Cell Surface Chemokine Receptor Research Articles

A Phase 2, Open-Label Study to Evaluate the Efficacy and Safety of Mgta-145 in Combination with Plerixafor for the Mobilization of Hematopoietic Stem Cells in Patients with Sickle Cell Anemia

Background: In sickle cell anemia (SCA), a point mutation in the β-globin gene results in abnormal hemoglobin prone to polymerization under hypoxic conditions. Erythrocytes become sickle-shaped and rigid, leading to painful vaso-occlusion, ischemic tissue damage, and endothelial dysfunction. Despite significant, life-threatening risks, allogeneic hematopoietic cell transplant (allo-HCT) is the only cure for SCA. Results of autologous HCT, using the addition of a non-sickling, post-transcriptional gene silencing or gene editing to increase fetal hemoglobin, are promising and suggest an alternative option for those who lack a suitable donor. Procuring hematopoietic stem and progenitor cells (HSPCs) is challenging in patients with SCA, as granulocyte colony-stimulating factor is contraindicated due to severe adverse effects. Peripheral blood mobilization with Plerixafor (Mozobil®, Genzyme) has recently been shown to be safe and effective in SCA. While mobilization with plerixafor allows collection of an HSPC product, many SCA patients require ≥2 cycles of multiday plerixafor doses and apheresis to collect sufficient quantities of HSCs for genetic manipulation. Reports suggest 30% of patients experience vaso-occlusive events (VOEs) of grade ≥3 during and after apheresis; therefore, a considerable unmet need exists for agents that augment stem cell (SC) yields and reduce apheresis time. MGTA-145 is an agonist of cell-surface chemokine receptor 2 (CXCR2) under development for the mobilization of CD34+ cells in combination with plerixafor. MGTA-145 binds to CXCR2, resulting in HSC release, which when given with plerixafor, increases mobilization of HSCs. Preclinical studies in animal models and clinical trials in healthy subjects and patients with multiple myeloma demonstrated that MGTA-145 with plerixafor results in more rapid and robust mobilization of CD34+ cells, including primitive HSC populations, compared to plerixafor alone. Objective: To evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of 1 or 2 doses of MGTA-145 combined with plerixafor for the mobilization of HSCs in individuals with SCA. Study Design and Methods: This phase 2, open-label, 2-part study (NCT05445128) will enroll approximately 10-14 adults ages 18-35 years with SCA, at 3 sites in the US. Participants must have a documented diagnosis of SCA, have a hydroxyurea washout period, and have adequate white blood cell counts and cardiopulmonary, renal, and liver function. Key exclusion criteria include history of VOE requiring a visit to a healthcare facility within 30 days of screening, poorly controlled healthcare conditions, including but not limited to cardiopulmonary or hepatorenal events ≤6 months prior to screening, and cerebrovascular accident or retinal infarct ≤2 years prior to screening. In the 1st cohort of Part A, subjects will receive a single 0.24 mg/kg subcutaneous dose of plerixafor followed by a single 0.03 mg/kg IV dose of MGTA-145, with apheresis occurring within approximately 30-45 minutes of MGTA-145 dosing. After a 1-week follow-up visit and an Independent Data Monitoring Committee (DMC) review, the 2nd cohort with additional subjects may be enrolled and given plerixafor and a 0.03 or 0.015 mg/kg or lower dose of MGTA-145 (FIGURE). Following a 1-week follow-up visit and a 2nd DMC review, patients will be enrolled in Part B and will receive, on 2 consecutive days, plerixafor and the same dose of MGTA-145 as the 2nd Part A cohort, followed by apheresis on each dosing day. The primary endpoint is total yield of CD34+ cells (CD34+ cells/kg). Safety outcomes include incidence of treatment-emergent adverse events (TEAEs), drug-related TEAEs, Grade ≥3 TEAEs, treatment-emergent serious adverse events, and TEAEs leading to study drug discontinuation. Core exploratory endpoints include characterization of the phenotype and function of cells collected by apheresis and assessment of the gene-modifying potential of mobilized CD34+ cells. The results of this trial will establish if MGTA-145 with plerixafor will rapidly and safely mobilize robust numbers of high-quality SCs for HSCT in SCA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1327: RUXOLITINIB COMBINED WITH GLUCOCORTICOID IN THE FIRST-LINE TREATMENT OF GRAFT-VERSUS-HOST DISEASE IN CHILDREN: A SINGLE CENTER RETROSPECTIVE STUDY

Background: Ruxolitinib has certain advantages and potential in the treatment of GVHD. It can inhibit the secretion of inflammatory factors by T cells, increase the number of regulatory T cells（Tregs）in the recipient, and then mediate immune tolerance. It can also reduce T cell surface related chemokines or chemokine receptors, and then change T cell migration. A multicenter retrospective study showed that the effective rate of ruxolitinib on glucocorticoid resistant aGVHD was 81.5%, and the effective rate of glucocorticoid resistant cGVHD was 85.4%. At present, there is no report on the first-line treatment of GVHD in children with glucocorticoid combined with glucocorticoid.Our center tried to use this combined scheme as the first-line treatment option in the early stage, in order to reduce the application time of glucocorticoid and reduce glucocorticoid related complications. Aims: To investigate the efficacy and safety of ruxolitinib combined with glucocorticoid in the treatment of graft-versus-host disease (GVHD) in children. Methods: A retrospective analysis of 17 cases of graft-versus-host disease（GVHD） after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in our center was conducted. The clinical efficacy and adverse reactions of early application of ruxolitinib combined with glucocorticoid were observed. Results: The median treatment time of 17 children was 47 (24-380) days, and the median onset time was 7 (4-90) days. The overall effective rate was 94.1% (16/17), the complete remission rate was 58.8% (10/17), the partial remission rate was 35.3% (6/17). The incidence of hemocytopenia during the combination therapy was 41.1% (7 /17).The incidence of EBV infection was 35.2% (6 /17), and the incidence infection of CMV was 29.4% (5/17).The incidence of hemorrhagic cystitis was 11.8% (2/17). The median follow-up time was 230 (74-545) days. One child died of grade IV acute graft versus host disease (aGVHD), and the other 16 children survived. The 6-month overall survival rate was 94.1%. Summary/Conclusion: Ruxolitinib combined with corticosteroid is effective and safe in the treatment of children with GVHD. It is expected to become a first-line anti GVHD therapy.

CXCL2-CXCR2 axis mediates \u03b1V integrin-dependent peritoneal metastasis of colon cancer cells

Peritoneal metastasis is an insidious aspect of colorectal cancer. The aim of the present study was to define mechanisms regulating colon cancer cell adhesion and spread to peritoneal wounds after abdominal surgery. Mice was laparotomized and injected intraperitoneally with CT-26 colon carcinoma cells and metastatic noduli in the peritoneal cavity was quantified after treatment with a CXCR2 antagonist or integrin-αV-antibody. CT-26 cells expressed cell surface chemokine receptors CXCR2, CXCR3, CXCR4 and CXCR5. Stimulation with the CXCR2 ligand, CXCL2, dose-dependently increased proliferation and migration of CT-26 cells in vitro. The CXCR2 antagonist, SB225002, dose-dependently decreased CXCL2-induced proliferation and migration of colon cancer cells in vitro. Intraperitoneal administration of CT-26 colon cancer cells resulted in wide-spread growth of metastatic nodules at the peritoneal surface of laparotomized animals. Laparotomy increased gene expression of CXCL2 at the incisional line. Pretreatment with CXCR2 antagonist reduced metastatic nodules by 70%. Moreover, stimulation with CXCL2 increased CT-26 cell adhesion to extracellular matrix (ECM) proteins in a CXCR2-dependent manner. CT-26 cells expressed the αV, β1 and β3 integrin subunits and immunoneutralization of αV abolished CXCL2-triggered adhesion of CT-26 to vitronectin, fibronectin and fibrinogen. Finally, inhibition of the αV integrin significantly attenuated the number of carcinomatosis nodules by 69% in laparotomized mice. These results were validated by use of the human colon cancer cell line HT-29 in vitro. Our data show that colon cancer cell adhesion and growth on peritoneal wound sites is mediated by a CXCL2-CXCR2 signaling axis and αV integrin-dependent adhesion to ECM proteins.

Antitumor and Radiosensitization Effects of a CXCR2 Inhibitor in Nasopharyngeal Carcinoma.

CXCR2, a member of the G-protein-coupled cell surface chemokine receptor family, is commonly found on leukocytes, endothelial cells and tumor cells including nasopharyngeal carcinoma cells. However, how the activity of CXCR2 and its ligand CXCL8 affects the development of nasopharyngeal carcinoma (NPC) remains unknown. Here, we found that CXCR2 and CXCL8 were both predicted poor prognosis in NPC patients. Furthermore, we identified that treatment with CXCR2 antagonist SB225002 of nasopharyngeal carcinoma cell lines resulted tumorigenesis inhibition in vitro and in vivo. In addition, we found that SB225002 could enhance NPC cells radiosensitivity through regulating cell circle distribution and interfering with cellular DNA damage repair. SB225002 also exhibited an efficient radiosensitization effect in C666-1 and HONE-1 bearing mice. Functionally, we showed that SB225002 reduced microvessel density and proliferation and induced tumor apoptosis. Furthermore, changes in the tumor microenvironment were also observed in this study. We observed that SB225002 reduced tumor-associated neutrophils (TANs) in the tumors tissue which were recruited especially after irradiation. Taken together, our results suggested that targeting the CXCL8-CXCR2 pathway is a promising therapeutic strategy for comprehensive NPC treatment.

Potential Benefit of Soluble Thrombomodulin in Coping with COVID-19/SARS-CoV-2-Induced Coagulopathy

Researchers should investigate the coagulopathy that accompanies Coronavirus Disease 2019 (COVID-19), as the incidence, as well as the mortality associated with coagulopathy, is higher than expected in COVID-19 patients. We read with interest the article published by Tang N et al. suggesting that elevated plasma levels of the fibrin degradation product D-dimer might be a predictive marker for mortality of patients with COVID-19 [1]. The same authors also reported that the use of heparin improved the prognosis of COVID-19 patients with severe coagulopathy and D-dimer levels >6-fold of the normal upper limit [2]. The high plasma levels of D-dimer suggest that the fibrinolytic system is not inhibited; nonetheless, thrombotic events are frequently noted in COVID-19 patients transferred to intensive care units [3]. Curiously, severe cases of COVID-19 are complicated by cytokine release syndrome in conjunction with Hemophagocytic Lymphohistiocytosis (HLH) [4], suggesting that extremely exaggerated hypercoagulability is induced in COVID-19. The pathophysiology of COVID-19-induced coagulopathy has not been fully elucidated. However, recently, postmortem examination of patients with COVID-19 who died of respiratory failure found thrombi in parallel with the deposition of the C5b-9 complex in the microvasculature, indicating terminal complement activation [5]. These pathological findings are reminiscent of the pathogenesis of Transplant-Associated Thrombotic Microangiopathy (TA-TMA) and atypical Hemolytic Uremic Syndrome (aHUS), in which activation of the complement system plays a role. Other possible factors involved in the pathogenesis of COVID-19-induced coagulopathy may be related to the tropism of Severe Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, to vascular endothelial cells [6]. SARS-CoV-2 infects vascular endothelial cells via the Angiotensin-Converting Enzyme 2 (ACE2) receptor, leading to vascular derangement, so-called endotheliitis, which may result in coagulopathy and TMA. Thrombomodulin (TM) is a cell-surface expressed glycoprotein comprising six distinct domains, including an NH2-terminal lectinlike region, six tandem Epidermal Growth Factor (EGF)-like structures, an O-glycosylation site-rich domain, a transmembrane domain, and a cytoplasmic tail domain [7]. TM binds to thrombin via its EGF-like region and Converts Protein C (PC) to Activated PC (APC), which inhibits coagulant factors FVIIIa and FVa in the presence of protein S, thereby inhibiting further thrombin generation. Interestingly, TM has an anti-inflammatory effect; the lectin-like domain of TM binds and degrades High Mobility Group Box-1 (HMGB1) and attenuates inflammation. Additionally, after interacting with thrombin, TM converts carboxypeptidase Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to activated TAFI (TAFIa), which inactivates C3 and C5. Furthermore, the fifth domain of EGF-like region of TM (TME5) interacts with the cell-surface chemokine receptor G protein-coupled receptor protein 15 (GPR15) and then exerts cytoprotective effects on vascular endothelial cells in association with upregulation of antiapoptotic MCl-1 proteins, which is mediated by the activation of extracellular signal-regulated kinase [7]. Recombinant human soluble TM (rhTM) comprises extracellular domains of TM. A clinical trial comparing the efficacy and safety of rhTM with Unfractionated Heparin (UFH) in patients with Intravascular Coagulation (DIC) caused by hematological malignancies or infection found that a greater number of patients treated with rhTM recovered from DIC with acceptable toxicity profiles than those who received UFH. Based on these results, the use of rhTM has been approved for the treatment of DIC in Japan since 2008. The use of rhTM effectively treated many patients with DIC with various types of underlying diseases, including transplant-associated complications such as TA-TMA, hepatic sinusoidal obstruction syndrome/veno-occlusive disease and engraftment syndrome [8]. Importantly, endothliitis plays a role in the development of all of these conditions, suggesting the possible cytoprotective roles of rhTM in vascular endothelial cells. Moreover, prompt improvement of DIC caused by HLH developed in association with immune reconstitution syndrome after initiation of highly active antiretroviral therapy for acquired immunodeficiency syndrome was noted in the individual who received rhTM [9]. Thus, the use of anticoagulant rhTM with additional activities for immune systems and vascular endothelial cells could counteract severe COVID-19 with coagulopathy, although a systematic review and meta-analysis of rhTM therapy for sepsis-induced coagulopathy failed to prove the significant improvement of clinical outcomes of patients with sepsis-induced coagulopathy [10].

Functional Heterogeneity and Therapeutic Targeting of Tissue-Resident Memory T Cells.

Tissue-resident memory T (TRM) cells mediate potent local innate and adaptive immune responses and provide long-lasting protective immunity. TRM cells localize to many different tissues, including barrier tissues, and play a crucial role in protection against infectious and malignant disease. The formation and maintenance of TRM cells are influenced by numerous factors, including inflammation, antigen triggering, and tissue-specific cues. Emerging evidence suggests that these signals also contribute to heterogeneity within the TRM cell compartment. Here, we review the phenotypic and functional heterogeneity of CD8+ TRM cells at different tissue sites and the molecular determinants defining CD8+ TRM cell subsets. We further discuss the possibilities of targeting the unique cell surface molecules, cytokine and chemokine receptors, transcription factors, and metabolic features of TRM cells for therapeutic purposes. Their crucial role in immune protection and their location at the frontlines of the immune defense make TRM cells attractive therapeutic targets. A better understanding of the possibilities to selectively modulate TRM cell populations may thus improve vaccination and immunotherapeutic strategies employing these potent immune cells.

Response of chemokine receptors CXCR2 and integrin β2 after Streptococcus agalactiae and Aeromonas hydrophila challenge in GIFT strain of Nile tilapia Oreochromis niloticus

CXCR2 is a G-protein-coupled cell surface chemokine receptor, and integrins are heterodimeric transmembrane (TM) glycoproteins. These proteins work together to activate neutrophils in the immune defense, but knowledge of their function in tilapia is limited. RACE technology was used to clone the full length of the Nile tilapia Oreochromis niloticus Cxcr2 gene, which included a 954 bp open reading frame encoding 318 amino acids, and the integrin β2 gene, with a 2373 bp open reading frame and 791 amino acids. Sequence analyses showed that Cxcr2 and integrin β2 are conserved among species. Expression profile was performed using qRT-PCR and indicated that Cxcr2 and integrin β2 were distributed throughout the examined organ tissues, with highest expression observed in the immune tissues. Expression of Cxcr2 and integrin β2 were increased after challenged with Streptococcus agalactiae or Aeromonas hydrophila. Results suggest that Cxcr2 and integrin β2 genes play a role in immune response in Nile tilapia and provide basic data for molecular-assistant selection of disease-resistant bloodstock to improve the production.

Circulating Leukocyte Alterations and the Development/Progression of Diabetic Retinopathy in Type 1 Diabetic Patients - A Pilot Study

ABSTRACT Background/Aims The aim of this study was to investigate the relationship between alterations in circulating leukocytes and the initiation and progression of DR in people with type 1 diabetes (T1D). Methods Forty-one patients with T1D [13 mild non-proliferative DR (mNPDR), 14 active proliferative DR (aPDR) and 14 inactive PDR (iPDR)], and 13 age- and gender-matched healthy controls were recruited prospectively. Circulating leukocytes, including CD4+ and CD8+ T-cells, CD14+CD16−, CD14−CD16+ and CD14+CD16+ monocytes; CD16+HLA-DR− neutrophils, CD19+ B-cells and CD56+ natural killer cells and their cell surface adhesion molecules and chemokine receptors (HLA-DR, CD62L, CCR2, CCR5, CD66a, CD157 and CD305) were examined by flow cytometry. Results In DR patients, compared to healthy controls, increased proportions of neutrophils (p = .0152); reduced proportions of lymphocytes (p = .0002), HLA-DR+ leukocytes (p = .0406) and non-classical monocytes (p = .0204); and reduced expression of CD66a (p = .0048) and CD157 (p = .0007) on CD4+ T cells were observed. Compared to healthy controls, CD19+ B cells were reduced at the mNPDR but not aPDR patients. Total lymphocytes, CD4+ T cells and CD8+ T cells progressively decreased whereas neutrophils, the neutrophil/lymphocyte ratio and the neutrophil/CD4+ ratio progressively increased from early to late stages of DR, reaching statistical significance at the aPDR stage. Longer diabetes duration was associated with a reduced proportion of CD8+ T cells (p = .002) and increased neutrophil/CD8+ ratio (p = .033). Conclusions In this pilot study, DR is associated with increased innate cellular immunity especially neutrophils and reduced adaptive cellular immunity particularly lymphocytes. Impaired B-cell immunity may play a role in the initiation of DR; whereas impaired T-cell immunity with increased neutrophil response may contribute to progression of DR from non-proliferative to proliferative stages in T1D patients. Large multicenter studies are needed to further understand the immune dysregulation in DR initiation and progression.

Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells

Background: Interleukin-8 (IL-8) released from mechanosensitive cancer cells plays a key role in the inflammation and metastasis of solid carcinomas. In this study, we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS. Methods: After the fluid shear stress was loaded, LightCycler™ system and quantitative sandwich ELISA were employed to assay the IL-8 mRNA expression and protein production, respectively. IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-κB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IκB phosphorylation and degradation. RT-PCR, Northern blot and immunofluorescence were used to determine the expression of a cell-surface chemokine receptor CXCR2 at mRNA and protein levels. Results: IL-8 mRNA expression and protein production had biphasic responses to low shear stress (1.5 dyne/cm 2 ), with the peaks at 1 and 2 h respectively. There was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm 2 for 2 h. Following the application of shear stress of 1.5 dyne/cm 2 , NF-κB p65 became detectable in the cell nuclei, and Phosphorylated IκB in cell lysates increased significantly. CXCR2, which was constitutively present on the surface of SKOV3 cells, increased following exposure to fluid shear stress for 60 min. Conclusions: Fluid shear stress triggered IL-8/CXCR2 signaling of SKOV3 cells is an early gene activation, and the activation can be mediated through NF-κB. This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have an important contribution to the pathogenesis and development of both inflammation and metastasis of ovarian carcinomas.

Fluid shear stress-induced IL-8/CXCR signaling in human ovarian cancer cells.

BackgroundInterleukin-8 (IL-8) released from mechanosensitive cancer cells plays a key role in the inflammation and metastasis of solid carcinomas. In this study, we have explored IL-8 and its receptors signal transduction process of human ovarian cancer cells under conditions of FSS.MethodsAfter the fluid shear stress was loaded, LightCycler™ system and quantitative sandwich ELISA were employed to assay the IL-8 mRNA expression and protein production, respectively. IL-8 reporter gene pEGFP1-IL8USCS was constructed for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-κB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IκB phosphorylation and degradation. RT-PCR, Northern blot and immunofluorescence were used to determine the expression of a cell-surface chemokine receptor CXCR2 at mRNA and protein levels.ResultsIL-8 mRNA expression and protein production had biphasic responses to low shear stress (1.5 dyne/cm2), with the peaks at 1 and 2 h respectively. There was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected SKOV3 cells subjected to a fluid shear stress of 1.5 dyne/cm2 for 2 h. Following the application of shear stress of 1.5 dyne/cm2, NF-κB p65 became detectable in the cell nuclei, and Phosphorylated IκB in cell lysates increased significantly. CXCR2, which was constitutively present on the surface of SKOV3 cells, increased following exposure to fluid shear stress for 60 min.ConclusionsFluid shear stress triggered IL-8/CXCR2 signaling of SKOV3 cells is an early gene activation, and the activation can be mediated through NF-κB. This observation suggested that fluid shear stress-induced IL-8 activation and the downstream signal pathways may have an important contribution to the pathogenesis and development of both inflammation and metastasis of ovarian carcinomas.

Evolution of CCL16 in Glires (Rodentia and Lagomorpha) shows an unusual random pseudogenization pattern

BackgroundThe C-C motif chemokine ligand 16 (CCL16) is a potent pro-inflammatory chemokine and a chemoattractant for monocytes and lymphocytes. In normal plasma, it is present at high concentrations and elicits its effects on cells by interacting with cell surface chemokine receptors. In the European rabbit and in rodents such as mouse, rat and guinea pig, CCL16 was identified as a pseudogene, while in the thirteen-lined ground squirrel it appears to be potentially functional. To gain insight into the evolution of this gene in the superorder Glires (rodents and lagomorphs), we amplified the CCL16 gene from eleven Leporidae and seven Ochotonidae species.ResultsWe compared our sequences with CCL16 sequences of twelve rodent species retrieved from public databases. The data show that for all leporid species studied CCL16 is a pseudogene. This is primarily due to mutations at the canonical Cys Cys motif, creating either premature stop codons, or disrupting amino acid replacements. In the Mexican cottontail, CCL16 is pseudogenized due to a frameshift deletion. Additionally, in the exon 1 (signal peptide), there are frameshift deletions present in all leporids studied. In contrast, in Ochotona species, CCL16 is potentially functional, except for an allele in Hoffmann’s pika. In rodents, CCL16 is functional in a number of species, but patterns of pseudogenization similar to those observed in lagomorphs also exist.ConclusionsOur results suggest that while functional in the Glires ancestor, CCL16 underwent pseudogenization in some species. This process occurred stochastically or in specific lineages at different moments in the evolution of Glires. These observations suggest that the CCL16 had different evolutionary constrains in the Glires group that could be associated with the CCL16 biological function.

Design and evaluation of a CXCR4 targeting peptide 4DV3 as an HIV entry inhibitor and a ligand for targeted drug delivery

The feasibility of utilizing the cell surface chemokine receptor CXCR4 for human immunodeficiency virus (HIV) entry inhibition and as an intracellular portal for targeted drug delivery was evaluated. Novel DV3 ligands (1DV3, 2DV3, and 4DV3) were designed, synthesized and conjugated to various probes (fluorescein isothiocyanate (FITC) or biotin) and cargos with sizes ranging from 10 to 50 nm (polyethylene glycol (PEG), streptavidin, and a polymeric nanoparticle). 4DV3 conjugated probes inhibited HIV-1 entry into the CXCR4-expressing reporter cell line TZM-bl (IC50 at 553 nM) whereas 1DV3 and 2DV3 did not. 4DV3 also inhibited binding of anti-CXCR4 antibody 44,708 to TZM-bl cells with nanomolar potency, while the small-molecule CXCR4 antagonist AMD3100 did not. Molecular modeling suggested simultaneous binding of a single 4DV3 molecule to four CXCR4 molecules. Differences in CXCR4-binding sites could explain the discrete inhibitory effects observed for 4DV3, the 44,708 antibody and AMD3100. In the Sup-T1 cell chemotaxis assay, the 4DV3 ligand functioned as a CXCR4 allosteric enhancer. In addition, 4DV3 ligand-conjugated cargos with sizes ranging from 10 to 50 nm were taken up into CXCR4-expressing Sup-T1 and TZM-bl cells, demonstrating that CXCR4 could serve as a drug delivery portal for nanocarriers. The uptake of 4DV3 functionalized nanocarriers combined with the allosteric interaction with CXCR4 suggests enhanced endocytosis occurs when 4DV3 is the targeting ligand. The current results indicate that 4DV3 might serve as a prototype for a new type of dual function ligand, one that acts as a HIV-1 entry inhibitor and a CXCR4 drug delivery targeting ligand.

Switching cell penetrating and CXCR4-binding activities of nanoscale-organized arginine-rich peptides

Arginine-rich protein motifs have been described as potent cell-penetrating peptides (CPPs) but also as rather specific ligands of the cell surface chemokine receptor CXCR4, involved in the infection by the human immunodeficiency virus (HIV). Polyarginines are commonly used to functionalize nanoscale vehicles for gene therapy and drug delivery, aimed to enhance cell penetrability of the therapeutic cargo. However, under which conditions these peptides do act as either unspecific or specific ligands is unknown. We have here explored the cell penetrability of differently charged polyarginines in two alternative presentations, namely as unassembled fusion proteins or assembled in multimeric protein nanoparticles. By this, we have observed that arginine-rich peptides switch between receptor-mediated and receptor-independent mechanisms of cell penetration. The relative weight of these activities is determined by the electrostatic charge of the construct and the oligomerization status of the nanoscale material, both regulatable by conventional protein engineering approaches.

Chemokine receptor co-expression reveals aberrantly distributed TH effector memory cells in GPA patients

BackgroundPersistent expansion of circulating CD4+ effector memory T cells (TEM) in patients with granulomatosis with polyangiitis (GPA) suggests their fundamental role in disease pathogenesis. Recent studies have shown that distinct functional CD4+ TEM cell subsets can be identified based on expression patterns of chemokine receptors. The current study aimed to determine different CD4+ TEM cell subsets based on chemokine receptor expression in peripheral blood of GPA patients. Identification of particular circulating CD4+ TEM cells subsets may reveal distinct contributions of specific CD4+ TEM subsets to the disease pathogenesis in GPA.MethodPeripheral blood of 63 GPA patients in remission and 42 age- and sex-matched healthy controls was stained immediately after blood withdrawal with fluorochrome-conjugated antibodies for cell surface markers (CD3, CD4, CD45RO) and chemokine receptors (CCR4, CCR6, CCR7, CRTh2, CXCR3) followed by flow cytometry analysis. CD4+ TEM memory cells (CD3+CD4+CD45RO+CCR7-) were gated, and the expression patterns of chemokine receptors CXCR3+CCR4-CCR6-CRTh2-, CXCR3-CCR4+CCR6-CRTh2+, CXCR3-CCR4+CCR6+CRTh2-, and CXCR3+CCR4-CCR6+CRTh2- were used to distinguish TEM1, TEM2, TEM17, and TEM17.1 cells, respectively.ResultsThe percentage of CD4+ TEM cells was significantly increased in GPA patients in remission compared to HCs. Chemokine receptor co-expression analysis within the CD4+ TEM cell population demonstrated a significant increase in the proportion of TEM17 cells with a concomitant significant decrease in the TEM1 cells in GPA patients compared to HC. The percentage of TEM17 cells correlated negatively with TEM1 cells in GPA patients. Moreover, the circulating proportion of TEM17 cells showed a positive correlation with the number of organs involved and an association with the tendency to relapse in GPA patients. Interestingly, the aberrant distribution of TEM1 and TEM17 cells is modulated in CMV- seropositive GPA patients.ConclusionsOur data demonstrates the identification of different CD4+ TEM cell subsets in peripheral blood of GPA patients based on chemokine receptor co-expression analysis. The aberrant balance between TEM1 and TEM17 cells in remission GPA patients, showed to be associated with disease pathogenesis in relation to organ involvement, and tendency to relapse.

Variants in CXCR4 associate with juvenile idiopathic arthritis susceptibility.

BackgroundJuvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease among children, the etiology of which involves a strong genetic component, but much of the underlying genetic determinants still remain unknown. Our aim was to identify novel genetic variants that predispose to JIA.MethodsWe performed a genome-wide association study (GWAS) and replication in a total of 1166 JIA cases and 9500 unrelated controls of European ancestry. Correlation of SNP genotype and gene expression was investigated. Then we conducted targeted resequencing of a candidate locus, among a subset of 480 cases and 480 controls. SUM test was performed to evaluate the association of the identified rare functional variants.ResultsThe CXCR4 locus on 2q22.1 was found to be significantly associated with JIA, peaking at SNP rs953387. However, this result is subjected to subpopulation stratification within the subjects of European ancestry. After adjusting for principal components, nominal significant association remained (p < 10−4). Because of its interesting known function in immune regulation, we carried out further analyses to assess its relationship with JIA. Expression of CXCR4 was correlated with CXCR4 rs953387 genotypes in lymphoblastoid cell lines (p = 0.014) and T-cells (p = 0.0054). In addition, rare non-synonymous and stop-gain sequence variants in CXCR4, putatively damaging for CXCR4 function, were significantly enriched in JIA cases (p = 0.015).ConclusionOur results suggest the association of CXCR4 variants with JIA, implicating that this gene may be involved in the pathogenesis of autoimmune disease. However, because this locus is subjected to population stratification within the subjects of European ancestry, additional replication is still necessary for this locus to be considered a true risk locus for JIA. This cell-surface chemokine receptor has already been targeted in other diseases and may serve as a tractable therapeutic target for a specific subset of pediatric arthritis patients with additional replication and functional validation of the locus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0285-3) contains supplementary material, which is available to authorized users.

A New Series of Orally Bioavailable Chemokine Receptor 9 (CCR9) Antagonists; Possible Agents for the Treatment of Inflammatory Bowel Disease

Chemokine receptor 9 (CCR9), a cell surface chemokine receptor which belongs to the G protein-coupled receptor, 7-trans-membrane superfamily, is expressed on lymphocytes in the circulation and is the key chemokine receptor that enables these cells to target the intestine. It has been proposed that CCR9 antagonism represents a means to prevent the aberrant immune response of inflammatory bowel disease in a localized and disease specific manner and one which is accessible to small molecule approaches. One possible reason why clinical studies with vercirnon, a prototype CCR9 antagonist, were not successful may be due to a relatively poor pharmacokinetic (PK) profile for the molecule. We wish to describe work aimed at producing new, orally active CCR9 antagonists based on the 1,3-dioxoisoindoline skeleton. This study led to a number of compounds that were potent in the nanomolar range and which, on optimization, resulted in several possible preclinical development candidates with excellent PK properties.

Mechanisms of the BCR-Mediated CXCR4 Down-Regulation and Its Clinical Relevance in Chronic Lymphocytic Leukemia Progression

Abstract Antigen-driven signals are involved in the pathogenesis and progression of CLL with a particular occurrence within the lymph node microenvironment. We have previously shown that ex-vivo BCR engagement promoted simultaneous down-regulation of CXCR4 and CD62L membrane expressions in CLL cells from patients at risk of disease progression only. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR triggering displayed both a reduction in migration toward CXCL12 and in adhesion to lymphatic endothelial cells. We studied a cohort of 73 previously untreated patients, 36 of whom were IGHV mutated and 37 were IGHV unmutated. The distribution of the extent of CXCR4 down-regulation was identical among CXCR4 responders, whether IGHV mutated (median 49% [Q1:28-Q3:62]) or unmutated (median 43% [Q1:31-Q3:66]). We also studied the expression of another chemokine receptor, CXCR5, which is also expressed at the membrane of CLL cells and plays an important role in the homing and trafficking of lymphocytes to the lymphoid follicles. Sustained antigenic stimulation of CLL cell samples (n=25) promoted down-regulation of CXCR4 and CXCR5 in a strictly correlated manner (r= 0,9) i.e. in the same subsets of cells. The extent of BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the B-CLL cells to respond to BCR engagement in a given patient. Moreover, treatment with the specific PKD inhibitor CID755673 blocked significantly both CXCR4 and CXCR5 BCR-mediated decrease (n=11), demonstrating that PKDs specifically target these 2 molecules, whereas CD62L down-regulation was not significantly affected by the PKD inhibitor. At the functional level, treatment with the PKD inhibitor restored CLL cell migration capacity in response to CXCL12. PKD phosphorylation/activation in response to BCR stimulation, which involvesPI3K-d, was required for CXCR4-phosphorylation and its down-regulation. We then studied the clinical relevance of CXCR4 down-regulation after BCR engagement. The capacity to down-regulate CXCR4 was significantly related to shorter PFS (p=0.043). This cohort allowed exploring the link with IGHV mutational status. 36/37 unmutated IGHV cases had a significant CXCR4 down-regulation after in vitro BCR stimulation, in concordance with the ultimately constant progression of the disease. Conversely, IGHV mutated cases fell into two groups: a) one group of 14/36 patients, with very low (&lt;5%) or no CXCR4 decrease in response to BCR ligation. None of these patients had any lymph node enlargement with a median follow up of 8.4 years [Q1:4.4-Q3:9.9]. Four patients among them required treatment but all for other reasons than nodal progression. b) the other group of 22/36 patients, for which there was a variable but significant CXCR4 down-regulation after BCR engagement. Among these cases, with a median follow up of 9.2 years [Q1:6.4-Q3:10.7], 14 patients developed clinical lymphadenopathy and 7 patients required treatment, all of them for tumor progression. The difference in lymphadenopathy was highly significant between these 2 IGHV mutated groups (p&lt; 0.001). In conclusion, the capacity of cells to down-regulate cell surface chemokine receptors and L-selectin is reflecting the size of the cell subset responding to BCR signaling. Interestingly, BCR signaling capacity to down-regulate CXCR4 is variable among IGHV mutated samples and strongly linked to the presence of lymph nodes as well as disease progression with time. It is tempting to speculate that depending on the BCR-mediated CXCR4/CXCR5 down-regulation capacity, some IGHV mutated patients might not benefit from Btk or PI3K-d inhibitors. Given the crucial importance of down-regulation of these two chemokine receptors after BCR engagement, we suggest that PKD could be promising new therapeutic target in CLL. Disclosures Levy: Roche: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Cymbalista:Janssen: Honoraria, Research Funding; Gilead: Honoraria; Roche: Honoraria; Karyopharm: Honoraria.

Successes and failures of chemokine-pathway targeting in rheumatoid arthritis.

Chemokines and chemokine receptors are involved in leukocyte recruitment and angiogenesis underlying the pathogenesis of rheumatoid arthritis (RA) and other inflammatory rheumatic diseases. Numerous chemokines, along with both conventional and atypical cell-surface chemokine receptors, are found in inflamed synovia. Preclinical studies carried out in animal models of arthritis involving agents targeting chemokines and chemokine receptors have yielded promising results. However, most human trials of treatment of RA with antibodies and synthetic compounds targeting chemokine signalling have failed to show clinical improvements. Chemokines can have overlapping actions, and their activities can be altered by chemical modification or proteolytic degradation. Effective targeting of chemokine pathways must take acount of these properties, and can also require high levels of receptor occupancy by therapeutic agents to prevent signalling. CCR1 is a promising target for chemokine-receptor blockade.

31-OR

Objectives Natural Killer (NK) cells are key regulators of normal placental development. In contrast to peripheral blood NK (pNK) cells, NK cells located at the maternal-fetal interface constitute the main local lymphocyte population in early pregnancy, are non-cytotoxic and pro-angiogenic. Decidual NK cells (dNK) regulate remodeling of uterine spiral arteries (SA), a process leading to increased blood supply to the developing fetal–placental unit. When SA remodeling is impaired, reduced placental perfusion results in elevated plasma sFlt1 levels, triggering preeclampsia systemic symptoms, (hypertension, edema and proteinuria). We aim to model a NK cell based therapy for preeclampsia. Methods Ex vivo manipulation of human pNK cells by a combination of hypoxia, TGFb-1 and 5-aza-2′-deoxycytidine (AZA) yields cells, termed idNK cells, with phenotypic and in vitro functional similarities to dNK cells including expression of dNK cell surface markers and chemokine receptors, proangiogenic capacity and reduced cytotoxicity. idNK cells were further characterized by microarray gene expression profiling, and their capacity to remodel SA was evaluated in immunodeficient mice presenting narrow SA. Results Although distinct from dNK cells, idNK cells acquired the gene expression signature that differentiates dNK cells from pNK cells. Most importantly, i-dNK cell injection increased placental perfusion in a mouse model with narrow SA as evidenced by decreased uterine artery resistance evaluated by Doppler ultrasound, and ameliorated SA remodeling. Conclusions Ex vivo conversion of peripheral blood NK cells into i-dNK cells may be a potential approach for the prevention or treatment of preeclampsia and related disorders. Disclosures R. de Carvalho Cavalli: None. A. Cerdeira: None. H. Korkes: None. S. Burke: None. A. Rajakumar: None. M. Bhasin: None. S. Karumanchi: Consultant, Commercial Interest: Aggamin LLC. H. Kopcow: None.

Expression Profile of CXCL3 Gene in Peripheral Blood Mononuclear Cells Challengedin vitrowithTheileria annulatain Crossbred Cattle

Bovine tropical theileriosis is a major haemoprotozoan disease associated with high rates of morbidity and mortality particularly in exotic and crossbred cattle. Recent studies suggest that a number of immune response genes, expressed differentially in exotic and indigenous breeds play an important role in breed specific resistance to tropical theileriosis. In the present study, expression of CXCL3 gene which has chemotactic activity for neutrophils, controls migration and adhesion of monocytes and ultimately mediates its effects on target cells by interacting with a cell surface chemokine receptor called CXCR2 was studied in crossbred cattle. The in vitro experimental result revealed significant difference in CXCL3 gene expression in Theileria annulata challenged peripheral blood mononuclear cells of crossbred animals as compared to healthy controls and a 2.53 fold increase (p < 0.05) was recorded. The results of current study indicate that CXCL3 may be involved in host-pathogen interaction during tropical theileriosis.