Mild cold stress induced by housing mice with a 4T1 triple-negative breast cancer (TNBC) cell implantation model at 22 °C increases tumor growth rate with a pro-tumorigenic immune microenvironment (lower CD8 ⁺T cells, higher myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs)). Since cold stress also activates thermogenesis, we hypothesized that enhanced thermogenesis is associated with more aggressive cancer biology and unfavorable tumor microenvironment (TME) in TNBC patients. A total of 6479 breast cancer patients from METABRIC, TCGA, GSE96058, GSE20194, and GSE25066 cohorts were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) thermogenesis score. High-thermogenesis TNBC was associated with a trend towards worse survival and with angiogenesis, adipogenesis, and fatty acid metabolism pathways. On the other hand, low-thermogenesis TNBC enriched most of the hallmark cell-proliferation-related gene sets (i.e., mitotic spindle, E2F targets, G2M checkpoint, MYC targets), as well as immune-related gene sets (i.e., IFN-α and IFN-γ response). Favorable cytotoxic T-cell-attracting chemokines CCL5, CXCL9, CXCL10, and CXCL11 were lower; while the MDSC- and Treg-attracting chemokine CXCL12 was higher. There were higher M2 but lower M1 macrophages and Tregs. In conclusion, high-thermogenesis TNBC is associated with pro-tumor immune microenvironment and may serve as biomarker for testing strategies to overcome this immunosuppression.

Keywords: GSE96058; METABRIC; TCGA; cold stress; thermogenesis; triple-negative breast cancer; tumor microenvironment.

Mast cells reside in many tissues, including the lungs, and might play a role in enhancing influenza virus infections in animals. In this study, we cultured porcine mast cells from porcine bone marrow cells with IL-3 and stem cell factor to study the infectivity and activation of the 2009 pandemic H1N1 influenza virus of swine origin. Porcine mast cells were infected with H1N1 influenza virus, without the subsequent production of infectious viruses but were activated, as indicated by the release of histamines. Inflammatory cytokine- and chemokine-encoding genes, including IL-1α, IL-6, CXCL9, CXCL10, and CXCL11, were upregulated in the infected porcine mast cells. Our results suggest that mast cells could be involved in enhancing influenza-virus-mediated disease in infected animals.

The chemokines and their receptors play a key role in immune and inflammatory responses by promoting recruitment and activation of different subpopulations of leukocytes. The membrane receptor CXCR3 binds three chemokines, CXCL9, CXCL10, and CXCL11, and its involvement is recognized in many inflammatory diseases and cancers. Therefore, the inhibition of CXCR3 pathway through interactions with three ligands was indicated as putative therapeutic target for the treatment of these diseases, and some inhibitory compounds have already been described in the literature. Recently, we studied the interaction between CXCR3 and its three natural ligands and showed that three CXCR3 ligands bound the receptor mainly by their N-terminal regions using aromatic and electrostatic interactions, and, in particular, CXCL11 had the highest affinity for CXCR3. In light of these results, we focused our attention on what structural region(s) of CXCL11 interacted with CXCR3 and what were the structural features. Therefore, we have synthesized three peptides, corresponding to the N-terminal region of CXCL11, but with different aromatic amino acids, analyzed their conformations by circular dichroism, NMR, and molecular dynamics simulations, simulated their complexes with CXCR3 by docking methods, and validated these data by in vitro studies. The results showed that two peptides were able to bind CXCR3 and to mimic the molecular recognition of CXCL11 and demonstrated that N-terminal region of CXCL11 can be used as template and starting point to obtain new molecules by de novo design approaches.

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString Pancancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e metastasis occupied >90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. This article is protected by copyright. All rights reserved.

BACKGROUND

Although initial infection with Helicobacter pylori may occur before 5 years of age, the pediatric mucosal immune response against H. pylori is not clear. The aim of the present study was to evaluate immune responses in the H. pylori-infected gastric mucosa of children using microarray and real-time polymerase chain reaction (PCR) analysis of pediatric gastric samples.

METHODS

Gastric samples were obtained from 12 patients undergoing routine endoscopy of chronic abdominal complaints. Six patients (three boys, three girls) aged 10.1-14.6 years had evidence of H. pylori infection, and the remaining six (three boys, three girls) aged 10.3-15.5 years had no evidence of infection and presented no histological changes associated with gastritis. Microarray and real-time PCR analyses were performed, and the changes in gene expression-related immune response were also analyzed.

RESULTS

Using microarray analysis, the total number of significantly upregulated and downregulated genes (fold change >5, P < 0.01) was 21 in the antrum and 16 in the corpus when comparing patients with or without infection. Using real-time PCR, the expression of lipocalin-2 (Lcn2), C-C motif chemokine ligand (CCL) 18, C-X-C motif chemokine ligand (CXCL) 9 and CXCL11 was upregulated, while the expression of pepsinogen (PG) I and PGII was downregulated when comparing patients with or without infection.

CONCLUSIONS

Lcn2, CCL18, CXCL9, CXCL11, PGI and PGII play important roles in childhood H. pylori infection.

BACKGROUND

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

METHODS

Eight healthy smokers aged 40-65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

RESULTS

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

CONCLUSIONS

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

BACKGROUND

NHS Health Research Authority (13/LO/0256).

Mast cells and their products are likely to be involved in regulating orbital fibroblast activity in Graves' Ophthalmopathy (GO). Histamine is abundantly present in granules of mast cells and is released upon mast cell activation. However, the effect of histamine on orbital fibroblasts has not been examined so far. Orbital tissues from GO patients and controls were analyzed for the presence of mast cells using toluidine blue staining and immunohistochemical detection of CD117 (stem cell factor receptor). Orbital fibroblasts were cultured from GO patients and healthy controls, stimulated with histamine and cytokines (IL-6, IL-8, CCL2, CCL5, CCL7, CXCL10 and CXCL11) were measured in culture supernatants. Also hyaluronan levels were measured in culture supernatants and hyaluronan synthase (HAS) and hyaluronidase (HYAL) gene expression levels were determined. In addition, histamine receptor subtype gene expression levels were examined as well as the effect of the histamine receptor-1 (HRH1) antagonist loratadine and NF-κB inhibitor SC-514 on histamine-induced cytokine production. Mast cell numbers were increased in GO orbital tissues. Histamine stimulated the production of IL-6, IL-8 and CCL2 by orbital fibroblasts, while it had no effect on the production of CCL5, CCL7, CXCL10, CXCL11 and hyaluronan. Orbital fibroblasts expressed HRH1 and loratadine and SC-514 both blocked histamine-induced IL-6, IL-8 and CCL2 production by orbital fibroblasts. In conclusion, this study demonstrates that histamine can induce the production of NF-κB controlled-cytokines by orbital fibroblasts, which supports a role for mast cells in GO.

Understanding the cellular interactions within the tumor microenvironment (TME) of melanoma paved the way for novel therapeutic modalities, such as T cell-targeted immune checkpoint inhibitors (ICI). However, only a limited fraction of patients benefits from such therapeutic modalities, highlighting the need for novel predictive and prognostic biomarkers. As myeloid cells orchestrate the tumor-specific immune response and influence the efficacy of ICI, assessing their activation state within the TME is of clinical relevance. Here, we characterized a myeloid activation (MA) signature, comprising the three genes Cxcl11, Gbp1, and Ido1, from gene expression data of human myeloid cells stimulated with poly(I:C) or cGAMP. This MA signature positively correlated to overall survival in melanoma. In addition, increased expression of the MA signature was observed in melanoma patients responding to ICI (anti-PD-1), as compared to non-responders. Furthermore, the MA signature was validated in the murine B16F10 melanoma model where it was induced and associated with decreased tumor growth upon intratumoral administration of poly(I:C) and cGAMP. Finally, we were able to visualize co-expression of the MA signature genes in myeloid cells of human melanoma tissues using RNAscope in situ hybridization. In conclusion, the MA signature indicates the activation state of myeloid cells and represents a prognostic biomarker for the overall survival in melanoma patients.

Keywords: innate immunity; melanoma; myeloid cells; prognostic gene signature; tumor immunity.

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3-68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05-0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03-3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3-68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3-68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A>> CXCL12-S-S4V, CXCL12-R47E>> CXCL122>> CXCL12(3-68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

ISCOM vaccines induce a balanced Th1/Th2 response, long-lasting antibody responses and cytotoxic T lymphocytes. The mode of action for the adjuvant component, the ISCOM-Matrix, is known to some extent but questions remain regarding its mechanism of action. The Affymetrix GeneChip® Porcine Genome Array was applied to study the global transcriptional response to ISCOM-Matrix in pigs at the injection site and in the draining lymph node 24h after i.m. injection. Gene enrichment analysis revealed inflammation, innate immunity and antigen processing to be central in the ISCOM-Matrix response. At the injection site, 594 genes were differentially expressed, including up-regulation of the cytokines osteopontin (SPP1), IL-10 and IL-18 and the chemokines CCL2, CCL19 and CXCL16. Of the 362 genes differentially expressed in the lymph node, IL-1β and CXCL11 were up-regulated whereas IL18, CCL15 and CXCL12 were down-regulated. ISCOM-Matrix also modulated genes for pattern recognition receptors at the injection site (TLR2, TLR4, MRC1, PTX3, LGALS3) and in the lymph node (TLR4, RIG-I, MDA5, OAS1, EIF2AK2, LGALS3). A high proportion of up-regulated interferon-regulated genes indicated an interferon response. Thus, several genes, genetic pathways and biological processes were identified that are likely to shape the early immune response elicited by ISCOM-based vaccines.

Fibroblast-like synoviocytes (FLS) are major contributors to joint inflammation in rheumatoid arthritis (RA). Forkhead box O 3 (FOXO3) perturbations in immune cells are increasingly linked to RA pathogenesis. Here, we show that FOXO3 is distinctly inactivated/phosphorylated in the FLS of rheumatoid synovitis. In vitro, stimulation of FLS with tumor necrosis factor-alpha α (TNFα) induced a rapid and sustained inactivation of FOXO3. mRNA profiling revealed that the inactivation of FOXO3 is important for the sustained pro-inflammatory interferon response to TNFα (CXCL9, CXCL10, CXCL11, and TNFSF18). Mechanistically, our studies demonstrate that the inactivation of FOXO3 results from TNF-induced downregulation of phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1). Thus, we identified FOXO3 and its modulator PIK3IP1 as a critical regulatory circuit for the inflammatory response of the resident mesenchymal cells to TNFα and contribute insight into how the synovial tissue brings about chronic inflammation that is driven by TNFα.

Our data give evidence that CXCR3 ligands exhibit pronociceptive properties and play an important role in the initiation, development and maintenance of neuropathic pain. Moreover, intrathecal administration of each CXCR3 ligand induced hypersensitivity reactions in naive mice and of its neutralizing antibodies diminished neuropathic pain syndrome in CCI-exposed mice. Furthermore, our results indicate that selective CXCR3 antagonist (±)-NBI-74330 reduced the neuropathic pain-related behaviour and also enhanced morphine analgesic potency in CCI-exposed rats. Interestingly, our data show that (±)-NBI-74330 administration diminished the spinal IBA1 and, in parallel, downregulated CXCL4, CXCL9 and CXCL10. In addition, CXCR3 antagonist increased the spinal GFAP, what correlates with upregulation of CXCR3 and CXCL11. Moreover, in DRG (±)-NBI-74330 did not change IBA1 and GFAP positive cells activation, however downregulated also CXCL9. CXCR3 and CXCL10 were co-localized predominantly with neuronal marker in the spinal cord. Summing up, chronic (±)-NBI-74330 intrathecal injection promotes beneficial analgesic effects in rat neuropathic pain model, as described in details in "Pharmacological blockade of CXCR3 by (±)-NBI-74330 reduces neuropathic pain and enhances opioid effectiveness - evidence from in vivo and in vitro studies" (Piotrowska et al., 2018).

OBJECTIVE

We previously reported that prostaglandin (PG) E2 acts as a ligand for prostaglandin E receptor 3 (EP3) in conjunctival epithelial cells, that it downregulates the progression of experimental murine allergic conjunctivitis, and that in human conjunctival epithelial cells it modulates the expression of polyI:C-induced proinflammatory genes via prostaglandin E receptor 2 (EP2) and EP3, suggesting that PGE2 might have important roles in ocular surface inflammation such as allergic conjunctivitis. Here, we investigated whether PGE2 also downregulates polyI:C-induced cytokine production in human corneal epithelial cells.

METHODS

We used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction to examine the effects of PGE2 on polyI:C-induced cytokine expression by immortalized human corneal-limbal epithelial cells (HCLE). Using reverse transcription-polymerase chain reaction, we examined the messenger RNA (mRNA) expression of the PGE2 receptor, EP1-4.

RESULTS

PGE2 significantly attenuated the expression of CC chemokine ligand (CCL)5 (P < 0.0005), CCL20 (P < 0.0005), C-X-C chemokine (CXCL)10 (P < 0.0005), CXCL11 (P < 0.05), and interleukin (IL)-6 (P < 0.005) in human corneal-limbal epithelial cells. Human corneal epithelial cells manifested the mRNA expression of EP2, EP3, and EP4, but not EP1. The EP2 agonist significantly suppressed the polyI:C-induced expression of CCL5 (P < 0.005), CXCL10 (P < 0.0005), and CXCL11 (P < 0.05) but not of CCL20 and IL-6. The EP3 agonist significantly suppressed the expression of CCL5 (P < 0.05), CCL20 (P < 0.005), CXCL10 (P < 0.0005), CXCL11 (P < 0.0005), and IL-6 (P < 0.005). The EP4 agonist failed to suppress cytokine production induced by polyI:C stimulation.

CONCLUSIONS

Our results show that in human corneal epithelial cells, PGE2 attenuated the mRNA expression and production of CCL5, CXCL10, and CXCL11 via both EP2 and EP3, and that the mRNA expression and production of CCL20 and IL-6 was attenuated only by EP3.

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over β-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

The appendix contains abundant lymphoid tissue and is constantly exposed to gut flora. When completed at a young age, appendicitis followed by appendectomy (AA) prevents or significantly ameliorates Inflammatory Bowel Diseases (IBDs) in later life. Inflammatory bowel disease comprises Crohn's disease and ulcerative colitis. Our murine AA model is the only existing experimental model of AA. In our unique model, AA performed in the most proximal colon limits colitis pathology in the most distal colon by curbing T-helper 17 cell activity, diminishing autophagy, modulating interferon activity-associated molecules, and suppressing endothelin vaso-activity-mediated immunopathology. In the research presented in this paper, we have examined the role of chemokines in colitis pathology with our murine AA model. Chemokines are a family of small cytokines with four conserved cysteine residues. Chemokines induce chemotaxis in adjacent cells with corresponding receptors. All 40 known chemokine genes and 24 chemokine receptor genes were examined for gene expression levels in distal colons three days post-AA and 28 days post-AA. At 28 days post-AA, the chemokine gene CCL5 was significantly upregulated. Furthermore, Gene Set Enrichment Analysis (GSEA) showed upregulation of seven CCL5-associated gene-sets 28 days post-AA in contrast to just one gene-set downregulated at the same time-point. The chemokine gene CXCL11 was significantly upregulated three days post-AA and 28 days post-AA. Evaluation using GSEA showed upregulation of six CXCL11-associated gene sets but no downregulation of any gene set. At 28 days post-AA, CCL17 gene expression was significantly downregulated. There was no expression of any chemokine receptor gene three days post-AA, but CCR10 was the only chemokine receptor gene that displayed differential gene expression (upregulation) 28 days post-AA. No CCR10-associated gene set was upregulated in GSEA in contrast to one downregulated gene set. Our analysis resulted in identifying three new therapeutic targets towards ameliorating colitis: CCL5, CXCL11, and CCL17. While CCL5 and CXCL11 are good therapeutic chemokine candidates to be exogenously administered, CCL17 is a good candidate chemokine to competitively inhibit or limit colitis pathology.

CXC chemokine receptor 3 (CXCR3), a receptor for the C-X-C motif chemokines (CXCL) CXCL9, CXCL10, and CXCL11, which not only plays a role in chemotaxis but also regulates differentiation and development of memory and effector T cell populations. Herein, we explored the function of CXCR3 in the modulation of different organ-specific autoimmune diseases in interleukin (IL)-2 receptor deficiency (CD25-/-) mice, a murine model for both cholangitis and colitis. We observed higher levels of CXCL9 and CXCL10 in the liver and colon and higher expression of CXCR3 on T cells of the CD25-/- mice compared with control animals. Deletion of CXCR3 resulted in enhanced liver inflammation but alleviated colitis. These changes in liver and colon pathology after CXCR3 deletion were associated with increased numbers of hepatic CD4+ and CD8+ T cells, in particular effector memory CD8+ T cells, as well as decreased T cells in mesenteric lymph nodes and colon lamina propria. In addition, increased interferon-γ response and decreased IL-17A response was observed in both liver and colon after CXCR3 deletion. CXCR3 modulated the functions of T cells involved in different autoimmune diseases, whereas the consequence of such modulation was organ-specific regarding to their effects on disease severity. Our findings emphasize the importance of extra caution in immunotherapy for organ-specific autoimmune diseases, as therapeutic interventions aiming at a target such as CXCR3 for certain disease could result in adverse effects in an unrelated organ.

With overuse of the broad-spectrum antibiotics, the pulmonary fungal infection increasingly becomes the most common complication associated with senile pulmonary tuberculosis (TB) and attracts intensive attentions from clinicians. Here we presented the retrospective analysis of impact of fluconazole treatment on the clinical outcome and immune response in fungal co-infected tuberculosis patients. A randomized, double-blind, placebo-controlled trial of fluconazole (100 mg per day for consecutive weeks) in fungal-positive senile tuberculosis patients was conducted in our hospital. Peripheral eosinophil counts were computed by the automatic hematology analyzer. The secretory inflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α and chemokines chemokine C-X-C motif ligand (CXCL)9, CXCL10, CXCL11 were determined with enzyme-linked immunosorbent assay kits. The peripheral T helper 1 cells (Th1) and regulatory T cells (Treg) population were analyzed by flow cytometry. None of significant difference in respect to baseline TB score was observed between placebo and fluconazole groups. Administration of fluconazole significantly stimulated eosinophils population and secretion of inflammatory cytokines IFN-γ and TNF-α. Simultaneously, the peripheral Th1% and chemokines including CXCL9, CSCL10, CXCL11 were markedly induced in response to fluconazole treatment. Fungal infection significantly affected host immunity during tuberculosis which was effectively reversed by fluconazole treatment.

BACKGROUND

Non-celiac wheat sensitivity is an emerging wheat-related syndrome showing peak prevalence in Western populations. Recent studies hypothesize that new gliadin alleles introduced in the human diet by replacement of ancient wheat with modern varieties can prompt immune responses mediated by the CXCR3-chemokine axis potentially underlying such pathogenic inflammation. This cultural shift may also explain disease epidemiology, having turned European-specific adaptive alleles previously targeted by natural selection into disadvantageous ones.

METHODS

To explore this evolutionary scenario, we performed ultra-deep sequencing of genes pivotal in the CXCR3-inflammatory pathway on individuals diagnosed for non-celiac wheat sensitivity and we applied anthropological evolutionary genetics methods to sequence data from worldwide populations to investigate the genetic legacy of natural selection on these loci.

RESULTS

Our results indicate that balancing selection has maintained two divergent CXCL10/CXCL11 haplotypes in Europeans, one responsible for boosting inflammatory reactions and another for encoding moderate chemokine expression.

CONCLUSIONS

This led to considerably higher occurrence of the former haplotype in Western people than in Africans and East Asians, suggesting that they might be more prone to side effects related to the consumption of modern wheat varieties. Accordingly, this study contributed to shed new light on some of the mechanisms potentially involved in the disease etiology and on the evolutionary bases of its present-day epidemiological patterns. Moreover, overrepresentation of disease homozygotes for the dis-adaptive haplotype plausibly accounts for their even more enhanced CXCR3-axis expression and for their further increase in disease risk, representing a promising finding to be validated by larger follow-up studies.

To investigate the effect of sinomenine on the expression of chemokines and chemokine receptors of dendritic cells in patients with rheumatoid arthritis in vitro.

METHODS

The peripheral blood mononuclear cells isolated from 8 patients with rheumatoid arthritis were induced to differentiate into dendritic cells with GM-CSF and IL-4. The dendritic cells were exposed to sinomenine at high (5 mmol/L), moderate (2 mmol/L), and low (1 mmol/L) concentrations or treated with the control medium. The expression of CCR5 and CCR7 on the surface of the dendritic cells were measured by flow cytometry, and the CCR5 and CCR7 mRNA expressions were detected by semi-quantitative PCR. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (ITAC).

RESULTS

Compared with the control cells, the dendritic cells treated with sinomenine, especially at high and moderate concentrations, showed significantly lowered mRNA and protein expressions of CCR5 and CCR7. Similar results were observed in the expressions of CXCL9 (MIG) and CXCL10 (IP-10), but not in CXCL11 (ITAC).

CONCLUSIONS

Sinomenine produces therapeutic effect on rheumatoid arthritis possibly by inhibiting the expression of chemokines and chemokine receptors in the dendritic cells to suppress the chemotactic migration of the dendritic cells.

Tuberculosis pleural effusion (TPE) is common in clinical practice, and its diagnosis remains a challenge for clinicians. Ziehl-Neelsen staining, PE Mycobacterium tuberculosis culture, and biopsy are the gold standards for TPE diagnosis; however, they are time-consuming, invasive, observer-dependent, and insensitive. PE markers represent a rapid, low-cost, and non-invasive objective diagnostic tool for TPE. In the past decades, several PE biomarkers have been developed, and their diagnostic accuracy has been evaluated in many studies. Here, we reviewed the literature to summarize the diagnostic accuracy of these biomarkers, especially using the evidence from systematic review and meta-analysis. The current research strongly suggests that adenosine deaminase (ADA), interferon-gamma (IFN-γ), and interleukin 27 (IL-27) have extremely higher diagnostic accuracy for TPE, while the diagnostic accuracy of interferon gamma release assays (IGRAs), tumor necrosis factor-α (TNF-α), and interferon-γ-induced protein 10 kDa (IP-10) is moderate. Although some evidence supports C-X-C motif chemokine ligand 9 (CXCL9), CXCL11, CXCL12, sFas ligand, angiotensin-converting enzyme (ACE), calpain-1, spectrin breakdown products (SBDP), matrix metalloproteinase-1 (MMP-1), soluble CD26 (sCD26), soluble interleukin 2 receptor (sIL-2R) as useful diagnostic markers for TPE, more support is needed to validate their diagnostic accuracy. Finally, nucleic acid amplification tests (NAATs) have extremely high diagnostic specificity, but their sensitivity is low. Taken together, ADA is the preferred marker for TPE because its low cost and suitability for standardization.

Keywords: Tuberculosis pleural effusion (TPE); diagnostic accuracy; pleural markers; sensitivity; specificity.

Dual-specific antibodies are characterized by an antigen-combining site mediating specific interactions with two different antigens. We have generated five dual-specific single chain variable fragments (scFv) that neutralize the activity of the two chemokines, CXCL9 and CXCL10, to bind to their receptor CXCR3. To better understand how these dual-specific scFvs bind these two chemokines that only share a 37% sequence identity, we mapped their epitopes on human CXCL9 and CXCL10 and identified serine 13 (Ser(13)) as a critical residue. It is conserved between the two chemokines but not in the third ligand for CXCR3, CXCL11. Furthermore, Ser(13) is exposed in the tetrameric structure of CXCL10, which is consistent with our finding that the scFvs are able to bind to CXCL9 and CXCL10 immobilized on glycosaminoglycans. Overall, the data indicate that these dual-specific scFvs bind to a conserved surface involved in CXCR3 receptor interaction for CXCL10 and CXCL9. Thus, structural mimicry between the two targets is likely to be responsible for the observed dual specificity of these antibody fragments.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The aim of the present study was to investigate the expression of microRNA-144 (miR-144) and C-X-C motif chemokine ligand 11 (CXCL11) in CRC and their association. Data from Gene Expression Omnibus (GEO) DataSets were analyzed to obtain the expression profile of CXCL11 in CRC. Subsequently, serum samples were collected from 65 subjects, including 39 patients with CRC and 26 controls; CRC and adjacent normal tissues were collected from all 39 CRC patients and the expression of CXCL11 was measured in these specimens. After searching for the potential regulator of CXCL11 through bioinformatics analysis, the levels of miR-144 in the clinical specimens were also detected. Finally, the regulatory association between miR-144 and CXCL11 was certified via the dual-luciferase reporter assay. Microarray data and bioinformatics analysis demonstrated that CXCL11 was significantly upregulated in CRC tissues and miR-144 was a potential regulator of CXCL11. In line with this finding, the expression of CXCL11 was significantly increased in the serum and tumor samples of patients with CRC, while that of miR-144 was downregulated. Dual-luciferase reporter assay revealed that miR-144 directly targets the 3'-untranslated region of CXCL11 mRNA to regulate its expression. These results demonstrated that enhanced CXCL11 expression in patients with CRC was associated with reduced miR-144 expression. The results of the present study may indicate a novel regulatory role of miR-144 in CRC through CXCL11 downregulation.