations and a TFH-phenotype in AITL and PTCL-NOS patients. Interestingly, BCL-6 expression was the only TFH marker differentially expressed between AITL and PTCL-NOS cases. There were many fewer mutated cases than there were cases with a TFH phenotype. Overall, these data suggest alternative ways by which neoplastic T-cells overexpress these proteins. On the other hand, no clinical or survival differences were found between any of the recognized subgroups of patients with respect to their immunohistochemistry or mutational profile.

Proliferation and survival of chronic lymphocytic leukemia (CLL) cells depend on microenvironmental signals coming from lymphoid organs. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the B-cell receptor (BCR). Syk protein, a tyrosine kinase essential for BCR signaling, is therefore a rational candidate for targeted therapy in CLL. Against this background, we tested the efficacy of the highly specific Syk inhibitor TAK-659 in suppressing the favorable signaling derived from the microenvironment. To ex vivo mimic the microenvironment found in the proliferation centers, we co-cultured primary CLL cells with BM stromal cells (BMSC), CD40L and CpG ODN along with BCR stimulation. In this setting, TAK-659 inhibited the microenvironment-induced activation of Syk and downstream signaling molecules, without inhibiting the protein homologue ZAP-70 in T cells. Importantly, the pro-survival, proliferative, chemoresistant and activation effects promoted by the microenvironment were abrogated by TAK-659, which furthermore blocked CLL cell migration toward BMSC, CXCL12, and CXCL13. Combination of TAK-659 with other BCR inhibitors showed synergistic effect in inducing apoptosis, and the sequential addition of TAK-659 in ibrutinib-treated CLL cells induced significantly higher cytotoxicity. These findings provide a strong rationale for the clinical development of TAK-659 in CLL.

Signaling activated by binding of the CXC motif chemokine ligand 12 (CXCL12) to its cognate G protein-coupled receptor (GPCR), chemokine CXC motif receptor 4 (CXCR4), is linked to metastatic disease. However, the mechanisms governing CXCR4 signaling remain poorly understood. Here, we show that endocytosis and early endosome antigen 1 (EEA1), which is part of the endosome fusion machinery, are required for CXCL12-mediated AKT Ser/Thr kinase (Akt) signaling selective for certain Akt substrates. Pharmacological inhibition of endocytosis partially attenuated CXCL12-induced phosphorylation of Akt, but not phosphorylation of ERK-1/2. Similarly, phosphorylation of Akt, but not ERK-1/2, stimulated by CXCL13, the cognate ligand for the chemokine receptor CXCR5, was also attenuated by inhibited endocytosis. Furthermore, siRNA-mediated depletion of the Rab5-effector EEA1, but not of adaptor protein, phosphotyrosine-interacting with PH domain and leucine zipper 1 (APPL1), partially attenuated Akt, but not ERK-1/2, phosphorylation promoted by CXCR4. Attenuation of Akt phosphorylation through inhibition of endocytosis or EEA1 depletion was associated with reduced signaling to Akt substrate forkhead box O1/3a but not the Akt substrates TSC complex subunit 2 or glycogen synthase kinase 3β. This suggested that endocytosis and endosomes govern discrete aspects of CXCR4- or CXCR5-mediated Akt signaling. Consistent with this hypothesis, depletion of EEA1 reduced the ability of CXCL12 to attenuate apoptosis in suspended, but not adherent, HeLa cells. Our results suggest a mechanism whereby compartmentalized chemokine-mediated Akt signaling from endosomes suppresses the cancer-related process known as anoikis. Targeting this signaling pathway may help inhibit metastatic cancer involving receptors such as CXCR4.

BACKGROUND

Thoracic aortic aneurysms (TAA) and abdominal aortic aneurysms (AAA) represent related but distinct disease processes. Interleukin-6 (IL-6) is known to be significantly upregulated in human TAA and AAA. We hypothesize that loss of IL-6 is protective in experimental TAA and AAA.

METHODS

Murine TAAs or AAAs were created using a novel model in C57/B6 mice by treating the intact aorta with elastase. Cytokine profiles were analyzed with antibody arrays (n = 5 per group). Separately, to determine the role of IL-6, thoracic (n = 7) or abdominal (n = 7) aortas of wild type mice and IL-6 knockout (KO) mice were treated with elastase. Additionally, thoracic animals treated with either the IL-6 receptor antagonist tocilizumab (n = 8) or vehicle (n = 5). Finally, human TAA and AAA were analyzed with human cytokine array.

RESULTS

Elastase treatment of thoracic aortas yielded dilation of 86.8% ± 9.6%, and abdominal aortas produced dilation of 85.6% ± 16.2%. Murine IL-6, CXCL13, and matrix metalloproteinase-9 were significantly elevated in TAA compared with AAA (p = 0.004, 0.028, and 0.001, respectively). The IL-6KO mice demonstrated significantly smaller TAA size relative to wild type mice (wild type 100.1% versus IL-6KO 76.5%, p = 0.04). The IL-6KO mice did not show protection from AAA (p = 0.732). Pharmacologic inhibition of IL-6 resulted in significant reduction in TAA size (tocilizumab 71.5% ± 13.2% versus vehicle 103.6% ± 20.7%, p = 0.005). Human TAA showed significantly greater IL-6 (p < 0.0001) compared with AAA and normal thoracic and abdominal aorta.

CONCLUSIONS

Interleukin-6 is significantly greater in both murine and human TAA compared with AAA, suggesting fundamental differences in these disease processes. Interleukin-6 receptor antagonism attenuates experimental TAA formation, indicating that IL-6 may be a potential target for human thoracic aneurysmal disease.

Death receptor 3 (DR3; TNFRSF25) and its tumor necrosis factor-like ligand TL1A (TNFSF15) control several processes in inflammatory diseases through the expansion of effector T cells and the induction of proinflammatory cytokines from myeloid and innate lymphoid cells. Using wild-type (DR3+/+) and DR3-knockout (DR3-/-) mice, we show that the DR3/TL1A pathway triggers the release of multiple chemokines after acute peritoneal inflammation initiated by a single application of Staphylococcus epidermidis supernatant, correlating with the infiltration of multiple leukocyte subsets. In contrast, leukocyte infiltration was not DR3 dependent after viral challenge with murine cytomegalovirus. DR3 expression was recorded on connective tissue stroma, which provided DR3-dependent release of chemokine (C-C motif) ligand (CCL) 2, CCL7, CXCL1, and CXCL13. CCL3, CCL4, and CXCL10 production was also DR3 dependent, but quantitative RT-PCR showed that their derivation was not stromal. In vitro cultures identified resident macrophages as a DR3-dependent source of CCL3. Whether DR3 signaling could contribute to a related peritoneal pathology was then tested using multiple applications of S. epidermidis supernatant, the repetitive inflammatory episodes of which lead to peritoneal membrane thickening and collagen deposition. Unlike their DR3+/+ counterparts, DR3-/- mice did not develop fibrosis of the mesothelial layer. Thus, this work describes both a novel function and essential requirement for the DR3/TL1A pathway in acute, resolving, and chronic inflammation in the peritoneal cavity.

BACKGROUND

Lyme neuroborreliosis (LNB) is a frequent manifestation of Lyme disease in children and its current diagnosis has limitations. The elevation of the chemokine CXCL13 in the cerebrospinal fluid (CSF) of adult patients with LNB has been demonstrated and suggested as a new diagnostic marker. Our aim was to evaluate this marker in the CSF of children with suspected LNB and to determine a CXCL13 cut-off concentration that would discriminate between LNB and other central nervous system (CNS) infections.

METHODS

For this single-center retrospective case-control study we used a diagnostic-approved ELISA to measure CXCL13 concentrations in the CSF of 185 children with LNB suspicion at presentation. Patients were classified into definite LNB (cases), non-LNB (controls with other CNS affections), and possible LNB. A receiver-operating characteristic curve was generated by comparison of cases and controls.

RESULTS

CXCL13 was significantly elevated in the CSF of 53 children with definite LNB (median 774.7 pg/ml) compared to 91 control patients (median 4.5 pg/ml, p < 0.001). A cut-off of 55 pg/ml resulted in a sensitivity of 96.7% and a specificity of 98.1% for the diagnosis of definite LNB and the test exhibited a diagnostic odds ratio of 1525.3. Elevated CSF CXCL13 levels were also detected in three controls with viral meningitis (enterovirus n = 1, varicella-zoster virus n = 2) while other CNS affections such as idiopathic facial palsy did not lead to CXCL13 elevation. Of the 41 patients with possible LNB, 27% had CXCL13 values above the cut-off of 55 pg/ml (median 16.7 pg/ml).

CONCLUSIONS

CSF CXCL13 is highly elevated in children during early LNB as previously shown in adults. CXCL13 is a highly sensitive and specific marker that helps to differentiate LNB from other CNS affections in children.

OBJECTIVE

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system characterized by a variable clinical course. Different pathogenic mechanisms responsible for relapsing remitting (RRMS) and primary progressive multiple sclerosis (PPMS) are modulated by immunological process with important role of chemokine network. CXCL10 and CXCL13 chemokines act as chemoattractants and modulators of proinflammatory reactions promoting process of demyelination. In the present study, we investigated the concentrations of CXCL10 and CXCL13 in serum and cerebrospinal fluid (CSF) of patients with RRMS and PPMS.

METHODS

The study groups comprised 25 RRMS patients (39,5±12years), 24 PPMS patients (49,9±10,5years), 31 healthy individuals (36±10,4years) with tension headache without symptoms of inflammatory diseases. A quantitive test kit based on ELISA has been used for chemokines measurement. Correlations analysis between the levels of CXCL10, CXCL13 and patient age, duration of MS, EDSS and IgG index were done.

RESULTS

The mean concentration of CXCL10 in the CSF was statistically significantly higher in RRMS in comparison with the control group. The mean concentration of CXCL13 in the CSF was significantly higher in RRMS and PPMS than in the control group. The results have shown that in the stable phase of MS without relapse, mean concentration of CXCL10 and CXCL13 in CSF did not differ significantly between RRMS and PPMS. In PPMS a positive correlation between IgG index and CSF CXCL10 level or CSF CXCL13 level was observed. In RRMS a positive correlation between IgG index and CSF CXCL13 level was observed.

CONCLUSIONS

These data indicate involvement of CXCL10 and CXCL13 chemokines in immunopathogenetic mechanisms in MS. There was no significant difference between mean CXCL10 or CXCL13 concentrations in the CSF in both RRMS and PPMS patients. No significant correlations were found between patient age and chemokines levels in theCSF in all groups. It suggest that these chemokines play similar role in inflammatory process despite more pronounced neurodegenerative process in PPMS.

BACKGROUND Long-non-coding RNA (lncRNA) SNHG15 has been reported to be an aberrantly expressed lncRNA in patients with ischemic stroke, but its role in neuronal injury following ischemic stroke remains unclear. We hypothesized that this lncRNA is associated with the pathogenesis of ischemic stroke. MATERIAL AND METHODS A mouse model of ischemic stroke was established by middle cerebral artery occlusion (MCAO). A neurogenic mouse cell line Neuro-2a (N2a) was subjected to oxygen-glucose deprivation (OGD) for in vitro experiments. Expression of SNHG15, microRNA-18a (miR-18a), and CXCL13 in mouse brain and in OGD-treated N2a cells was determined. Altered expression of SNHG15 and miR-18a was introduced to detect their roles in N2a cell viability and apoptosis. Targeting relationships between miR-18a and SNHG15 or CXCL13 were validated by luciferase assays. Cells were treated with the ERK/MEK antagonist U0126 to assess the role of the ERK/MEK signaling pathway in N2a cell growth. RESULTS SNHG15 and CXCL13 were overexpressed and miR-18a was underexpressed in MCAO-induced mice and OGD-treated N2a cells. Silencing of SNHG15 or overexpression of miR-18a promoted cell viability, while decreased cell apoptosis induced by OGD; however, subsequent disruption of the ERK/MEK signaling pathway reversed these effects. SNHG15 was found to bind to miR-18a, which could further target CXCL13. CONCLUSIONS Silencing of SNHG15 led to CXCL13 upregulation through sequestering miR-18a and the following ERK/MEK activation, thus enhancing viability while reducing apoptosis of N2a cells. SNHG15 may serve as a novel target for ischemic stroke treatment.

Clear cell renal carcinoma (ccRC) is a highly heterogeneous and progressively malignant disease. Analyzing ccRC progression in terms of modifications at the molecular and genetic level may help us to develop a broader understanding of its patho-physiology and may give us a glimpse toward improved therapeutics. In this work, by using TCGA data, we studied the molecular progression of the four main ccRC stages (i, ii, iii, iv) in two different yet complementary approaches: (a) gene expression and (b) gene co-expression. For (a) we analyzed the differential gene expression between each stage and the control non-cancer group. We compared the progression molecular signature between stages, and observed those genes that change their expression patterns through progression stages. For (b) we constructed and analyzed co-expression networks for the four ccRC progression stages, as well as for the control phenotype, to observe whether and how the co-expression landscape changes with progression. We separated genomic interactions into intra-chromosome (cis-) and inter-chromosome (trans-). Finally, we intersected those networks and performed functional enrichment analysis. All calculations were made over different network sizes, from the top 100 edges to top 1,000,000. We show that differential expression is quite similar between ccRC progression stages. However, interestingly, two genes, namely SLC6A19 and PLG show a significant progressive decrease in their expression according to ccRC stage, meanwhile two other genes, SAA2-SAA4 and CXCL13 show progressive increase. Despite the high similarity between gene expression profiles, all networks are substantially different between them in terms of their topological features. Control network has a larger proportion of trans- interactions, meanwhile for any stage, the amount of cis- interactions is higher, independent of the network cut-off. The majority of interactions in any network are phenotype-specific. Only 189 interactions are shared between the five networks, and 533 edges are ccRC-specific, independent of the stage. The small resulting connected components in both cases are formed by genes with the same differential expression trend, and are associated with important biological processes, such as cell cycle or immune system, suggesting that activity of these categories follows the differential expression trend. With this approach we have shown that, even if the expression program is similar during ccRC progression, the co-expression programs strongly differ. More research is needed to understand the delicate interplay between expression and co-expression, but this is a first approach to enclose both approaches in an integrative view aimed at a deeper understanding in gene regulation in tumor evolution.

Keywords: CXCL13 progressive overexpression; PLG progressive underexpression; SAA2-SAA4 progressive overexpression; SLC6A19 progressive underexpression; cancer progression stages; clear cell renal carcinoma; gene co-expression networks; loss of long-range co-expression.

The chemokine, C-X-C motif ligand 13 (CXCL13), is constitutively expressed in lymphoid organs and controls the recruitment and compartmentalization of lymphocytes and antigen presenting cells within these specialized structures. Recent data, however, also show induction of this molecule under a variety of circumstances during central nervous system (CNS) inflammation. While its role(s) in the pathogenesis of neoplastic, infectious and autoimmune disorders of the CNS remain incompletely understood, growing evidence suggests that CXCL13 could become a relevant therapeutic target in at least some of these conditions. This review focuses on the diseases, cellular sources and external factors known to regulate CXCL13 production in the inflamed CNS.