C-C Motif) Ligand 2 Research Articles

Suppression of androgen receptor signaling induces prostate cancer migration via activation of the CCL20-CCR6 axis.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR-controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C-C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4-2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20-treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C-C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti-CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti-CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration-resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6.

27: PRE-CARDIOPULMONARY BYPASS BIOMARKERS PREDICT POOR OUTCOMES IN NEONATAL CONGENITAL HEART DISEASE

Introduction: Neonates requiring cardiopulmonary bypass (CPB) for congenital heart disease have higher rates of postoperative mortality and morbidity than older children. However, identified risk factors for poor outcome to date have been limited to non-modifiable demographic or surgical factors. We aimed to identify novel biomarkers measured preoperatively that correlate with poor outcomes. Methods: Plasma samples were collected immediately prior to CPB in 65 consented subjects. Twenty-eight soluble analytes were measured via individual or multiplexed ELISA and tested for association with the validated “ICU-30” composite poor outcome, defined as (1) mortality within 30 days of CPB, (2) ICU stay >30 days, or (3) ICU readmission within 30 days. Differences in biomarker concentrations between subjects with and without ICU-30 were assessed using a Wilcoxon rank sum test. Associations of standardized biomarkers and ICU-30 was tested with logistic regression, adjusting for surgical complexity. Results: Samples for 63 patients were included for analysis (2 subjects did not undergo CPB after blood collection); 18 subjects (28%) had the ICU-30 outcome. Patients with ICU-30 were more likely to have a chromosomal abnormality, with no difference in gestational age or birth weight. There was no difference between age at CPB surgery and CPB time during operation. Seven biomarkers differed between those with and without ICU-30: angiopoietin 2, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, growth differentiation factor-15, C-C motif ligand 2 (CCL2), and neutrophil gelatinase-associated lipocalin (NGAL) were higher in patients with ICU-30, and ADAMTS13 (A disintegrin and metalloproteinase with thrombospondin motifs 13) was lower in those with ICU-30. When controlling for STAT category, TNF-alpha (aOR 5.4, 95% CI 1.6-19.0), IL-6 (aOR 3.0, 95% CI 1.3-6.8), CCL2 (aOR 2.6, 95% CI 1.3-5.2), and NGAL (aOR 2.6, 95% CI 1.3-5.1) were associated with ICU-30. Conclusions: Four novel preoperative plasma biomarkers implicating innate immunity and renal function were associated with worse outcomes in neonates requiring CPB independent of STAT operation. Biomarkers have the potential to identify preoperative risk factors and mechanistic pathways for prognostication and targeted therapeutic interventions.

CircCYP24A1 facilitates esophageal squamous cell carcinoma progression through binding PKM2 to regulate NF-κB-induced CCL5 secretion

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common gastrointestinal malignant tumor, while the molecular mechanisms have not been fully elucidated. Multiple circular RNAs have been reported to involve in the onset and progression of malignant tumors through various molecular mechanisms. However, the clinical significance and functional mechanism of most circRNAs involved in the progression of ESCC remains obscure.MethodsRNA-Seq was used to explore potential circRNAs in participated in 5 pairs of ESCC and their corresponding normal esophageal tissues. The up-regulated circCYP24A1 was selected. Fluorescence in situ hybridization was cunducted to verificated the expression and intracellular localization of circCYP24A1 by using the tissue microarray. The Kaplan–Meier method and Cox proportional hazards model was used to examine the potential prognostic value of circCYP24A1 on overall survival of ESCC patients. The biological function were confirmed by gain- and loss-of-function approaches in vivo. mRNA expression profile microarray was proformed to investigate the downstream signaling pathways involved in circCYP24A1. RNA pull-down assay and mass spectrometry were performed to identify the proteins associated with circCYP24A1. Rescue experiments were carried out to identified hypothetical regulatory role of circCYP24A1 on ESCC progression in vivo and in virto.ResultsIn this study, we identified circCYP24A1 in ESCC tissues by RNA sequencing, which is up-regulated in 114 cases of ESCC tissues and acts as a novel prognosis-related factor. Moreover, circCYP24A1 promoted the ability of proliferation, migration, invasion and clone formation in vitro, as well as tumor growth in vivo. Mechanistically, chemokine (C-Cmotif) ligand 5 (CCL5) is functional downstream mediator for circCYP24A1, which is screened by mRNA microarray. Moreover, circCYP24A1 physically interacts with M2 isoform of pyruvate kinase (PKM2). Rescue experiments showed that PKM2 knockdown partly reverses the promotional effects of circCYP24A1. It was revealed that circCYP24A1 increases secretion of CCL5 through the mechanism mainly by interacting with PKM2, an activator of NF-κB pathway, and thereby accelerate malignant progression of ESCC.ConclusionsUp-regulated circCYP24A1 could activate NF-κB pathway by binding PKM2, which promotes the secretion of CCL5 and accelerate malignant progression of ESCC. Our fndings recommended a novel function for circCYP24A1 as a potential effective biomarker for judging prognosis and a therapeutic target in ESCC.

Oxaliplatin-Resistant Hepatocellular Carcinoma Drives Immune Evasion Through PD-L1 Up-Regulation and PMN-Singular Recruitment

Previously, we showed the inhibitor of differentiation or DNA binding 1 (ID1)/Myc signaling is highly expressed in oxaliplatin-resistant hepatocellular carcinoma (HCC). This study sought to investigate the role of ID1/Myc signaling on immune evasion in oxaliplatin-resistant HCC. The oxaliplatin (OXA)-resistant HCC cell lines (Hepa 1-6-OXA, 97H-OXA, and 3B-OXA) were established and their oxaliplatin tolerance was confirmed invitro and invivo. The relationship between ID1/Myc and programmed death-ligand 1 (PD-L1) up-regulation and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) accumulation was explored. The underlying mechanism in which ID1/Myc signaling regulated PD-L1 expression and PMN-MDSC accumulation was investigated invitro and vivo. Increased ID1/Myc expression was identified in oxaliplatin-resistant HCC and correlated with PD-L1 up-regulation and PMN-MDSC accumulation. The knockdown of Myc sensitized oxaliplatin-resistant HCC cells to oxaliplatin and resulted in a decrease of PMN-MDSCs and an increase of interferon-γ+ CD8+ T cells in a tumor microenvironment. Polymerase chain reaction array, enzyme-linked immunosorbent assay, and MDSC Transwell migration assay indicated that oxaliplatin-resistant HCC cells recruited PMN-MDSCs through chemokine (C-C motif) ligand 5 (CCL5). The dual luciferase reporter assay and chromatin immunoprecipitation assay indicated that Myc could directly increase the transcriptions of PD-L1 and CCL5. Furthermore, anti-PD-L1 antibody combined with CCL5 blockade showed significant antitumor effects in oxaliplatin-resistant HCC. ID1/Myc signaling drives immune evasion in oxaliplatin-resistant HCC via PD-L1 up-regulation and PMN-MDSC recruitment. Blocking the ID1/Myc-induced immune tolerance represents a promising treatment target to conquer chemoresistance in HCC.

Extended access to fentanyl vapor self-administration leads to addiction-like behaviors in mice: Blood chemokine/cytokine levels as potential biomarkers

Rodent models are useful for understanding the mechanisms that underlie opioid addiction, but most preclinical studies have focused on rewarding and consummatory aspects of opioids without components of dependence-induced escalation of drug taking or seeking. We characterized several opioid-related behaviors in mice using a model of vaporized fentanyl self-administration. Male and female C57BL/6J mice were assigned to short-access (ShA; 1 h, nondependent) or long-access (LgA; 6 h, dependent) fentanyl vapor self-administration and subsequently tested in a battery of behavioral tests, followed by blood collection during withdrawal. Compared with mice in the ShA group, mice in the LgA group escalated their fentanyl intake, were more motivated to work to obtain the drug, exhibited greater hyperalgesia, and exhibited greater signs of naloxone-precipitated withdrawal. Principal component analysis indicated the emergence of two independent behavioral constructs: “intake/motivation” and “hyperalgesia/punished seeking.” In mice in the LgA condition only, “hyperalgesia/punished seeking” was associated with plasma levels of proinflammatory interleukin-17 (IL-17), chemokine (C-C motif) ligand 4 (CCL-4), and tumor necrosis factor α (TNF-α). Overall, the results suggest that extended access to opioids leads to addiction-like behavior, and some constructs that are associated with addiction-like behavior may be associated with levels of the proinflammatory cytokines/chemokines IL-17, TNF-α, and CCL-4 in blood.

Nanotherapy: New Approach for Impeding Hepatic Cancer Microenvironment via Targeting Multiple Molecular Pathways.

Hepatocellular carcinoma (HCC) microenvironment has been recognized as a key contributor for cancer progression, metastasis, and drug resistance. The crosstalk between tumor cells, the vascular endothelial growth factor (VEGF), and the chemokine (C-C motif) ligand 2 (CCL2) signaling networks mediates immunoinhibitory impact and facilitates tumor angiogenesis. The current investigation aimed at exploring the potent anti-cancer activity of the newly designed nano-based anti-cancer therapy comprising anti-VEGF drug, avastin (AV), and CCR2 antagonist (CR) to counteract HCC and tracking its mode of action in vivo. The prepared AV, CR, and AVCR nanoprototypes were characterized by nanoscale characterization techniques in our previous work. Here, they are applied for unearthing their anti-cancer properties / mechanisms in hepatic cancer-induced rats via analyzing protein levels and genetic expression of the elements incorporated in the angiogenesis, apoptosis, and metastasis signalling pathways. The present results revealed a significant down-regulation in the angiogenesis, survival and metastasis indices along with up-regulation in the pro-apoptotic mediators upon treatment of hepatic cancer-bearing rats with the novel synthesized nanomaterials when compared with the untreated counterparts. We showed across HCC model that anti-VEGF in combination with CCR2 antagonism therapy leads to sensitization and enhanced tumor response over anti-VEGF or CCR2 antagonism monotherapy, particularly in its nanoscale formulation. The present approach provides new mechanistic insights into the powerful anti-hepatic cancer advantage of the novel nanoprototypes which is correlated with modulating critical signal transduction pathways implicated in tumor microenviroment such as angiogenesis, apoptosis and metastasis. This research work presents a substantial foundation for future studies focused on prohibiting cancer progression and recovery by targeting tumor microenviroment.

Chinese Herbal Medicine 'Diwu' Suppresses Myeloma Bone Disease Via ERK/MAPK Signaling Pathway By Targeting CCL3

Background： Multiple myeloma (MM) associated bone disease (MBD) is largely dependent on the bone marrow microenvironment (BME). The cross-talk between MM cells, bone marrow mesenchymal cells and myeloid progenitors affects the osteoclastogenesis. Traditional Chinese medicine 'Diwu' is effective for patients with arthritis and inflammation, also occasionally uses as one ingredient treating MM. But the effects of 'Diwu' in MBD are largely unknown. The aim of this study is to investigate the role and underlying mechanism of 'Diwu' in regulating BME and osteoclast differentiation. Methods： Myeloma cell lines RPMI-8226, NCI-H929, U266 and MM1 were treated with 'Diwu' extract. Killing effects was evaluated by the half inhibitory dose (IC50) value and proportion of apoptotic MM cells. RNA-seq was applied in 'Diwu' treated myeloma cells to identify MBD associated signaling pathways. RPMI-8226 was co-cultured with mesenchymal stromal cell (MSC) line HS5. Human monocyte cell line THP-1 was treated with CCL3 and M-CSF to form osteoclasts determined by tartrate-resistant acid phosphatase (TRACP) staining. Primary human MSCs were cultured into osteogenic or adipogenic differentiation, determined by alizarin red staining and oil red staining, respectively. RNAi technology was applied to knock down CCL3. The expression of osteoclast-specific genes TRACP and MAPK pathway-related genes was tested using quantitative real-time PCR. Phosphorylation and expression of MEK1, ERK, FOS were measured by Western Blot. The myeloma-bearing NOD/SCID mice were treated with intragastric 'Diwu'. Serum C-terminal telopeptide of type 1 collagen (CTXI) and propeptide of type I procollagen (PINP) was tested by ELISA. The tibia of mice was fixed and stained to analyze the differentiation and maturation of osteoblasts and osteoclasts. Micro-CT was used for bone destruction measurement. Results： 'Diwu' inhibited MM cells proliferation (IC50, 29.22-364.7μg/ml) in a dose-dependent manner and promoted apoptosis by upregulating protein expression of CASP3, BCL2. 'Diwu' decreased the secretion of chemokine (C-C motif) ligand 3 (CCL3) and receptor activator of nuclear factor-kappa B ligand (RANKL) in myeloma-MSC co-cultured system by diminishing the direct attachment and interaction of these two cells. The mRNA and protein levels of osteoclast-specific genes such as TRACP, and the phosphorylation and expression of MEK1, ERK, FOS in osteoclast were all reduced after 'Diwu' treatment. We observed improved osteogenic activation rather than lipid formation in hMSC differentiation. The level of TRACP isoform 5b (TRACP-5b) was significantly downregulated in mouse serum after 'Diwu' treatment for 14 days. Conclusions: Altogether, our results demonstrated that effective extract of Chinese herbal medicine 'Diwu' could simultaneously inhibit myeloma cells and suppress osteoclast formation, indicating an important role of 'Diwu' in regulating BM microenvironment. It is possible to translate 'Diwu' into clinical application as an oral agent for myeloma bone disease. Key words： 'Diwu'; Myeloma Bone Disease; Bone Marrow Microenvironment; CCL3; ERK/MAPK

Safety and Feasibility of Anti-CD19 CAR-T Expressing IL-7 and CCL19 in Patients with Relapsed or Refractory Large B-Cell Lymphoma

Chimeric antigen receptor (CAR) T cell therapy provides a potential curative option for patients with relapsed or refractory large B-cell lymphoma (R/R LBCL). However, there are major limitations of this therapy which result in treatment resistance in B-cell malignancies, including inadequate CAR-T persistence, poor trafficking and tumor infiltration, the immunosuppressive microenvironment and frequent tumor antigen escape. Recently, several armored CAR T-cells are being developed for potential improved efficacy relative to conventional CAR T cells. Here, we engineered a CD19-spectific CAR-T cell capable of constitutive secretion of interleukin (IL)-7 and chemokine (C-C motif) ligand (CCL)-19 (7 × 19 CAR-T) and performed a multicenter phase 1b clinical trial of 7 × 19 CAR-T cell therapy in patients with R/R LBCL (NCT03929107 and NCT04833504). The 7 × 19 CAR-T cells showed that a favorable safety profile, with grade 3 cytokine release syndrome occurred in 5 (12.8%) patients and grade 3 or higher neurotoxicity occurred in 4 (10.3%) patients. The overall response rate evaluated at 3 months post-single infusion was 79.5% (complete remission, 56.4%; partial response, 23.1%). With a median follow-up of 32 months, the median progression free survival was 13 months and median overall survival was not reached, with the estimated rate of 53.8% (95% CI, 40.3% to 72.0%) at two years. Together, this long-term follow-up data from a multicenter study suggests that 7 × 19 CAR-T cells can induce durable responses with a median overall survival of greater than 2 years, and has a manageable safety profile in patients with R/R LBCL.

Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity.

Chemokines such as C-C motif ligand 5 (CCL5) regulate immune cell trafficking in the tumor microenvironment (TME) and govern tumor development, making them promising targets for cancer therapy. However, short half-lives and toxic off-target effects limit their application. Oncolytic viruses (OVs) have become attractive therapeutic agents. Here, we generate an oncolytic herpes simplex virus type 1 (oHSV) expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (EGFR) antibody cetuximab linked to CCL5 by an Fc knob-into-hole strategy that produces heterodimers (OV-Cmab-CCL5). OV-Cmab-CCL5 permits continuous production of CCL5 in the TME, as it is redirected to EGFR+ glioblastoma (GBM) tumor cells. OV-Cmab-CCL5 infection of GBM significantly enhances the migration and activation of natural killer cells, macrophages and T cells; inhibits tumor EGFR signaling; reduces tumor size; and prolongs survival of GBM-bearing mice. Collectively, our data demonstrate that OV-Cmab-CCL5 offers a promising approach to improve OV therapy for solid tumors.

Ccr2 Gene Ablation Does Not Influence Seizure Susceptibility, Tissue Damage, or Cellular Inflammation after Murine Pediatric Traumatic Brain Injury.

Pediatric traumatic brain injury (TBI) is a major public health issue, and a risk factor for the development of post-traumatic epilepsy that may profoundly impact the quality of life for survivors. As the majority of neurotrauma research is focused on injury to the adult brain, our understanding of the developing brain's response to TBI remains incomplete. Neuroinflammation is an influential pathophysiological mechanism in TBI, and is thought to increase neuronal hyperexcitability, rendering the brain more susceptible to the onset of seizures and/or epileptogenesis. We here hypothesized that peripheral blood-derived macrophages, recruited into the injured brain via C-C motif ligand 2 (CCL2) chemokine/C-C chemokine receptor type 2 (CCR2) signaling, contributes to neuroinflammation and thus seizure susceptibility after experimental pediatric TBI. Using Ccr2 gene-deficient mice in the controlled cortical impact (CCI) model of TBI, in 3-week-old male mice we found that TBI led to an increase in susceptibility to pentylenetetrazol (PTZ)-evoked seizures, associated with considerable cortical tissue loss, a robust cellular neuroinflammatory response, and oxidative stress. Intriguingly, although Ccr2-deficiency increased CCL2 levels in serum, it did not exacerbate seizure susceptibility or the neuroinflammatory cellular response after pediatric TBI. Similarly, acute post-injury treatment with a CCR2 antagonist did not influence seizure susceptibility or the extent of tissue damage in wild-type (WT) mice. Together, our findings suggest that CCR2 is not a crucial driver of epileptogenesis or neuroinflammation after TBI in the developing brain. We propose that age may be an important factor differentiating our findings from previous studies in which targeting CCL2/CCR2 has been reported to be anti-inflammatory, neuroprotective or anti-seizure.

Src Family Kinases Facilitate the Crosstalk between CGRP and Cytokines in Sensitizing Trigeminal Ganglion via Transmitting CGRP Receptor/PKA Pathway.

The communication between calcitonin gene-related peptide (CGRP) and cytokines plays a prominent role in maintaining trigeminal ganglion (TG) and trigeminovascular sensitization. However, the underlying regulatory mechanism is elusive. In this study, we explored the hypothesis that Src family kinases (SFKs) activity facilitates the crosstalk between CGRP and cytokines in sensitizing TG. Mouse TG tissue culture was performed to study CGRP release by enzyme-linked immunosorbent assay, cytokine release by multiplex assay, cytokine gene expression by quantitative polymerase chain reaction, and phosphorylated SFKs level by western blot. The results demonstrated that a SFKs activator, pYEEI (YGRKKRRQRRREPQY(PO3H2)EEIPIYL) alone, did not alter CGRP release or the inflammatory cytokine interleukin-1β (IL-1β) gene expression in the mouse TG. In contrast, a SFKs inhibitor, saracatinib, restored CGRP release, the inflammatory cytokines IL-1β, C-X-C motif ligand 1, C-C motif ligand 2 (CCL2) release, and IL-1β, CCL2 gene expression when the mouse TG was pre-sensitized with hydrogen peroxide and CGRP respectively. Consistently with this, the phosphorylated SFKs level was increased by both hydrogen peroxide and CGRP in the mouse TG, which was reduced by a CGRP receptor inhibitor BIBN4096 and a protein kinase A (PKA) inhibitor PKI (14-22) Amide. The present study demonstrates that SFKs activity plays a pivotal role in facilitating the crosstalk between CGRP and cytokines by transmitting CGRP receptor/PKA signaling to potentiate TG sensitization and ultimately trigeminovascular sensitization.

Characterization of immunomodulatory responses induced by manuka honey.

Manuka honey (MH) is known for its wound-healing, anti-microbial, anti-oxidant and anti-tumor properties. However, there is conflicting evidence regarding the role of MH in inflammatory responses, with some studies highlighting its pro-inflammatory capacity and others showing that it has a predominantly anti-inflammatory activity. The current study is aimed at characterizing the immunomodulatory capacity of MH using both in vitro and in vivo approaches, focusing on the underlying mechanisms. Treatment of RAW 264.7 macrophages with 1% MH (w/v) resulted in a significant increase in the gene expression (~26-fold) and secretion (~27-fold) of tumor necrosis factor-alpha (TNF-α). Similarly, an increase was observed in the gene expression of other inflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), as well as the chemokines; (C-X-C motif) ligand 2 (CXCL2) and (C-C) motif ligand 2 (CCL2). Using an in vivo model, intraperitoneal (i.p.) administration of MH in C57BL/6 mice elicited a peritoneal response characterized by a significant expansion in the number of peritoneal exudate cells (PECs), which was mainly due to a 35-fold increase in the recruitment of neutrophils. Importantly, this response was evident in toll-like receptor 4 (TLR4)-defective C3H/HeJ mice, indicating that the observed stimulatory effect occurs independently of TLR4 and unlikely to be mediated by any lipopolysaccharide (LPS) contaminant. MH administration also led to changes in the phenotypic expression and functional maturation of peritoneal macrophages, as evidenced by a shift towards the CD11blo F4/80lo phenotype and an increase in the expression of major histocompatibility complex (MHC) class II proteins. In contrast, the MH-initiated peritoneal response was largely abrogated in mice deficient in myeloid differentiation primary response 88 (MyD88) protein, a critical adaptor of most TLR signaling pathways. Thus, the current findings help to characterize the immunostimulatory properties of MH and their dependence on TLR signaling, and highlight the potential utility of MH as an immunomodulatory agent in a variety of disorders.

Therapeutic effect of adipose-derived mesenchymal stem cells (AD-MSCs) compared to pirfenidone on corticosteroid resistance in a mouse model of acute exacerbation of idiopathic pulmonary fibrosis.

Acute exacerbation-idiopathic pulmonary fibrosis (AE-IPF) is a life-threatening condition. In the treatment of AE-IPF, corticosteroid medication is commonly utilized. However, there is insufficient evidence to justify its usage. Pirfenidone (PFD) has recently been discovered to be effective in the treatment of AE-IPF patients. However, regenerative therapy, such as stem cell therapy or tissue engineering, is necessary due to ineffective and limited therapies. Combining MSC transplantation with pharmacological therapy may also give additional benefits; nevertheless, its use must be proven experimentally. As a result, the goal of this study was to assess the therapeutic effects of adipose-derived mesenchymal stem cells (AD-MSCs) on corticosteroid resistance in an animal model of AE-IPF caused by bleomycin compared to PFD. Seventy C57BL/6J male mice were randomly divided into seven groups, control, BLM, methylprednisolone (MP), PFD, AD-MSCs, PFD +MP, and AD-MSCs +MP. In terms of survival, collagen deposition, the acute lung injury score (ALI), and the Ashcroft score, AD-MSCs exceeded PFD. AD-MSCs + MP provided protection and preserved the lung's architecture in BLM-induced AE. In addition, AD-MSCs successfully decreased chemokine (CC motif) ligand-2 (CCL2) positive cells and lower pro-fibrotic and pro-inflammatory cytokines. AD-MSCs enhanced histological structure, Ashcroft and ALI scores, lung collagen deposition, survival, and cytokines in an animal model of AE-IPF. As a result, we believe that AD-MSCs may be more therapeutically helpful for AE-IPF than presently available therapies, either alone or in conjunction with MP.

Follistatin-like 1 and Biomarkers of Neutrophil Activation Are Associated with Poor Short-Term Outcome after Lung Transplantation on VA-ECMO.

Simple SummaryThe determination of biomarkers linked to adverse outcome after lung transplantation is crucial to identify vulnerable recipients and to define targeted therapeutic options. In this study, we investigated the perioperative course of a cytokine that has been related to graft-versus-host disease named Follistatin-like 1 and cytokines associated with neutrophil activation. We included 42 consecutive patients with different etiologies of end-stage pulmonary disease undergoing double lung transplantation. We found that all cytokines increased immediately after surgery. Follistatin-like 1 was significantly reduced at baseline in patients developing primary graft dysfunction. Serum concentrations of neutrophil-derived cytokines were related to prolonged extracorporeal membrane oxygenation and increases sequential organ failure assessment. Follistatin-like 1 serum concentrations seem exhausted in patients developing primary graft dysfunction. This finding could be of great importance, especially since this protein appears to contribute to allograft tolerance. However, larger trials are necessary to assess the predictive power of each of these cytokines.The investigation of biomarkers associated with undesired outcome following lung transplantation (LuTX) is essential for a better understanding of the underlying pathophysiology, an earlier identification of susceptible recipients and the development of targeted therapeutic options. We therefore determined the longitudinal perioperative course of putative cytokines related to neutrophil activation (chemokine CC motif ligand 4 (CCL-4), interleukin (IL)-23 and Lipocalin 2 (LCN2)) and a cytokine that has been implicated in graft-versus-host disease (Follistatin-like 1 (FSTL1)) in 42 consecutive patients undergoing LuTX. We plotted receiver-operating curves (ROC) to assess the predictive power of the measured cytokines for short-term outcomes namely primary graft dysfunction (PGD), early complications requiring extracorporeal membrane oxygenation (ECMO), and a high postoperative sequential organ failure assessment (SOFA). All cytokines increased immediately after surgery. ROC analyses determined significant associations between CCL4 and a high SOFA score (area under the curve (AUC) 0.74 (95%CI:0.5–0.9; p < 0.05), between LCN2 and postoperative ECMO support (AUC 0.73 (95%CI:0.5–0.9; p < 0.05), and between FSTL1 and PGD (AUC 0.70 (95%CI:0.5–0.9; p < 0.05). The serum concentrations of the neutrophil-derived cytokines LCN2 and CCL4 as well as FSTL1 were all related to poor outcome after LuTX. The specific predictive power, however, still has to be assessed in larger trials. The potential role of FSTL1 as a biomarker in the development of PGD could be of great interest particularly since this protein appears to play a crucial role in allograft tolerance.

Inflammatory biomarkers after an exercise intervention in childhood acute lymphoblastic leukemia survivors.

Cancer survivors show increased risk for non-communicable diseases and chronic low-grade inflammation characterizes the development of such diseases. We investigated inflammatory plasma protein profiles of survivors of childhood acute lymphoblastic leukemia (ALL) in comparison to healthy controls and after an intervention with a home-based exercise program. Survivors of childhood ALL aged 16-30 years (n = 21) with a median age at diagnosis 4.9 (1.6-12.9) years and a median time of 15.9 years from diagnosis, and sex- and age-matched healthy controls (n = 21) were studied. Stored plasma samples were analyzed with Olink's 92-protein-wide Inflammation panel in 21 ALL long-term survivors at baseline, after a previous 16-week home-based exercise intervention (n = 17) and in 21 age- and sex-matched controls at baseline. Protein expression levels were compared between the groups. Inflammatory protein levels did not differ between the survivors and controls at baseline. Significantly reduced levels after the intervention were found in 11 proteins related to either vascular inflammation, insulin resistance, or both: tumor necrosis factor superfamily member 14 (TNFSF14), oncostatin M (OSM), monocyte chemoattractant protein 1 (MCP-1), MCP-2, fibroblast growth factor 21 (FGF-21), chemokine (C-C motif) ligand 4 (CCL4), transforming growth factor alpha (TGF-α), tumor necrosis factor-related apoptosis-inducing ligand 10 (TRAIL), adenosine deaminase (ADA), chemokine (C-X-C motif) ligand 6 (CXCL6), and latency-associated peptide transforming growth factor beta 1 (LAP TGF-β1). The ALL survivors were not significantly more affected by inflammation than controls at baseline. The survivors' 16-week exercise intervention led to significant reduction in inflammatory protein levels. Physical exercise should be promoted for survivors of childhood cancer.

Evaluation of VEGF-A and CCL2 in dogs with brachycephalic obstructive airway syndrome or canine idiopathic pulmonary fibrosis and in normocephalic dogs

Brachycephalic obstructive airway syndrome (BOAS) and canine idiopathic pulmonary fibrosis (CIPF) of West Highland White Terriers (WHWTs) often cause intermittent or chronic hypoxemia. Our objective was to evaluate serum and bronchoalveolar lavage fluid (BALF) concentrations of hypoxemia-related proinflammatory mediators vascular endothelial growth factor A (VEGF-A) and chemokine (CC motif) ligand 2 (CCL2) in brachycephalic dogs (BDs) and WHWTs with and without CIPF. Additionally, effects of BOAS severity and ageing on these mediators were assessed.114 BDs (28 English Bulldogs (EBs), 37 French Bulldogs, 49 Pugs), 16 WHWTs with CIPF, 26 healthy WHWTs, and 39 normocephalic control dogs were included. Fifty-four BDs were re-examined after two to three years. Bead-based immunoassay was used for proinflammatory mediator measurements.Compared with controls, significantly higher serum concentrations of VEGF-A were seen in EBs (P = 0.009) and of CCL2 in CIPF and healthy WHWTs (P < 0.001; P = 0.002). BALF samples were available from controls, EBs, and WHWTs. VEGF-A was significantly lower in EBs (P < 0.001) and in CIPF and healthy WHWTs (P = 0.006; P = 0.007) and CCL2 was higher in CIPF WHWTs (P = 0.01) compared with controls. Between visits, only serum VEGF-A significantly decreased in BDs (P < 0.001), but breed, BOAS severity, or its change had no significant effect.In conclusion, in EBs with BOAS proinflammatory changes in VEGF-A were detected in both serum and BALF. Ageing reduced serum VEGF-A in BDs. In WHWTs, our results confirmed earlier findings of CCL2 as an important biomarker for CIPF.

Serum CCL2 Is a Prognostic Biomarker for Non-Metastatic Castration-Sensitive Prostate Cancer.

Purpose: Prostate-specific antigen (PSA) is a useful prostate cancer (PC) biomarker, but some cases reported that PSA does not correlate with the Gleason score. Serum chemokine (CC motif) ligand 2 (CCL2) has been reported to be a potential complementary PSA biomarker, but it remains unclear whether it can be applied to non-metastatic castration-sensitive prostate cancer (nmCSPC) or each section of the stages. Serum CCL2′s usefulness was investigated as a prognostic nmCSPC biomarker in this study. Methods: Serum samples were collected from 379 patients who underwent prostate biopsy at Kanazawa University Hospital from 2007 to 2013. A total of 230 patients with nmCSPC were included in this study of the 255 patients with histologically diagnosed prostate cancer. The serum CCL2 efficacy as a prognostic nmCSPC biomarker was investigated retrospectively. Results: An independent significant predictor of worse OS was CCL2 ≥ 280 pg/dL and CRP ≥ 0.5 mg/dL in multivariate analysis. Gleason score ≥ 8 and CCL2 ≥ 280 pg/dL were independent significant predictors of CRPC-free survival (CFS) worsening in multivariate analysis. Serum CCL2 was a predictive biomarker for OS and CFS in nmCSPC. Furthermore, CCL2 ≥ 280 pg/mL patients had significantly worse visceral metastasis-free survival than those with CCL2 < 280 pg/mL. Conclusion: This study is the first to demonstrate serum CCL2 utility as a biomarker to predict OS and CFS in nmCSPC.

OS10.7.A Activation of the CCR8-ACP5 axis by human microglia/macrophage derived CCL18 promotes glioma growth

Abstract Background Glioblastoma multiforme is a highly malignant primary brain tumor with an average survival of 14 months and very limited therapeutic options. Glioma associated microglia and macrophages (GAMs) foster tumor growth by releasing several cytokines, which have only partly been identified. Here, we studied the chemokine (C-C motif) ligand 18 (CCL18), a chemokine which is only expressed in human, but not rodent GAMs, in a novel ex-vivo brain slice model including transplantation of human induced pluripotent stem cells (iPSC) derived human microglia (iMGL) and human glioma cells in to murine brain slices, which had been depleted of intrinsic murine microglia before. Material and Methods After establishing the humanized ex-vivo brain slice model, we performed immunohistochemical analysis (IHC) of growth and invasiveness, qrtPCR on glioma cells isolated by magnetic-activated cell sorting (MACS), functional assays measuring invasiveness, proliferation, migration and colony formation of glioma cells in vitro and in slice experiments. Corresponding studies on tumor growth and invasiveness were performed after treatment with a CCL18 neutralizing antibody, a CCR8 neutralizing antibodies and knockdown of CCR8, ACP5 (Acid Phosphatase 5) and PITPNM3 with small interfering RNA (siRNA) and short hairpin RNA (shRNA). QrtPCR, IHC and Westernblot analysis were performed on primary glioma specimens. We also conducted bioinformatic analyses, based on the TCGA GBM, GLIOVIS and GEPIA databases. Results We observed that CCL18 was highly expressed in GAMs, whereas CCR8 was only expressed in glioma cells. We identified the chemokine (C-C motif) receptor 8 (CCR8) as a functional receptor for CCL18 and ACP5 as an important down-stream signaling component in glioma cells. Activation of the CCL18/CCR8/ACP5 signaling pathway in human glioblastoma was associated with enhanced tumor growth and invasiveness. Conclusion GAMs derived CCL18 promoted glioma growth by activation of the CCR8/ACP5 axis in human glioma cells and therefore is a potential therapeutic target.

CCL5 Levels Predict Stroke Volume Growth in Acute Ischemic Stroke and Significantly Diminish in Hemorrhagic Stroke Patients.

Stroke remains an important health challenge. Here, we study whether circulating chemokine (C-C motif) ligand 5 (CCL5) levels may predict clinical outcomes for stroke patients. A total of 100 consecutive stroke patients (36 acute ischemic and 64 hemorrhagic) were admitted to the stroke unit. Clinical history data and monitoring parameters were recorded. Blood serum was collected at days 0, 1, and hospital discharge to measure CCL5 levels by ELISA. Infarct or hemorrhagic volume, neurological severity (NIHSS), and functional prognosis (mRankin scale) were measured as clinical outcomes. CCL5 levels were lower in patients with hemorrhagic stroke than in patients with acute ischemic stroke. No differences were found between females and males in both types of stroke. Ischemic stroke patients whose infarct volume grew had lower CCL5 levels at day 0. Levels of CCL5 in ischemic and hemorrhagic patients were not associated with more severe symptoms/worse prognosis (NIHSS > 3; mRankin > 2) at admission or at 3 months. CCL5 could be used as a diagnostic marker to distinguish between ischemic and hemorrhagic strokes. Furthermore, CCL5 levels could predict the infarct volume outcomes in ischemic patients.

Differential roles of NMDAR subunits 2A and 2B in mediating peripheral and central sensitization contributing to orofacial neuropathic pain

The spinal N-methyl-d-aspartate receptor (NMDAR), particularly their subtypes NR2A and NR2B, plays pivotal roles in neuropathic and inflammatory pain. However, the roles of NR2A and NR2B in orofacial pain and the exact molecular and cellular mechanisms mediating nervous system sensitization are still poorly understood. Here, we exhaustively assessed the regulatory effect of NMDAR in mediating peripheral and central sensitization in orofacial neuropathic pain. Von-Frey filament tests showed that the inferior alveolar nerve transection (IANX) induced ectopic allodynia behavior in the whisker pad of mice. Interestingly, mechanical allodynia was reversed in mice lacking NR2A and NR2B. IANX also promoted the production of peripheral sensitization-related molecules, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, brain-derived neurotrophic factor (BDNF), and chemokine upregulation (CC motif) ligand 2 (CCL2), and decreased the inward potassium channel (Kir) 4.1 on glial cells in the trigeminal ganglion, but NR2A conditional knockout (CKO) mice prevented these alterations. In contrast, NR2B CKO only blocked the changes of Kir4.1, IL-1β, and TNF-α and further promoted the production of CCL2. Central sensitization-related c-fos, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) were promoted and Kir4.1 was reduced in the spinal trigeminal caudate nucleus by IANX. Differential actions of NR2A and NR2B in mediating central sensitization were also observed. Silencing of NR2B was effective in reducing c-fos, GFAP, and Iba-1 but did not affect Kir4.1. In contrast, NR2A CKO only altered Iba-1 and Kir4.1 and further increased c-fos and GFAP. Gain-of-function and loss-of-function approaches provided insight into the differential roles of NR2A and NR2B in mediating peripheral and central nociceptive sensitization induced by IANX, which may be a fundamental basis for advancing knowledge of the neural mechanisms’ reaction to nerve injury.