Rabies remains a public health threat in most parts of the world, and approximately 99% of the cases are transmitted by dogs. There is an urgent need to develop an efficacious and affordable vaccine to control canine-transmitted rabies in developing countries. Our previous studies demonstrate that overexpression of chemokines/cytokines such as CCL-3 (MIP-1α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can enhance the immunogenicity of rabies vaccines. In the present study, the chemokine CXCL13 was inserted into the genome of the recombinant rabies virus (rRABV) strain LBNSE, and the effect of the chemokine CXCL13 on the immunogenicity of RABV was investigated. It was found that LBNSE-CXCL13 recruited follicular helper T (Tfh) and germinal center (GC) B cells, promoted the formation of GCs, and increased the population of plasma cells in immunized mice. Further studies showed that mice immunized with LBNSE-CXCL13 produced more rabies virus-neutralizing antibodies (VNAs) and developed better protection than those immunized with the parent virus LBNSE or the GM-CSF-expressing RABV (LBNSE-GM-CSF). Collectively, these findings provide a better understanding of the role of CXCL13 expression in the immunogenicity of the RABV, which may help in designing more-efficacious rabies vaccines.

OBJECTIVE

Rabies is endemic in most parts of the world, and more effort is needed to develop affordable and effective vaccines to control or eliminate this disease. The chemokine CXCL13 recruits both Tfh and B cells, which is essential for the homing of Tfh cells and the development of B cell follicles. In this study, the effect of the overexpression of CXCL13 on the immunogenicity of the RABV was evaluated in a mouse model. We found that CXCL13 expression promoted humoral immunity by recruiting Tfh and GC B cells, facilitating the formation of GCs, and increasing the number of plasma cells. As expected, the overexpression of CXCL13 resulted in enhanced virus-neutralizing antibody (VNA) production and protection against a virulent RABV challenge. These findings provide a better understanding of the role of CXCL13 in RABV-induced immune responses, which will help in designing more efficacious rabies vaccines.

Mice infected with relapsing fever (RF) spirochaetes survive recurrent waves of high-level bacteraemia with little, if any, clinical complications or tissue injury. In the absence of B-cells, peak bacteraemia does not resolve, resulting in multi-organ complications. During peak bacteraemia, large amounts of interleukin-10 (IL-10) are produced in blood and tissues. In mice unable to clear peak bacteraemia, exogenous IL-10 greatly reduced the clinical manifestations, serum levels of CXCL13, cerebral microgliosis, and the pathogen load. In contrast, IL-10 deficiency in mice unable to clear peak bacteraemia resulted in microvascular complications with distinct severities, depending on the serotype: serotype 2 (Bt2), which causes peak bacteraemia of c. 10(8)/mL, resulted in rapid death from subarachnoid and intraparenchymal haemorrhage; in contrast, serotype 1, which causes peak bacteraemia of c. 10(7)/mL, resulted in milder multi-organ haemorrhage and thrombosis. IL-10 deficiency also resulted in multi-organ haemorrhage and thrombosis with infarction in wild-type mice despite lower peak bacteraemia. Two mechanisms for pathogen control have been identified: antibody clearance of peak bacteraemia, and antibody-independent lowering of bacteraemia via phagocytosis in the spleen. IL-10 plays opposite roles in pathogen control, depending on the severity of bacteraemia: during persistent high bacteraemia, IL-10 helps to control it by protecting innate immune cells from apoptosis; in contrast, during transient peak bacteraemia, IL-10 slows down antibody-mediated clearance. A successful outcome from RF depends on a balanced immune response to clear bacteraemia while avoiding microvascular injury, in which production of IL-10, in response to the pathogen load, plays a critical role.

Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.

Analysis of RNA expression in mixed cell populations often requires laborious and costly cell sorting. Here we describe a flow cytometric assay that combines antibody staining and in situ hybridization for multi-parametric analysis of single cells. This method, referred to as the PrimeFlow™ RNA Assay, enables simultaneous detection of protein markers and RNA targets in mixed cell populations. Both coding and non-coding RNA sequences can be measured with a limit of detection of approximately 10 copies of mRNA and 20 copies of microRNA per cell. In this study, we used mouse bone marrow-derived macrophages to demonstrate that our method allows for analysis of the activation and polarization status of cells using expression patterns of protein and RNA. We then performed analysis of four cell subsets of mouse resident peritoneal cells and showed that the two macrophage populations present in this compartment are relatively heterogeneous in terms of expression of two M2 markers: Arg1, Retnla, and a B-cell attractant chemokine Cxcl13. In addition, we profiled the expression of a panel of microRNA in the four peritoneal cell subsets, showing that the assay can be readily adapted to parallel, high-throughput screening of multiple cell populations. This new method allows for single cell analysis of multiple RNA targets without the need for cell sorting, enables direct correlation between RNA and protein expression, and promises to accelerate biomarker and drug discovery.

Background: Although immunotherapy has achieved success in treating various refractory malignancies including gastric cancers (GCs) with DNA mismatch repair deficiency, only a subset of cancer patients are responsive to immunotherapy. Therefore, the identification of useful biomarkers or interventional targets for improving cancer immunotherapy response is urgently needed.

Methods: We investigated the associations between various molecular features and immune signatures using three multi-omics GC datasets. These molecular features included genes, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), proteins, and pathways, and the immune signatures included CD8+ T cell infiltration, immune cytolytic activity (ICA), and PD-L1 expression. Moreover, we investigated the association between gene mutations and survival prognosis in a gastrointestinal (GI) cancer cohort receiving immunotherapy and two GC cohorts not receiving such a therapy.

Results: The mutations of some important oncogenes and tumor suppressor genes were appreciably associated with immune signatures in GC, including PIK3CA, MTOR, RNF213, TP53, ARID1A, PTEN, ATM, and CDH1. Moreover, a number of genes exhibited a significant expression correlation with immune signatures in GC, including CXCL9, CXCL13, CXCR6, CCL5, GUCY2C, MAP3K9, NEK3, PAK6, STK35, and WNK2. We identified several proteins whose expression had a significant positive correlation with immune signatures in GC. These proteins included caspase-7, PI3K-p85, PREX1, Lck, Bcl-2, and transglutaminase. In contrast, acetyl-CoA carboxylase (ACC) had a significant inverse expression correlation with immune signatures in GC, suggesting that inhibiting ACC could enhance antitumor immunity in GC. Furthermore, we identified numerous miRNAs and lncRNAs with a significant expression correlation with GC immunity, including hsa-miR-150, 155, 142, 342, 146, 101, 511, 29, AC022706.1, LINC01871, and AC006033.2. We also identified numerous cancer-associated pathways whose activity was associated with GC immunity, including mTOR, PI3K-AKT, MAPK, HIF-1, and VEGF signaling pathways. Interestingly, we found seven genes (ARID1A, BCOR, MTOR, CREBBP, SPEN, NOTCH4, and TET1) whose mutations were associated with better OS in GI cancer patients receiving anti-PD-1/PD-L1 immunotherapy but were not associated with OS in GC patients without immunotherapy.

Conclusions: The molecular features significantly associated with GC immunity could be useful biomarkers for stratifying GC patients responsive to immunotherapy or intervention targets for promoting antitumor immunity and immunotherapy response in GC.

Keywords: Gastric cancer (GC); biomarker; multi-omics data analysis; tumor immunity; tumor immunotherapy.

The prognosis of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains unsatisfactory and, despite major advances in genomic studies, the biological mechanisms underlying chemoresistance are still poorly understood. We conducted for the first time a large-scale differential multi-omics investigation on DLBCL patient's samples in order to identify new biomarkers that could early identify patients at risk of R/R disease and to identify new targets that could determine chemorefractoriness. We compared a well-characterized cohort of R/R versus chemosensitive DLBCL patients by combining label-free quantitative proteomics and targeted RNA sequencing performed on the same tissues samples. The cross-section of both data levels allowed extracting a sub-list of 22 transcripts/proteins pairs whose expression levels significantly differed between the two groups of patients. In particular, we identified significant targets related to tumor metabolism (Hexokinase 3), microenvironment (IDO1, CXCL13), cancer cells proliferation, migration and invasion (S100 proteins) or BCR signaling pathway (CD79B). Overall, this study revealed several extremely promising biomarker candidates related to DLBCL chemorefractoriness and highlighted some new potential therapeutic drug targets. The complete datasets have been made publically available and should constitute a valuable resource for the future research.

Innate lymphoid cells (ILCs) are responsible for mucosal tissue homeostasis and are involved in the progression and suppression of several types of cancer. However, the effects of ILCs on colorectal cancer (CRC) are poorly understood. We characterized human ILCs in normal colon and CRC tissue, investigating their role in the tumor immune microenvironment. Normal mucosa and tumor tissues were obtained from CRC patients and the cells were isolated by enzymatic digestion. NKp44+ ILC3s with high expression of tertiary lymphoid structure (TLS) formation-related genes, including LTA, LTB, and TNF, accumulated in the normal colonic mucosa and T1/T2 tumors. However, the number of NKp44+ ILC3s was significantly reduced in T3/T4 tumors compared to normal colonic mucosa and T1/T2 tumors. NKp44+ ILC3s present in T3/T4 tumors had decreased expression of TLS formation-related genes, whereas stromal cells had decreased expression of CXCL13, CCL19, and CCL21. The decreasing number of NKp44+ ILC3s during tumor progression correlated with the TLS density in tumors. Thus, our results indicate that NKp44+ ILC3s infiltrate CRC tissue but the number of cells decreases in T3/T4 tumors, with associated decreases in TLS induction.

Lysophosphatidic acid (LPA) is a bioactive phospholipid playing an important role in various inflammatory diseases by inducing expression and secretion of many inflammatory cytokines/chemokines. Here we report in a murine air pouch model of inflammation that LPA induced CXCL13 secretion in a time-dependent manner and with exacerbation of the response when LPA was administered after a pretreatment with TNF-α, a key inflammatory cytokine. LPA mediates recruitment of leukocytes, including that of CD3(+) cells into unprimed and TNF-α-primed air pouches. CXCL13 neutralization using a blocking antibody injected into air pouches prior to administration of LPA into TNF-α-primed air pouches decreased CD3(+) cell influx. Our data highlight that LPA-mediated CXCL13 secretion plays a role in T cell recruitment and participates in regulation of the inflammatory response.

Interleukin 17 (IL-17) family members are key players in regulating the immune response in mammals. Here, we identified the IL-17C and IL-17D homologs from large yellow croaker (Larimichthys crocea), named LcIL-17C and LcIL-17D, respectively. The deduced LcIL-17C and LcIL-17D proteins possessed the typical IL-17 domain and shared a conserved arrangement of eight cysteine residues. Both LcIL-17C and LcIL-17Dc genes were constitutively expressed in all tissues examined, although at different levels. After challenge with Aeromonas hydrophila, the expression of LcIL-17C and LcIL-17D was significantly increased in gills, head kidney, and spleen. In the peripheral blood leukocytes (PBLs), the recombinant LcIL-17C (rLcIL-17C) could strongly promote the expression of chemokines (CXCL8, CXCL12, and CXCL13), proinflammatory factors (TNF-α, IL-1β, IL-6, and IFNg), and antibacterial peptide hepcidin, whereas rLcIL-17D induced a weaker expression of these chemokines. Consistently, the culture supernatants from the PBLs treated by rLcIL-17C showed a stronger ability to induce the migration of PBLs than those treated by rLcIL-17D. Furthermore, both rLcIL-17C and rLcIL-17D could activate the NF-κB signalling in the epithelioma papulosum cyprini (EPC) cells. Taken together, these results indicated that LcIL-17C and LcIL-17D, although differing in their ability to mediate chemotaxis for PBLs, may promote the inflammatory response and host defence via activating NF-κB signalling. To our knowledge, this is the first report on functional identification of a IL-17C in teleost.

CXCL13 is a B lymphocyte chemoattractant and activates CXCR5 receptor in the immune system. Here we investigated whether CXCL13/CXCR5 mediates inflammatory pain in dorsal root ganglia (DRG) and the underlying mechanisms. Peripheral injection of complete Freund's Adjuvant (CFA) increased the expression of CXCL13 and CXCR5 in DRG neurons. In Cxcr5-/- mice, CFA-induced pain hypersensitivity were attenuated. Whole-cell patch-clamp recording showed that the excitability of dissociated DRG neurons was increased after CFA injection or CXCL13 incubation from wild-type (WT) mice, but not from Cxcr5-/- mice. Additionally, sodium channel Nav1.8 was co-expressed with CXCR5 in dissociated DRG neurons, and the increased neuronal excitability induced by CFA or CXCL13 was reduced by Nav1.8 blocker. Intrathecal injection of Nav1.8 blocker also attenuated intrathecal injection of CXCL13-induced pain hypersensitivity. Furthermore, CXCL13 increased Nav1.8 current density in DRG neurons, which was inhibited by p38 MAP kinase inhibitor. CFA and CXCL13 increased p38 phosphorylation in the DRG of WT mice but not Cxcr5-/- mice. Finally, intrathecal p38 inhibitor alleviated CXCL13-induced pain hypersensitivity. Taken together, these results demonstrated that CXCL13, upregulated by peripheral inflammation, acts on CXCR5 on DRG neurons and activates p38, which increases Nav1.8 current density and further contributes to the maintenance of inflammatory pain.

Nonalcoholic fatty liver disease (NAFLD) is a common disorder hallmarked by excessive lipid deposits. Based on our recent research on lipid droplet (LD) formation in hepatocytes, we investigated LD-associated gene regulations in NAFLD of different grades, that is, steatosis vs steatohepatitis by comparing liver biopsies from healthy controls (N = 13) and NAFLD patients (N = 102). On average, more than 700 differentially expressed genes (DEGs) were identified of which 146 are mechanistically linked to LD formation. We identified 51 LD-associated DEGs frequently regulated in patient samples (range ≥5 to ≤102) with the liver-receptor homolog-1(NR5A2), that is, a key regulator of cholesterol metabolism being commonly repressed among 100 patients examined. With bland steatosis, notable regulations involved hypoxia-inducible lipid droplet-associated-protein and diacylglycerol-O-acyltransferase-2 renowned for their role in LD-growth. Conversely, nonalcoholic steatohepatitis-associated DEGs coded for epidermal growth factor receptor and TLR4 signaling with decreased expression of the GTPase Rab5 and the lipid phosphohydrolase PPAP2B thus highlighting adaptive responses to inflammation, LDL-mediated endocytosis and lipogenesis, respectively. Studies with steatotic primary human hepatocyte cultures demonstrated induction of LD-associated PLIN2, CIDEC, DNAAF1, whereas repressed expression of CPT1A, ANGPTL4, and PKLR informed on burdened mitochondrial metabolism. Equally, repressed expression of the B-lymphocyte chemoattractant CXCL13 and STAT4 as well as induced FGF21 evidenced amelioration of steatosis-related inflammation. In-vitro/in-vivo patient sample comparisons confirmed C-reactive protein, SOCS3, NR5A2, and SOD2 as commonly regulated. Lastly, STRING network analysis highlighted potential "druggable" targets with PLIN2, CIDEC, and hypoxia-inducible lipid droplet-associated-protein being confirmed by immunofluorescence microscopy. In conclusion, steatosis and steatohepatitis specific gene regulations informed on the pathogenesis of NAFLD to broaden the perspective of targeted therapies.

Background: Ulcerative colitis (UC) is an inflammatory bowel disease that is difficult to diagnose and treat. To date, the degree of inflammation in patients with UC has mainly been determined by measuring the levels of nonspecific indicators, such as C-reactive protein and the erythrocyte sedimentation rate, but these indicators have an unsatisfactory specificity. In this study, we performed bioinformatics analysis using data from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) databases and verified the selected core genes in a mouse model of dextran sulfate sodium (DSS)-induced colitis.

Aim: To identify UC-related differentially expressed genes (DEGs) using a bioinformatics analysis and verify them in vivo and to identify novel biomarkers and the underlying mechanisms of UC.

Methods: Two microarray datasets from the NCBI-GEO database were used, and DEGs between patients with UC and healthy controls were analyzed using GEO2R and Venn diagrams. We annotated these genes based on their functions and signaling pathways, and then protein-protein interactions (PPIs) were identified using the Search Tool for the Retrieval of Interacting Genes. The data were further analyzed with Cytoscape software and the Molecular Complex Detection (MCODE) app. The core genes were selected and a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed. Finally, colitis model mice were established by administering DSS, and the top three core genes were verified in colitis mice using real-time polymerase chain reaction (PCR).

Results: One hundred and seventy-seven DEGs, 118 upregulated and 59 downregulated, were initially identified from the GEO2R analysis and predominantly participated in inflammation-related pathways. Seven clusters with close interactions in UC formed: Seventeen core genes were upregulated [C-X-C motif chemokine ligand 13 (CXCL13), C-X-C motif chemokine receptor 2 (CXCR2), CXCL9, CXCL5, C-C motif chemokine ligand 18, interleukin 1 beta, matrix metallopeptidase 9, CXCL3, formyl peptide receptor 1, complement component 3, CXCL8, CXCL1, CXCL10, CXCL2, CXCL6, CXCL11 and hydroxycarboxylic acid receptor 3] and one was downregulated [neuropeptide Y receptor Y1 (NYP1R)] in the top cluster according to the PPI and MCODE analyses. These genes were substantially enriched in the cytokine-cytokine receptor interaction and chemokine signaling pathways. The top three core genes (CXCL13, NYP1R, and CXCR2) were selected and verified in a mouse model of colitis using real-time PCR Increased expression was observed compared with the control mice, but only CXCR2 expression was significantly different.

Conclusion: Core DEGs identified in UC are related to inflammation and immunity inflammation, indicating that these reactions are core features of the pathogenesis of UC. CXCR2 may reflect the degree of inflammation in patients with UC.

Keywords: Bioinformatics analysis; C-X-C motif chemokine ligand 13; C-X-C motif chemokine receptor 2; Colitis model mice; Neuropeptide Y receptor Y1; Ulcerative colitis.

Background: Thymocyte selection-associated high mobility group box (TOX) plays a crucial role on the development of innate immunity and tumor microenvironment. This study aims to explore the prognostic potential of TOX and comprehensively analyze the correlations between TOX, immune infiltration, and T cells function in diverse cancers particularly lung adenocarcinoma (LUAD).

Methods: TIMER was used to analyze TOX expression in different cancers. Potential prognostic value of TOX was evaluated by the PrognoScan, Kaplan-Meier Plotter, and GEPIA2. The relationships between TOX, immune infiltration, and related gene marker sets were analyzed by TIMER and GEPIA2. Single-cell RNA-seq for T cells in LUAD was analyzed to further investigate the correlations between TOX expression and different T cells populations.

Results: TOX downregulates in most of the cancer types and correlates with poor prognosis in LUAD. TOX shows significant impacts on survival of LUAD with early stage, ever-smoking, or low-TMB status. Increased TOX expression positively correlates with high immune infiltration levels in most of the immune cells and functional T cells including exhausted T cells. Moreover, multiple key genes of exhausted T cells comprising PD-1, TIM-3, TIGHT, and CXCL13 have remarkable interaction with TOX. Specifically, TOX is observed with high enrichment in exhausted CD4⁺ and CD8⁺ T cells populations in single-cell RNA-seq analysis for LUAD.

Conclusion: TOX is a prognosis-related biomarker for multiple cancer types especially LUAD. Increased TOX expression significantly increase immune infiltration levels in most of the immune cells comprising CD8⁺ T cells, CD4⁺ T cells, mast cells, and functional T cells. Moreover, we verified that TOX highly correlates with exhausted T cells and is probable a critical regulator promoted T cells exhaustion in LUAD. Detection of TOX expression could help to predict prognosis and regulating TOX expression in exhausted T cells may offer a novel strategy in maximizing immunotherapy efficacy for LUAD.

Keywords: T cells exhaustion; TOX; immune infiltration; immunotherapy; lung adenocarcinoma; prognosis.

Neuropathic pain is an unneglectable pain condition with limited treatment options owing to its enigmatic underlying mechanisms. Long noncoding RNA small nucleolar RNA host gene 5 (SNHG5) is involved in the progression of a spectrum of human cancers. However, its role in neuropathic pain remains undiscovered. In the present study, we established a mouse spinal nerve ligation (SNL) model, and a significant upregulation of SNHG5 was observed. Then we knocked down SNHG5 level in mouse L5 dorsal root ganglion (DRG) by delivering specific short hairpin RNA against SNHG5 with adenovirus vehicle. Mouse paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) in response to mechanical stimuli was increased after SNHG5 knockdown, accompanied with decreased protein levels of glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule 1 (IBA-1). Besides, SNHG5 directly modulated the expression of miR-154-5p, which was downregulated in SNL mice. MiR-154-5p inhibition abolished the effect of SNHG5 knockdown on mouse behavioral tests and GFAP and IBA-1 levels. In addition, we validated that C-X-C motif chemokine 13 (CXCL13) was a novel downstream target of miR-154-5p, and CXCL13 level was positively related to that of SNHG5 in SNL mice. In conclusion, our study demonstrated that SNHG5 knockdown alleviated neuropathic pain and inhibited the activation of astrocytes and microglia by targeting the miR-154-5p/CXCL13 axis, which might be a novel therapeutic target for neuropathic treatment clinically.

The mechanisms that underlie the pathogenesis of epilepsy are still unclear. Recent studies have indicated that inflammatory processes occurring in the brain are involved in a common and crucial mechanism in epileptogenesis. C-X-C motif chemokine ligand 13 (CXCL13) and its only receptor, C-X-C motif chemokine receptor 5 (CXCR5), are highly expressed in the central nervous system (CNS) and participate in inflammatory responses. The present study aimed to assess the expression of CXCL13 and CXCR5 in the brain tissues of both patients with intractable epilepsy (IE) and a rat model (lithium-pilocarpine) of temporal lobe epilepsy (TLE) to identify possible roles of the CXCL13-CXCR5 signaling pathway in epileptogenesis. Real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical, double-labeled immunofluorescence and Western blot analyses were performed in this study. CXCL13 and CXCR5 mRNA expression and protein levels were found to be significantly up-regulated in the TLE patients and TLE rats. Further, CXCL13 and CXCR5 protein levels were altered during the different epileptic phases after onset of status epilepticus (SE) in the pilocarpine model rats, including the acute phase (6, 24, and 72 h), latent phase (7 and 14 days) and chronic phase (30 and 60 days groups). Moreover, double-labeled immunofluorescence analysis revealed that CXCL13 was mainly expressed in the cytomembranes and cytoplasm of neurons and astrocytes, while CXCR5 was mainly expressed in the cytomembranes and cytoplasm of neurons. Thus, the CXCL13-CXCR5 signaling pathway may play a possible pathogenic role in IE. CXCL13 and CXCR5 may represent potential biomarkers of brain inflammation in epileptic patients.

Introduction: Although the microbiome is altered in various esophageal diseases, there is no direct evidence for a link between the oral or esophageal microbiome and underlying esophageal tissue. Here, we aimed to address these gaps through use of an antimicrobial mouth rinse to modify the esophageal microbiome and tissue gene expression.

Methods: In this randomized controlled trial, patients scheduled to undergo endoscopy for clinical indications used chlorhexidine mouth rinse or no treatment for 2 weeks before endoscopy. Oral swabs and saliva were collected at baseline and at follow-up, and the esophagus was sampled on the day of endoscopy. The microbiome was analyzed by 16S rRNA gene sequencing, and esophageal tissue gene expression was ascertained by RNA-Seq.

Results: Twenty subjects were enrolled and included in the analyses. Within individuals, the oral and esophageal microbiome composition was significantly correlated. Chlorhexidine treatment associated with significant alterations to the relative abundance of several esophageal bacterial taxa, and to expression of genes in the esophagus including reductions in periostin, claudin-18, chemokines CXCL1 and CXCL13, and several members of the tumor necrosis factor receptor superfamily. A taxon in genus Haemophilus in the esophagus also associated with significant changes in tissue gene expression.

Discussion: The oral and esophageal microbiomes are closely related within individuals, and esophageal microbiome alterations correlate with tissue gene expression changes. The esophageal microbiome may act as an important cofactor that influences pathogenesis and outcomes of diseases such as eosinophilic esophagitis, gastroesophageal reflux, and Barrett's esophagus.

Lung cancer is one of the leading causes of death worldwide. Cell death pathways such as autophagy, apoptosis, and necrosis can provide useful clinical and immunological insights that can assist in the design of personalized therapeutics. In this study, variations in the expression of genes involved in cell death pathways and resulting infiltration of immune cells were explored in lung adenocarcinoma (The Cancer Genome Atlas: TCGA, lung adenocarcinoma (LUAD), 510 patients). Firstly, genes involved in autophagy (n = 34 genes), apoptosis (n = 66 genes), and necrosis (n = 32 genes) were analyzed to assess the prognostic significance in lung cancer. The significant genes were used to develop the cell death index (CDI) of 21 genes which clustered patients based on high risk (high CDI) and low risk (low CDI). The survival analysis using the Kaplan-Meier curve differentiated patients based on overall survival (40.4 months vs. 76.2 months), progression-free survival (26.2 months vs. 48.6 months), and disease-free survival (62.2 months vs. 158.2 months) (Log-rank test, p < 0.01). Cox proportional hazard model significantly associated patients in high CDI group with a higher risk of mortality (Hazard Ratio: H.R 1.75, 95% CI: 1.28-2.45, p < 0.001). Differential gene expression analysis using principal component analysis (PCA) identified genes with the highest fold change forming distinct clusters. To analyze the immune parameters in two risk groups, cytokines expression (n = 265 genes) analysis revealed the highest association of IL-15RA and IL 15 (> 1.5-fold, p < 0.01) with the high-risk group. The microenvironment cell-population (MCP)-counter algorithm identified the higher infiltration of CD8+ T cells, macrophages, and lower infiltration of neutrophils with the high-risk group. Interestingly, this group also showed a higher expression of immune checkpoint molecules CD-274 (PD-L1), CTLA-4, and T cell exhaustion genes (HAVCR2, TIGIT, LAG3, PDCD1, CXCL13, and LYN) (p < 0.01). Furthermore, functional enrichment analysis identified significant perturbations in immune pathways in the higher risk group. This study highlights the presence of an immunocompromised microenvironment indicated by the higher infiltration of cytotoxic T cells along with the presence of checkpoint molecules and T cell exhaustion genes. These patients at higher risk might be more suitable to benefit from PD-L1 blockade or other checkpoint blockade immunotherapies.

Keywords: LUAD; apoptosis; cell death; gene expression; immunotherapy; lung cancer; necrosis; prognostic genes; tumor microenvironment.

To evaluate the expression and role of serum CXC chemokine ligand 13 (CXCL13) and cytokine IL-31 in hepatocellular carcinoma (HCC) patients.

A case-control study including preoperative serum samples of 78 patients with HCC, 78 patients with chronic hepatitis B (CHB) and 36 healthy controls (HCs) was conducted. The levels of serum CXCL13 and IL-31 were measured by enzyme-linked immunosorbent assays. The correlation of serum cytokines and clinical characteristics, laboratory parameters were analyzed retrospectively.

Serum CXCL13, rather than IL-31, was significantly higher in patients with HCC compared with CHB patients or healthy controls. Moreover, there were no statistical differences between CHB patients and healthy controls. Serum CXCL13 was further increased in patients with large tumor size, metastasis and advanced HCC (TNM III-IV Stage). On correlation analysis, the levels of serum CXCL13 were related to HB, ALB, CHE, INR and Child-Pugh scores. The area under the ROC curve values for combination of CXCL13 and AFP was 0.938, whose sensitivity and specificity was 82.8% and 100%, respectively.

This study indicated that CXCL13 rather than IL-31 may have clinical values of diagnosis and prognosis in HCC.

IL-4 plays an important role in the pathogenesis of atopic dermatitis (AD), a common chronic inflammatory skin disease. We have generated IL-4 transgenic (Tg) mice by over-expressing IL-4 in the epidermis. These mice spontaneously develop chronic pruritic inflammatory skin lesions, which meet the clinical and histological diagnostic criteria for human AD. Systemic survey of immune-related genes in this mouse model, however, has not been performed. In this study, we utilize PCR array technique to examine hundreds of inflammation-related genes in the IL-4 Tg mice before and after the onset of skin lesions as well as in their wild type (WT) littermates. Only those genes with at least 2-fold up-regulation or down-regulation and with a P-value of less than 0.05 in comparison to WT controls were identified and analyzed. In the skin lesions, many chemokines, pro-inflammatory cytokines, and other AD-related factors are dysregulated compared to the wild type mice. Particularly, CXCL5, IL-1β, IL-24, IL-6, oncostatin M, PTGS2, FPR1 and REG3γ are up-regulated several hundred-fold. In the pre-lesional group that shows no obvious skin abnormality on clinical observation, 30 dysregulated genes are nevertheless identified though the fold changes are much less than that of the lesional group, including CCL6, CCL8, CCL11, CCL17, CXCL13, CXCL14, CXCR3 and IL-12Rβ2. Finally using ELISA, we demonstrate that 4 most dramatically up-regulated factors in the skin are also elevated in the peripheral blood of the IL-4 Tg mice. Taken together, our data have identified hundreds of dysregulated factors in the IL-4 Tg mice before and after the onset of skin lesions. Future detailed examination of these factors will shed light on our understanding of the development and progression of AD and help to discover important biomarkers for clinical AD diagnosis and treatment.

Central nervous system (CNS) relapse occurs in around 5% of diffuse large B-cell lymphoma (DLBCL) cases. No biomarkers to identify high-risk patients have been discovered. We evaluated the expression of lymphocyte-guiding chemokine receptors in systemic and CNS lymphomas. Immunohistochemical staining for CXCR4, CXCR5, CCR7, CXCL12, and CXCL13 was performed on 89 tissue samples, including cases of primary central nervous system lymphoma (PCNSL), secondary CNS lymphoma (sCNSL), and systemic DLBCL. Also, 10 reactive lymph node samples were included. Immunoelectron microscopy was performed on two PCNSLs, one sCNSL, one systemic DLBCL, and one reactive lymph node samples, and staining was performed for CXCR4, CXCR5, CXCL12, and CXCL13. Chi-square test was used to determine correlations between clinical parameters, diagnostic groups, and chemokine receptor expression. Strong nuclear CXCR4 positivity correlated with systemic DLBCL, whereas strong cytoplasmic CXCR5 positivity correlated with CNS involvement (P = 0.003 and P = 0.039). Immunoelectron microscopy revealed a nuclear CXCR4 staining in reactive lymph node, compared with cytoplasmic and membranous localization seen in CNS lymphomas. We found that CNS lymphoma presented a chemokine receptor profile different from systemic disease. Our findings give new information on the CNS tropism of DLBCL and, if confirmed, may contribute to more effective targeting of CNS prophylaxis among patients with DLBCL.

BACKGROUND

Henoch-Schönlein purpura (HSP) is an immune complex-mediated disease predominantly characterized by the deposition of circulating immune complexes containing immunoglobulin A (IgA) on the walls of small vessels. Although the pathogenesis of HSP is not yet fully understood, some researchers proposed that B-cell activation might play a critical role in the development of this disease.

OBJECTIVE

To investigate the serum levels of visfatin (pre-B-cell colony-enhancing factor), B-cell-activating factor (BAFF), and CXCL13, and to analyze their association with disease severity.

METHODS

The serum levels of visfatin, BAFF, and CXCL13 were measured by using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) in 43 patients with HSP and 45 controls. The serum levels of IgA anticardiolipin antibodies (ACA) were detected by using a double-antigen sandwich ELISA.

RESULTS

Levels of visfatin but not BAFF and CXCL13 were significantly elevated in the sera of patients with HSP in the acute stage, and restored to normal levels in the convalescent stage. Furthermore, serum levels of visfatin were significantly higher in patients with HSP having renal involvement than in those without renal involvement. Serum levels of visfatin were correlated with the severity of HSP and serum concentration of ACA-IgA.

CONCLUSIONS

We show for the first time that the serum levels of visfatin are abnormally elevated in patients with HSP. Visfatin may be associated with the pathogenesis of HSP.

In nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), little is known about the presence of intranodular clusters of cytologically activated lymphoid cells producing a moth-eaten pattern histologically. This pilot study of 32 NLPHL cases from Finland ascertained (1) the frequency of the intranodular clusters of activated lymphoid cells, (2) the immunophenotype of the activated cells, (3) the size and immunophenotype of the rosetting cells, and (4) the clinical significance of the activated cells. Histologically, intranodular clusters of activated cells produced a moth-eaten pattern in 100% (32 cases; subtle in 62.5%, overt in 37.5%). In immunostains, activated cells in subtle clusters (20 cases) were very difficult to identify. Twelve cases had overt clusters of activated cells, which were positive with CD3, CD4, PD1, CXCL13 (T follicular helper [T(FH)] phenotype), but rarely with Ki-67 and BCL2. Most activated rosetting cells had the same immunophenotype as the nonrosetting cells, except for CXCL13. Clinical presentation for all 32 Finnish patients was distinctive: 97% men, 97% with peripheral lymphadenopathy and 35.5% with stage III/IV disease. Only 22% relapsed; 97% were in remission. There was no significant clinical difference between cases with overt and subtle clusters. Intranodular activated TFH cells in NLPHL appeared to be nonproliferating and not long-living, and they were not associated with any adverse clinical outcome. Although most activated cells were TFH cells, it seemed that they were unable to increase the number of malignant cells. The pathogenetic role of the intranodular activated TFH and the small T cells in NLPHL needs further investigation.