Although tumor blood vessels have been a major therapeutic target for cancer chemotherapy, little is known regarding the stepwise development of the tumor microenvironment. Here, we use a multicolor Cre-dependent marker system to trace clonality within the tumor microenvironment to show that tumor blood vessels follow a pattern of dynamic clonal evolution. In an advanced melanoma tumor microenvironment, the vast majority of tumor vasculature clones are derived from a common precursor. Quantitative lineage analysis reveals founder clones diminish in frequency and are replaced by subclones as tumors evolve. These tumor-specific blood vessels are characterized by a developmental switch to a more invasive and immunologically silent phenotype. Gene expression profiling and pathway analysis reveals selection for traits promoting upregulation of alternative angiogenic programs such as unregulated HGF-MET signaling and enhanced autocrine signaling through VEGF and PDGF. Furthermore, we show a developmental switch in the expression of functionally significant primary lymphocyte adhesion molecules on tumor endothelium, such as the loss in expression of the mucosal addressin MAdCAM-1, whose counter receptor a4β7 on lymphocytes controls lymphocyte homing. Changes in adhesive properties on tumor endothelial subclones are accompanied by decreases in expression of lymphocyte chemokines CXCL16, CXCL13, CXCL12, CXCL9, CXCL10, and CCL19. These evolutionary patterns in the expressed genetic program within tumor endothelium will have both a quantitative and functional impact on lymphocyte distribution that may well influence tumor immune function and underlie escape mechanisms from current antiangiogenic pharmacotherapies.

Follicular dendritic cells (FDCs) in reactive germinal centres (GCs) show modulated expression of antigens indicative of step-wise maturation from more undifferentiated stroma. The present study compared the expression of FDC markers CD21, CD23, CD35, and chemokine CXCL13 and the stromal markers low-affinity nerve growth factor receptor (LNGFR) and CNA.42 in 35 follicular lymphoma (FL) cases with reactive lymphoic tissue. CXCL13 was expressed by follicular stroma in all FLs but most cases showed either partial (11/35 cases, 31%) or complete (10/35 cases, 29%) absence of other FDC antigens, most commonly CD23, followed by CD21 and CD35, with variable patterns of LNGFR and CNA.42 immunostaining. Only a minority of FL cases (14/35, 40%) showed stroma that resembled mature FDCs (CD23+, CD21+, CD35+) and these tumours were always associated with numerous intrafollicular T-cells, similar to reactive GCs. In the 25 FL cases that had identifiable extrafollicular tumour cells, the immunophenotype of follicular stroma showed the same variability but the extrafollicular stroma showed an absence of FDC markers, with the exception of frequent strong LNGFR staining. Stromal phenotypic changes in FL were not correlated with mean follicle size, percentage of diffuse growth, tumour mitotic rate or the proliferation index as determined by PCNA immunostaining. Serial biopsy specimens analysed in a subset of 15 patients showed either a stable stromal phenotype (seven cases, 47%) or loss of FDC antigens in tumour-associated stroma over time (seven cases, 47%). The GC-like subset of FLs, having both abundant intrafollicular T-cells and fully differentiated CD23+ FDCs, comprises a minority of FL cases that likely have different growth requirements from FLs that lack these features. The pattern of FDC antigen loss in stroma of FL is a readily assessable biological feature that appears independent of architectural growth pattern and may serve as a useful surrogate marker of tumour progression.

BACKGROUND

Autoimmune pancreatocholangitis (AIPC) is an emerging, not completely characterized disease. Aim of this study was the comprehensive evaluation of a series of AIPC patients, who were diagnosed and treated in a European institution between January 2003 and July 2006.

RESULTS

Thirty-three patients with histologically confirmed AIPC were analyzed and compared to 20 patients with non-autoimmune chronic pancreatitis (CP) and 14 patients with primary sclerosing cholangitis (PSC). Clinical features and conventional histopathology were taken into account. Immunohistochemistry and real-time quantitative PCR were used for the characterization of the inflammatory infiltrate and the stromal reaction. AIPC was localized in the pancreatic head in 94% of the patients. Intra- and/or extrapancreatic biliary tract involvement was present in 64% of the cases. The number of infiltrating T-lymphocytes, macrophages and total plasma cells was significantly higher in AIPC than in CP (3-, 4- and 8-fold increase, respectively). The absolute number of IgG4-positive plasma cells was higher in AIPC than in CP and PSC (7-fold and 35-fold increase, respectively), but significance was only reached in comparison with PSC. CXCR5- and CXCL13-positive cells were almost exclusively detected in AIPC.

CONCLUSIONS

AIPC is mainly a disease of the pancreatic head with possible extension into the periphery of the gland and/or into the biliary tract/gallbladder. The morphology of AIPC, as well as the immune- and stromal reaction is characteristic and comparable between cases with and without biliary tract involvement. Immunological markers (IgG4, CXCR5, CXCL13) can be of diagnostic relevance in specific settings.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.

OBJECTIVE

To investigate the effect of CXCL13 (C-X-C motif chemokine 13) on hepatocellular carcinoma and clarify the potential mechanisms.

METHODS

32 patients with hepatocellular carcinoma and 12 healthy controls were recruited for analyzing the expression of CXCL13 by RT-PCR (reverse transcription-polymerase chain reaction). ELISA (enzyme-linked immune-sorbent assay) was used to test the concentration of serum CXCL13. The interaction between CXCL13 and Wnt signaling was analyzed by western blot. In vitro PBMCs cultured with HepG2 supernatant, the levels of IL-12, IL4, IL-6, and IL-17, and four IgG subclasses were detected by ELISA.

RESULTS

The rate of high expression CXCL13 was 63.4% in advanced HCC patients, and the serum CXCL13 was also at a high level in stage IV HCC patients. Meanwhile CXCL13 level was positively correlated with serum ALT (Alanine Transaminase) and AST (Aspartate Aminotransferase). CXCL13 and Wnt/β-catenin signaling shared a positive feedback loop. Furthermore, CXCL13 could obviously promote the expressions of IL-12 and IL-17, and induce IgG4 secreted by B cells.

CONCLUSIONS

The effect of CXCL13 on promoting liver cancer is related to the activation of Wnt/β-catenin pathway and the facilitation of IL-12, IL-17 and IgG4. CXCL13 plays an important role in the progression of HCC, and it may act as a potential target for the diagnosis and treatment of HCC.

Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is a treatable autoimmune disorder characterized by prominent neuropsychiatric symptoms that predominantly affects children and young adults. In this review, we discuss the pathogenic mechanisms and immunologic triggers of anti-NMDAR encephalitis, and provide an overview of treatment and prognosis of this disorder, with specific focus on the management of common symptoms, complications, and patients during pregnancy. Most patients respond well to first-line treatment and surgical resection of tumors. When first-line immunotherapy fails, second-line immunotherapy can often improve outcomes. In addition, treatment with immunomodulators and tumor resection are effective treatment strategies for pregnant patients. Benzodiazepines are the preferred treatment for patients with catatonia, and electroconvulsive therapy (ECT) may be considered when pharmacological treatment is ineffective. Age, antibody titer, cerebellar atrophy, levels of biomarkers such as C-X-C motif chemokine 13 (CXCL13), cell-free mitochondrial (mt)DNA in cerebral serum fluid (CSF), and timing from symptom onset to treatment are the main prognostic factors. Patients without tumors or those who receive insufficient immunotherapy during the first episode are more likely to relapse.

The microvasculature of human spleens is still incompletely understood. Two enigmatic types of red pulp microvessels, penicillar arterioles and sheathed capillaries, have already been described in the nineteenth century without gaining much attention afterwards. We performed a detailed analysis of sheathed capillaries to clarify the cellular composition of their sheaths by immunohistological double-staining experiments. Capillary sheaths comprise three different cell types, namely specialized cuboidal CD271(++) inner sheath cells surrounded by CD271(-) macrophages and accumulations of B lymphocytes. The CD271(++) inner sheath cells express the chemokine CXCL13 in a unique single dot pattern. Sheath-associated B lymphocytes consist of IgM(+), IgD(++), and of "switched" cells. T lymphocytes do not accumulate in pericapillary sheaths. The predominant sheath-associated macrophage population is CD163(-)CD68(+) and thus differs from the majority of red pulp macrophages. The sheath-associated macrophages strongly express CD169 only in perifollicular sheaths, but not in sheaths located deeper in the red pulp. IgM(+), IgD(++), and "switched" B cells are also closely associated with red pulp arterioles characterized by the expression of smooth muscle actin in muscle cells and in branched periarteriolar stromal cells. Capillary sheaths are observed in a post-arteriolar position and appear to be of limited length. We suggest to change the term "Vagina periarteriolaris makrophagocytica" of the international histological and embryological terminologies to "Vagina pericapillaris."

The aim of the study was to test the hypothesis that B-cell repopulation following rituximab (anti-CD20) therapy is orchestrated by chemokines and non-chemokine cytokines. Twenty-five children with opsoclonus-myoclonus syndrome (OMS) received rituximab with or without conventional agents. A comprehensive panel of 40 chemokines and other cytokines were measured in serum by ELISA and multiplexed fluorescent bead-based immunoassay. Serum BAFF concentration changed dramatically (even after first infusion) and inversely with B-cell depletion/repopulation and CXCL13 concentration at 1, 3, and 6 months. Negative correlations were found for BAFF concentration vs blood B cell percentage and serum CXCL13 concentration; positive correlations with serum rituximab concentrations. Six months after initiation of therapy, no significant difference in the levels of APRIL, CXCL10, IL-6, or 17 other cytokines/chemokines were detected. These data reveal a major role for BAFF in peripheral B cell repopulation following rituximab-induced B-cell depletion, and novel changes in CXCL13. ClinicalTrials.gov NCT0024436.

Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that Foxp3+ T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus-associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident Foxp3+ T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident Foxp3+ T lymphocytes resulted in prolonged interactions between B and CD4+ T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory Foxp3+ T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses.

The purpose of this study was to determine levels of the cytokines IL-1β, IL-6, IL-21, IL-22, and IL-23 and the chemokines CXCL13, CCL19, CCL20, and CCL21 in aqueous humor (AH) samples from patients with specific uveitic entities.

Paired serum samples (n = 13) and AH samples (n = 111) from patients with active idiopathic granulomatous uveitis (IGU) or with uveitis associated with HLA-B27-related inflammation, Behçet's disease (BD), Vogt-Koyanagi-Harada (VKH) disease, or sarcoidosis and control patients were analyzed in two different multiplex assays.

Cytokines IL-1β, IL-21, IL-22, and IL-23 were not detected in any AH sample. Chemokine CCL21 concentrations in serum were significantly higher than those in AH. CCL19 levels in AH and serum were not significantly different. Levels of CCL20 and CXCL13 in AH were significantly higher than those in serum. IL-6 was not detected in serum samples. IL-6 AH levels were significantly higher in patients with HLA-B27-associated uveitis and in BD patients than in patients with VKH disease, sarcoidosis, and IGU (P < 0.0001). CCL20 AH levels were significantly higher in HLA-B27-associated uveitis than in BD, VKH, sarcoidosis, and IGU (P = 0.001), whereas CXCL13 AH levels were significantly higher in VKH disease and IGU than in HLA-B27-associated uveitis, BD, and sarcoidosis (P = 0.007).

IL-6-driven immune responses are more potent in HLA-B27-associated uveitis and BD than in VKH disease, sarcoidosis, and IGU. CCL20 appears to be a specific biomarker of HLA-B27-associated uveitis, whereas CXCL13 appears to be a biomarker of VKH disease and IGU. Our findings suggest that IL-6, CCL20, and CXCL13 could serve as drug targets for treatment of specific clinical entities of endogenous uveitis.

Orosomucoid (ORM, composed of two isoforms, ORM1 and ORM2) has been described as an inducer of M2 macrophages, which are cells that decrease host antibacterial innate immunities. However, it is unknown which phenotypes of M2 macrophages are induced by ORM. In this study, healthy donor monocytes stimulated with ORM (ORM-monocytes) were characterized phenotypically and biologically. CCL1 (a biomarker of M2b macrophages) and IL-10 were detected in monocyte cultures supplemented with ORM1; however, CCL17 (a biomarker of M2a macrophages) and CXCL13 (a biomarker of M2c macrophages) were not produced in these cultures. All of these soluble factors were not detected in the culture fluids of monocytes stimulated with ORM2. Monocytes stimulated with ORM1 were characterized as CD64(-)CD209(-)CD163(+)CCL1(+) cells. MRSA and Enterococcus faecalis infections were accelerated in chimeras (NOD/scid IL-2Rγ(null) mice reconstituted with white blood cells) after inoculation with monocytes stimulated with ORM1 or treatment with ORM1; however, the infections were greatly mitigated in both chimeras inoculated with ORM1-stimulated monocytes and treated with ORM1, after an additional treatment with an inhibitor of M2b macrophages (CCL1 antisense ODN). These results indicate that ORM1 stimulates quiescent monocytes to polarize to M2b monocytes. The regulation of M2b macrophages may be beneficial in controlling opportunistic infections in patients with a large amount of plasma ORM1.

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Bone marrow (BM) biopsy is often performed early in the evaluation of patients with angioimmunoblastic T-cell lymphoma (AITL), and may be the first diagnostic tissue sample; yet the BM histopathology associated with this disease has not been well described. In this study, BM specimens from 13 patients with AITL were reviewed. Seven (54%) were involved by AITL, which was characterised by paratrabecular and interstitial polymorphous infiltrates containing cytologically atypical lymphocytes, histiocytes and eosinophils. The neoplastic cells were positive for CD10 and CXCL13 by immunohistochemistry in a subset of cases. As in lymph nodes, the lymphomatous infiltrate in some BMs contained numerous small or scattered large B cells, resembling either benign lymphoid aggregates or T cell rich large B cell lymphoma, respectively. Secondary haematological changes were frequent and presented independent of BM involvement by AITL; these included trilineage haematopoietic hyperplasia and plasmacytosis. When BM biopsy preceded the diagnosis of AITL, these secondary changes were misinterpreted as chronic myeloproliferative disease (n = 2), or plasma cell dyscrasia (n = 2). In two cases, these changes obscured the presence of BM involvement by AITL. The spectrum of BM findings in AITL patients is important to recognise for early and accurate diagnosis in this disease.

We have previously shown that peripherally restricted acute phase response (APR) elicited by intraperitoneal (i.p.) injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), renders the brain hypersusceptible to excitotoxic insult as seen from profoundly exacerbated kainic acid (KA)-induced seizures. In the present study, we found that this hypersusceptibility was protracted for up to 72 h. RT-PCR profiling of hippocampal gene expression revealed rapid upregulation of 23 genes encoding cytokines, chemokines and chemokine receptors generally within 6 h after PIC challenge. The expression of most of these genes decreased by 24 h. However, two chemokine genes, i.e., Ccl19 and Cxcl13 genes, as well as two chemokine receptor genes, Ccr1 and Ccr7, remained upregulated for 72 h suggesting their possible involvement in the induction and sustenance of seizure hypersusceptibility. Also, 12 genes encoding proteins related to glutamatergic and GABAergic neurotransmission featured initial upregulation or downregulation followed by gradual normalization. The upregulation of the Gabrr3 gene remained upregulated at 72 h, congruent with its plausible role in the hypersusceptible phenotype. Moreover, the expression of ten microRNAs (miRs) was rapidly affected by PIC challenge, but their levels generally exhibited oscillating profiles over the time course of seizure hypersusceptibility. These results indicate that protracted seizure susceptibility following peripheral APR is associated with a robust polygenic response in the hippocampus.

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.

Chemokines are peptides that function as chemoattractant cytokines in cell activation, differentiation and trafficking. Endothelin B receptor (ETBR) is a receptor for endothelin, which is known to function as a vasoconstrictor. In the present study, to clarify the immune escape mechanism of primary central nervous system lymphomas (PCNSLs), the expression of ETBR and of subsets of chemokines (CXCL12, 13) in 24 PCNSLs was investigated. CXCL12 was expressed by lymphoma cells in different resident brain cell populations in 22/24 cases. CXCL13 expression was identified in tumor cells in 19/24 cases, but was only expressed by tumor cells and by proliferating vascular endothelial cells. In addition, tumor-infiltrated lymphocytes (TILs) accumulated in areas with expression of chemokines, particularly of CXCL13. ETBR expression was detected in 12/24 cases. Positive ETBR cases were associated with a paucity of TILs, particularly of cytotoxic T cells, whereas negative ETBR cases were associated with an abundance of TILs. The combined data indicate that CXCL12 and CXCL13 up-regulation may be differently linked to the development of PCNSLs and to the accumulation of TILs. In addition, ETBR expression by lymphoma and endothelial cells may mediate trafficking of TILs, which may explain the immune escape processes of PCNSLs.

In this study, we sought to identify definitive biomarkers associated with disease activity in primary Sjögren's syndrome (pSS).

Serum protein concentrations in pSS patients and healthy controls (HCs) were comprehensively screened using high-throughput proteomic analysis, and differentially expressed proteins were extracted. Correlation between differentially expressed proteins and European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (ESSDAI) scores was analyzed and disease activity-associated biomarkers were identified. These biomarkers were validated by enzyme-linked immunosorbent assay (ELISA) in a separate pSS cohort.

The serum concentrations of 1100 proteins were compared between 30 pSS patients and 30 HCs, with 82 differentially expressed proteins identified as pSS-associated proteins. Of these 82 proteins, 9 were identified as disease activity-associated biomarkers. These nine biomarkers underwent validation by ELISA in a separate pSS validation cohort (n = 58), with five proteins (CXCL13, TNF-R2, CD48, B-cell activating factor (BAFF), and PD-L2) subsequently being confirmed as candidate biomarkers. Of these five candidate biomarkers, CXCL13 exhibited the most significant correlation with the lymphadenopathy, glandular, and pulmonary domains of the ESSDAI. CXCL13, TNF-R2 and CD48 exhibited a positive correlation with the biological domain of the ESSDAI. TNF-R2 exhibited the most negative correlation with uptake in the submandibular gland on technetium 99m-pertechnetate salivary gland scintigraphy.

Our approach successfully identified serum biomarkers associated with disease activity in pSS patients. These markers might be potential therapeutic targets in pSS patients.

Chemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19(+) B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection.

Flow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation.

The chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19(+) cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact.

CXCR3 is the primary chemokine receptor on CD19(+) B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

The epimerization of glucuronic acid into iduronic acid adds structural variability to chondroitin/dermatan sulfate polysaccharides. Iduronic acid-containing domains play essential roles in processes such as coagulation, chemokine and morphogen modulation, collagen maturation, and neurite sprouting. Therefore, we generated and characterized, for the first time, mice deficient in dermatan sulfate epimerase 1 and 2, two enzymes uniquely involved in dermatan sulfate biosynthesis. The resulting mice, termed DKO mice, were completely devoid of iduronic acid, and the resulting chondroitin sulfate chains were structurally different from the wild type chains, from which a different protein binding specificity can be expected. As a consequence, a vast majority of the DKO mice died perinatally, with greatly variable phenotypes at birth or late embryological stages such as umbilical hernia, exencephaly and a kinked tail. However, a minority of embryos were histologically unaffected, with apparently normal lung and bone/cartilage features. Interestingly, the binding of the chemokine CXCL13, an important modulator of lymphoid organogenesis, to mouse DKO embryonic fibroblasts was impaired. Nevertheless, the development of the secondary lymphoid organs, including the lymph nodes and spleen, was normal. Altogether, our results indicate an important role of dermatan sulfate in embryological development and perinatal survival.

BACKGROUND

The clearance of hepatitis B surface antigen (HBsAg) loss, defined as functional cure, is a clinical target in patients with chronic hepatitis B (CH). To understand the immune responses underlying functional cure, we evaluated cytokine and chemokine expression profiles from patients with resolving and nonresolving acute hepatitis B (AH).

METHODS

We cross-sectionally evaluated 41 chemokines and cytokines at the peak of hepatitis in the sera from 41 self-limited AH patients who achieved HBsAg seroconversion, 8 AH patients who failed to clear HBsAg within 1 year after the diagnosis, 8 CH patients with hepatic flare, and 14 healthy volunteers. We longitudinally examined 41 chemokines and cytokines in the sera from 4 self-limited AH patients, 3 chimpanzees inoculated with hepatitis B virus (HBV), and 2 CH patients treated with nucleotide analogs and PEG-IFN-α, one resulting in functional cure.

RESULTS

In AH patients and HBV-inoculated chimpanzees with HBsAg loss, CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 were elevated at hepatitis with subsequent decline of HBsAg. Interestingly, IL-21 elevation was observed only in resolving AH patients but not in nonresolvers. CXCL13 and IL-21 elevation was not observed in CH patients who failed to attain HBsAg loss, even at hepatic flare. A concomitant increase of CXCL13 and IL-21 was significant in CH patients who attained HBsAg seroconversion with a sequential therapy.

CONCLUSIONS

Elevation of serum CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 might be a hallmark of functional cure of AH or CH patients.

BACKGROUND

B cells play an important role in allergic asthma. However, the mechanisms by which these cells are activated in the airways remain poorly understood.

METHODS

We used a mouse model of ovalbumin (OVA)-induced allergic inflammation to study CXCL13 and to investigate the concentration of this chemokine in the BAL fluid derived from asthmatic and normal control subjects.

RESULTS

We found that OVA-challenged mice upregulate the CXCL13/CXCR5 axis, which is associated with several changes in their airways, including recruitment of B and CD4(+) cells, development of bronchial-associated lymphoid tissue, and airway inflammation. Treating sensitized mice with an anti-CXCL13 antibody reduced cell recruitment, bronchial-associated lymphoid tissue formation, and airways inflammation. Interestingly, measurements of CXCL13 using enzyme-linked immunosorbent assay showed that levels of this cytokine were significantly elevated in BAL fluid from subjects with asthma compared with control subjects (median, 162 [range, 120-296] vs 31 [range, 120-156] pg/mL; P = .005).

CONCLUSIONS

All together, these findings suggest that CXCL13 is involved in the allergic airway inflammatory process, and targeting this chemokine may constitute a novel approach in asthma.

Recurrent airway obstruction (RAO) is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs). We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE) was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.