Interaction Of Chemokines Research Articles

Role of chemokines in aging and age-related diseases

Chemokines (chemotactic cytokines) play essential roles in developmental process, immune cell trafficking, inflammation, immunity, angiogenesis, cellular homeostasis, aging, and tumorigenesis. Chemokines also modulate response to immunotherapy, and consequently influence the therapeutic outcome. The mechanisms underlying these processes are accomplished by interaction of chemokines with their cognate cell surface G protein-coupled receptors (GPCRs) and subsequent cellular signaling pathways. Chemokines play crucial role in influencing aging process and age-related diseases across various tissues and organs, primarialy through inflammatory responses (inflammaging), recruitment of macrophages, and orchestrated trafficking of other immune cells. Chemokines are categorized in four distinct groups based on the position and number of the N-terminal cysteine residues; namely, the CC, CXC, CX3C, and (X)C. They mediate inflammatory responses, and thereby considerably impact aging process across multiple organ-systems. Therefore, understanding the underlying mechanisms mediated by chemokines may be of crucial importance in delaying and/or modulating the aging process and preventing age-related diseases. In this review, we highlight recent progress accomplished in understanding the role of chemokines and their cellular signaling pathways involved in aging and age-relaed diseases of various organs. Moreover, we explore potential therapeutic strategies involving chemokines and chemokine receptor antagonists aimed at reducing aging and mitigating age-related diseases. One of the modern methods in this direction involves use of chemokine receptor antagonists and anti-chemokines, which suppress the pro-inflammatory response, thereby helping in resolution of inflammation. Considering the wide-spectrum of functional involvements of chemokines in aging and associated diseases, several clinical trials are being conducted to develop therapeutic approaches using chemokine receptor antagonists to improve life span and promote healthy aging.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 prevents vascular dysfunction in angiotensin II-induced hypertension and selectively modulates aortic myeloid cell phenotype

Abstract Background Leukocyte migration to the vasculature and the heart plays a critical role in hypertensive immune responses and is a carefully orchestrated process, regulated by the interactions of inflammatory chemokines with their receptors, notably CCR1, CCR2, CCR3, and CCR5 (collectively termed inflammatory chemokine receptors (iCCRs)). However, the inflammatory chemokine/receptor system is characterised by redundancy, ligand sharing and overlapping expression patterns. Consequently, our understanding of the specific and combinatory roles of chemokine receptors in cardiovascular inflammation remains incomplete, thus hindering the development of targeted therapies. To address this challenge, we have utilised two novel mouse models: iREP mice, carrying fluorescent reporters of inflammatory chemokine receptor expression, and TACKO mice, in which the entire iCcr locus containing Ccr1, Ccr2, Ccr3, and Ccr5 has been deleted. Purpose To evaluate the expression pattern of iCCRs in experimental hypertension and understand how the global deletion of iCCRs impacts disease progression. Methods We induced hypertension in mice by subcutaneously implanting osmotic minipumps administering angiotensin II (Ang II) for a duration of 14 days. Single-cell RNA sequencing was utilised for investigating iCCR expression in aortas during hypertension. Additionally, we employed flow cytometry and confocal microscopy to track iCCR expression and localization in aortas and hearts obtained from iREP mice. TACKO mice were utilised to assess the effect of global iCCr deficiency on hypertensive phenotypes. Blood pressure was measured via telemetry and vascular function was assessed using myography. Vascular and heart remodelling were evaluated through histology. Molecular mechanisms were analysed using Western Blotting and RT-qPCR, while immune cell changes in the aorta were evaluated through cytometry of time-of-flight (CyTOF). Results Ang II infusion induced higher iCCRs expression levels in both the aorta and the heart in comparison with sham treatment, particularly in fibrotic areas. After Ang II-treatment, global deletion of iCCRs in TACKO mice resulted in the prevention of endothelial dysfunction (n=5/group, P<0.05) and perivascular fibrosis (n=6-9, P<0.01), and a reduction in cardiomyocyte size (n=12-16, P<0.01) compared to wild type mice. However, no significant improvements were observed in other hypertensive phenotypes, including blood pressure, heart fibrosis, vascular NO synthase, and oxidative stress. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta, accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Conclusion Deficiency in iCCRs confers protective effects against vascular dysfunction in experimental hypertension, primarily by selectively reducing inflammatory myeloid cell homing to the aorta.

Identification and analysis of chemokine-related and NETosis-related genes in acute pancreatitis to develop a predictive model.

Background: Chemokines and NETosis are significant contributors to the inflammatory response, yet there still needs to be a more comprehensive understanding regarding the specific molecular characteristics and interactions of NETosis and chemokines in the context of acute pancreatitis (AP) and severe AP (SAP). Methods: To address this gap, the mRNA expression profile dataset GSE194331 was utilized for analysis, comprising 87 AP samples (77 non-SAP and 10 SAP) and 32 healthy control samples. Enrichment analyses were conducted for differentially expressed chemokine-related genes (DECRGs) and NETosis-related genes (DENRGs). Three machine-learning algorithms were used for the identification of signature genes, which were subsequently utilized in the development and validation of nomogram diagnostic models for the prediction of AP and SAP. Furthermore, single-gene Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed. Lastly, an interaction network for the identified signature genes was constructed. Results: We identified 12 DECRGs and 7 DENRGs, and enrichment analyses indicated they were primarily enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, TNF signaling pathway, and T cell receptor signaling pathway. Moreover, these machine learning algorithms finally recognized three signature genes (S100A8, AIF1, and IL18). Utilizing the identified signature genes, we developed nomogram models with high predictive accuracy for AP and differentiation of SAP from non-SAP, as demonstrated by area under the curve (AUC) values of 0.968 (95% CI 0.937-0.990) and 0.862 (95% CI 0.742-0.955), respectively, in receiver operating characteristic (ROC) curve analysis. Subsequent single-gene GESA and GSVA indicated a significant positive correlation between these signature genes and the proteasome complex. At the same time, a negative association was observed with the Th1 and Th2 cell differentiation signaling pathways. Conclusion: We have identified three genes (S100A8, AIF1, and IL18) related to chemokines and NETosis, and have developed accurate diagnostic models that might provide a novel method for diagnosing AP and differentiating between severe and non-severe cases.

Chemokines in Cartilage Regeneration and Degradation: New Insights.

Cartilage plays a crucial role in the human body by forming long bones during development and growth to bear loads on joints and intervertebral discs. However, the increasing prevalence of cartilage degenerative disorders is a growing public health concern, especially due to the poor innate regenerative capacity of cartilage. Chondrocytes are a source of several inflammatory mediators that play vital roles in the pathogenesis of cartilage disorders. Among these mediators, chemokines have been explored as potential contributors to cartilage degeneration and regeneration. Our review focuses on the progress made during the last ten years in identifying the regulators and roles of chemokines and their receptors in different mechanisms related to chondrocytes and cartilage. Recent findings have demonstrated that chemokines influence cartilage both positively and negatively. Their induction and involvement in either process depends on the local molecular environment and is both site- and time-dependent. One of the challenges in defining the role of chemokines in cartilage pathology or regeneration is the apparent redundancy in the interaction of chemokines with their receptors. Hence, it is crucial to determine, for each situation, whether targeting specific chemokines or their receptors will help in developing effective therapeutic strategies for cartilage repair.

Metabolic changes with the occurrence of atherosclerotic plaques and the effects of statins.

Atherosclerosis is a common cardiovascular disease caused by the abnormal expression of multiple factors and genes influenced by both environmental and genetic factors. The primary manifestation of atherosclerosis is plaque formation, which occurs when inflammatory cells consume excess lipids, affecting their retention and modification within the arterial intima. This triggers endothelial cell (EC) activation, immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation and migration, foam cell formation, lipid streaks, and fibrous plaque development. These processes can lead to vascular wall sclerosis, lumen stenosis, and thrombosis. Immune cells, ECs, and VSMCs in atherosclerotic plaques undergo significant metabolic changes and inflammatory responses. The interaction of cytokines and chemokines secreted by these cells leads to the onset, progression, and regression of atherosclerosis. The regulation of cell- or cytokine-based immune responses is a novel therapeutic approach for atherosclerosis. Statins are currently the primary pharmacological agents utilised for managing unstable plaques owing to their ability to enhance endothelial function, regulate VSMC proliferation and apoptosis by reducing cholesterol levels, and mitigate the expression and activity of inflammatory cytokines. In this review, we provide an overview of the metabolic changes associated with atherosclerosis, describe the effects of inflammatory responses on atherosclerotic plaques, and discuss the mechanisms through which statins contribute to plaque stabilisation. Additionally, we examine the role of statins in combination with other drugs in the management of atherosclerosis.

The immunological and structural epidermal barrier dysfunction and skin microbiome in atopic dermatitis-an update.

Atopic dermatitis (AD) is a common, chronic and relapsing inflammatory skin disease with various clinical presentations and combinations of symptoms. The pathophysiology of AD is complex and multifactorial. There are several factors involved in the etiopathogenesis of AD including structural and immunological epidermal barrier defect, imbalance of the skin microbiome, genetic background and environmental factors. Alterations in structural proteins, lipids, proteases, and their inhibitors, lead to the impairment of the stratum corneum which is associated with the increased skin penetration and transepidermal water loss. The elevated serum immunoglobulin E levels and blood eosinophilia have been shown in the majority of AD patients. Type 2 T-helper cell immune pathway with increased expression of interleukin (IL)-4, IL-5, and IL-13, has an important role in the etiopathogenesis of AD. Both T cells and keratinocytes contribute to epidermal barrier impairment in AD via a dynamic interaction of cytokines and chemokines. The skin microbiome is another factor of relevance in the etiopathogenesis of AD. It has been shown that during AD flares, Staphylococcus aureus (S. aureus) colonization increased, while Staphylococcus epidermidis (S. epidermidis) decreased. On the contrary, S. epidermidis and species of Streptococcus, Corynebacterium and Propionibacterium increased during the remision phases. However, it is not clear whether skin dysbiosis is one of the symptoms or one of the causes of AD. There are several therapeutic options, targeting these pathways which play a critical role in the etiopathogenesis of AD. Although topical steroids are the mainstay of the treatment of AD, new biological therapies including IL-4, IL-13, and IL-31 inhibitors, as well as Janus kinase inhibitors (JAKi), increasingly gain more importance with new advances in the therapy of AD. In this review, we summarize the role of immunological and structural epidermal barrier dysfunction, immune abnormalities, impairment of lipids, filaggrin mutation and skin microbiome in the etiopathogenesis of AD, as well as the therapeutic options for AD and their effects on these abnormalities in AD skin.

Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Optimized CAR-T therapy based on spatiotemporal changes and chemotactic mechanisms of MDSCs induced by hypofractionated radiotherapy

Poor intratumoral infiltration is the major challenge for chimeric antigen receptor (CAR)-T cell therapy in solid tumors. Hypofractionated radiotherapy (HFRT) has been reported to induce immune cell infiltration and reshape the tumor immune microenvironment. Here, we showed that HFRT (5×5 Gy)-mediated an early accumulation of intratumoral myeloid-derived suppressor cells (MDSCs) and decreased infiltration of T cells in the tumor microenvironment (TME) of immunocompetent mice bearing triple-negative breast cancer (TNBC) or colon cancer, which was further confirmed in tumors from patients. RNA sequencing (RNA-seq) and cytokine profiling analysis revealed that HFRT-induced the activation and proliferation of tumor-infiltrated MDSCs, and which was mediated by the interactions of multiple chemokines and chemokine receptors. Further investigation showed that when combined with HFRT, CXCR2 blockade significantly inhibited MDSCs trafficking to tumors, and effectively enhanced the intratumoral infiltration and treatment efficacy of CAR-T cells. Our study demonstrates that MDSCs blockade combined with HFRT is promising for CAR-T cell therapy optimization in solid tumors.

Abstract 2654: Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein

Abstract Mantle Cell Lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma that frequently metastasizes. Lymphoma dissemination into distant sites is the primary cause of cancer mortality. Lymphoma metastasis requires development of supporting vasculature in which the interaction between malignant B cells and the vasculature or lymphatics is critical to tumor cell growth and dissemination. Human umbilical vein endothelial cells (HUVEC) or human lymphatic endothelial cells (HLEC) cultured on Matrigel, in vitro, assemble into networks of capillary like structures (CLS) or lymphatic like structures (LLS). When Mino or Jeko-1 MCL cells are co-cultured with vascular or lymphatic cells they migrate toward and anneal to nascent CLS or LLS. We constructed a novel antibody fusion protein, αCD20-IgG1-huEndoP125A, to inhibit such interactions. αCD20-IgG1-huEndoP125A links an αCD20 human IgG1 targeting domain and a mutant version of human endostatin, huEndoP125A, with enhanced anti-angiogenic properties. αCD20-IgG1-huEndoP125A completely inhibits CLS or LLS formation and prevents the alignment and annealing of MCL to CLS or LLS. αCD20-IgG1-huEndoP125A also significantly reduces both endothelial and MCL migration and invasion in vitro. Cell migration and trafficking is tightly regulated by chemokine interactions with corresponding chemokine receptors. We assayed for the presence of chemokines using a dot blot chemokine array. Jeko-1 and HUVEC cells were cultured on Matrigel, alone or together in co-culture, and either treated with αCD20-IgG1-huEndoP125A or left untreated. CXCL12 was expressed at high levels in HUVEC cultures undergoing CLS formation and expression was further increased in HUVEC and MCL co-cultures. HUVEC or HUVEC/MCL co-cultures treated with αCD20-IgG1-huEndoP125A showed markedly decreased CXCL12 expression. Flow cytometry showed that corresponding chemokine receptor CXCR4 was upregulated on MCL cells compared to normal B cells. CXCR4 was also upregulated on HUVEC undergoing CLS formation, and further increased on HUVEC in co-culture with MCL cells. αCD20-IgG1-huEndoP125A inhibited HUVEC CLS formation, reduced MCL migration to and alignment with nascent CLS or LLS, downregulated CXCL12 levels in HUVEC or coculture supernatants, and reduced CXCR4 expression on both HUVEC and MCL cells. Murine 38c13-huCD20 lymphoma cells were implanted s.c. into C3H mice and mice were treated i.p. with either PBS, Rituximab, or equimolar αCD20-IgG1-huEndoP125A. Mice treated with αCD20-IgG1-huEndoP125A showed significantly reduced tumor growth compared to mice treated with PBS or rituximab. αCD20-IgG1-huEndoP125A ability to inhibit angiogenesis, lymphangiogenesis, and lymphoma association with angiogenic or lymphatic vasculature may be a potent means of preventing lymphoma growth and metastasis. Citation Format: Christian Elledge, Seung-Uon Shin, Rathin Das, Yu Zhang, Hyun-Mi Cho, Sundaram Ramakrishnan, Ankita Sankar, Joseph Rosenblatt. Inhibition of lymphoma interactions withangiogenic and lymphangiogenic endotheliumusing αCD20-IgG1-huEndoP125A, an anti-CD20 endostatin fusion protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2654.

Abstract 586: Development of a novel pan-chemokine receptor inhibitor as therapeutic for breast cancer

Abstract The interaction of chemokines and their cognate receptors (CCRs) forms a key signaling network dedicated to promoting an inflammatory tumor microenvironment (TME), leukocyte recruitment and activation, angiogenesis, and growth in all stages of various cancers. Aberrant expressions of chemokines and CCRs are found to correlate with immunosuppression, metastasis, drug resistance, and poor prognosis in breast cancer patients. Several specific inhibitors for chemokines or CCRs are currently tested in clinical trials of multiple cancers. To date, no pan-CCR inhibitor has been developed as a cancer therapy, which is expected to have more clinical advantages than the agent with specificity for a single CCR. We collaborated with Dr. Filip Fratev's team (Micar21, Bulgaria) to develop potent pan-CCR inhibitors for breast cancer. By performing a virtual screen of 4 million compounds based on the analyses of Pharmacophore-based Docking, Induced-fit Docking, Molecular Dynamics, and Metadynamics, a novel pan-CCR inhibitor, SSFF-02, was identified, which showed strong inhibitory activities against CCR1, 3, 5. We tested SSFF-02 in xenografts bearing tumors derived from mouse breast cancer cell line 4T1. The treatments of SSFF-02 (5, 30, and 150 mg/kg) inhibited tumor growth on day 31 and significantly diminished the metastasis in the lung, liver, and ribs. We further examined the levels of serum INF-γ and 26 crucial cytokines and chemokines in the mice treated with SSFF-02 and the control group by enzyme-linked immunosorbent assay (ELISA) and chemokine array assay (RayBiotech). The SSFF-02-treated mice showed significant decreases in serum INF-γ and increases in CXCL5, CCL9, and CCL12. These data suggest that SSFF-02 can module the TME in breast cancer xenografts. The total mRNA of tumors collected from 4T1 xenografts have been harvested and are currently subjected to RNA sequencing (RNAseq) to characterize the chemokine-associated signature of breast cancer in response to SSFF-02. Furthermore, our MTT assays in human triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, Hs578T, and BT-20) and the hormone-dependent MCF7 cell line showed that 25 mM SSFF-02 significantly decreased over 50% of cell viabilities in all tested lines. Matrigel invasion assay demonstrated that the treatment of 5 or 10 mM SSFF-02 inhibited more than 60% of cancer cell migration in all lines. Our studies show that SSFF-02 is a promising targeted therapeutic and immune modulator for metastatic breast cancer. This study provides a foundation to pre-clinically develop SSFF-02 as therapeutics for breast cancer and other inflammatory cancers, such as lung, colorectal, and melanoma, with documented aberrant chemokine signaling. Citation Format: Mariana Grigoruta, Filip Fratev, Yong Qin. Development of a novel pan-chemokine receptor inhibitor as therapeutic for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 586.

Exploration of the underlying comorbidity mechanism in psoriasis and periodontitis: a bioinformatics analysis

BackgroundIncreasing evidence indicates that psoriasis (PSO) and periodontitis (PD) are likely to occur together, however, the underlying mechanism remains unclear.Materials and methodsThe expression profiles of PSO (lesion vs non-lesion, GSE30999, GSE14905) and PD (affected vs unaffected gingival tissue, GSE16134, GSE10334) were downloaded from the GEO database. First, we investigated the common differentially expressed genes (DEGs) of PSO and PD. Then, GO and KEGG enrichment analysis, protein interaction network (PPI) construction, and hub gene identification analysis were carried out. Finally, GO and KEGG enrichment analysis, miRNA interaction analysis, and transcription factors (TFs) interaction analysis for hub genes were performed.ResultsEighteen DEGs were identified for further analysis, including 15 up-regulated genes and 3 down-regulated genes. 9 hub genes were then identified via Cytohubba, including IL1B, CXCL1, CXCL8, MMP12, CCL18, SELL, CXCL13, FCGR3B, and SELE. Their functions are mainly enriched in two aspects: neutrophil chemotaxis and migration, chemokine activation and interaction. The enriched signaling pathways includes three categories: host defense, inflammation-related signaling pathways, and disease-related pathways. 9 common miRNAs based on experimental evidence and 10 common TFs were further identified in both PSO and PD.ConclusionOur study revealed possible comorbidity mechanisms in PSO and PD from the perspective of bioinformatics tentatively. The data can present new insight for joint prevention and treatment of in PSO and PD, as well as provide data support for further prospective studies.

“Glyco-sulfo barcodes” regulate chemokine receptor function

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

NMR Methods for Characterization of Glycosaminoglycan-Chemokine Interactions.

Humans express around 50 chemokines that play crucial roles in human pathophysiology from combating infection to immune surveillance by directing and trafficking leukocytes to the target tissue. Glycosaminoglycans (GAGs) regulate chemokine function by tuning monomer/dimer levels, chemotactic/haptotactic gradients, and how they are presented to their receptors. Knowledge of the structural features of the chemokine-GAG complexes and GAG properties that define chemokine interactions is essential not only to understand chemokine function, but also for developing drugs that disrupt chemokine-GAG crosstalk and thereby impart protection against dysregulated host defense. Nuclear magnetic resonance (NMR) spectroscopy has proven to be quite useful for providing residue-specific interactions, binding geometry and models, specificity, and affinity. Multiple NMR methods have been used including (1) chemical shift perturbation (CSP), (2) saturation transfer difference (STD), and (3) paramagnetic relaxation enhancement (PRE) techniques. In this chapter, we describe how NMR CSP, STD, and PRE can be best used for characterizing chemokine-GAG interactions.

Bioinformatic analysis of potential biomarkers and mechanisms of immune infiltration in mitral regurgitation complicated by atrial fibrillation.

Mitral regurgitation (MR) is one of the most prevalent valvular diseases. Degenerated MR-induced volume overload leads to left atrial enlargement and eventually, atrial fibrillation (AF). AF has a negative effect on patient prognosis despite recent advances in minimal invasive transcatheter devices for valve surgery. However, more effective strategies aimed at precisely treating from pathophysiology and genetic perspective are scarce. The gene expression datasets, GSE109744 and GSE79768, were obtained from the Gene Expression Omnibus database and analyzed to identify the differentially expressed genes (DEGs) in patients with mitral value prolapse (MVP) and AF. Subsequently, we predicted the extensive miRNA targets, and the protein-protein interaction (PPI) and miRNA-target gene regulatory networks were established. Functional enrichment analyses were performed for the DEGs. In addition, the co-expressed DEGs coupled with their predicted miRNAs and disease phenotypes involved in MVP and AF were assessed. Finally, the immune infiltration in both datasets was examined. A total of 491 and 180 DEGs were identified in the mitral valve and left atrial specimens, respectively. From these, 11 integrated co-expressed DEGs were identified, namely, PRG4, GPR34, RELN, CA3, IL1B, EPHA3, CHGB, TCEAL2, B3GALT2, ASB11, and CRISPLD1. The enriched Gene Ontology terms and KEGG pathways associated with the DEGs were determined, and the top 10 hub genes and top 3 gene clusters were selected from the PPI network. A prediction of target miRNAs was performed based on the co-expressed DEGs. The enrichment of the co-expressed DEGs suggested that immune and inflammatory responses might be involved in the disease development through multiple immune related pathways, including the interaction of cytokines and chemokines. Notably, this result was consistent with the immune infiltration analysis since the proportions of naïve B cells and memory B cells were significantly different in MVP and AF tissues compared to normal tissues. MR and AF are related, and 11 co-expressed DEGs were found to be significantly associated with MVP with AF, and indeed, these may represent novel biomarkers. Several immune cells were found to contribute to the process of MVP and AF via diverse mechanisms, in particular, antigen-presenting cells.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine–chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

Abstract A10: Radiolabelled AMD3100 for the management of malignant lymphoma: Translational study

Abstract Introduction: Malignant lymphomas are heterogeneous group of neoplasm comprising of proliferating lymphoid cells or their precursors. The two main types of lymphoma are Hodgkin Lymphoma and Non- Hodgkin Lymphoma. The current treatment modalities include chemotherapy; radiation therapy; passive immunotherapy; radio-immunotherapy and small-molecule inhibitors. Radiation therapy is the treatment of choice if disease is localized. Targeted therapy with antibody and small molecules may be more suitable for malignant lymphomas. Lymphoma cells are reported to over-express chemokine receptors that favors leukocyte infiltration, promotes tumorigenesis, angiogenesis, immune evasion, and metastasis. Human CXCL4 and CCL5 oligomerize with over 20 chemokine subfamilies to control leukocytes infiltration. CXCR4 antagonists disrupt the specific interaction of chemokines with CXCR4 and may elicit therapeutic potential. Chemokine receptor 4 (CXCR4) has been explored with radiolabelled CXCR4 antagonist (AMD3100) for imaging and future targeted radiation therapy for malignant lymphoma. Methods: Standardization of conjugation of AMD3100 with bifunctional chelating agents (DTPA, NOTA etc.). Optimization include radiolabelling of conjugated AMD3100 with 68Ga and 177Lu for imaging and therapeutic purpose respectively. The radionuclide and radiochemical purity of the product was determined. CXCR4 binding efficacy and toxicity studies were performed on CXCR4 expressing cancer cell-lines. The quality control of radiolabelled AMD3100 included, radiochemical purity, sterility, pyrogenicity, human serum and PBS stability. The biodistribution studies were performed in normal rats. After obtaining clearance from institutional ethics committee,68Ga-AMD3100-DTPA PET/CT was performed in lymphoma patients (n=2). The uptake was compared with 18FDG uptake for proof of concept. Results: DTPA conjugation of AMD3100 (1087 Da) and NOTA conjugation (1014 Da) was confirmed with MALDI-TOF. The radionuclide and radiochemical purity of 68Ga and 177Lu AMD3100 was >99%. Radio-ligand binding assay confirmed high specificity (Kd = 57.16 nM) of 177Lu-AMD3100-DTPA towards CXCR4 expressing cancer cells. Furthermore, the log absolute IC50 concentration of 177Lu-AMD3100-DTPA calculated for cytotoxicity assessment, via MTT assay, was 2.628 nM. Immunocytochemistry depicted positive nuclear staining of CXCR4 receptors in cancer cells. The synthesized radiopharmaceuticals were sterile and pyrogen-free. In-vivo physiological biodistribution of 68Ga-AMD3100-DTPA was found in liver, lung and spleen. The radiotracer showed faster renal clearance and low blood pool activity. In addition to physiological uptake, 68Ga-AMD3100-DTPA PET/CT showed similar lesions that were seen in 18FDG PET/CT of lymphoma patients. Conclusion: 68Ga/177Lu-AMD3100-DTPA demonstrated high target specificity towards CXCR4 expressing lymphoma cells. Cytotoxicity and patient study indicated its potential as theranostic agent. It can be used for the selection of patients, targeted therapy and response evaluation. Citation Format: Tamanna Lakhanpal, Jaya Shukla, Rajender Kumar, Harmanpreet Singh, Pankaj Malhotra, Alka Khadwal, Gaurav Prakash, Amanjit Bal, Yogesh Rathore, BR Mittal. Radiolabelled AMD3100 for the management of malignant lymphoma: Translational study [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A10.

636P Development of radiolabelled plerixafor as a theranostic tool for malignant lymphomas

Lymphomas are a heterogeneous group of neoplasm comprising proliferating lymphoid cells or their precursors. The two main types are Hodgkin Lymphoma and Non- Hodgkin Lymphoma. The current treatment modalities include radiation therapy; chemotherapy; radio-immunotherapy and small-molecule inhibitors. Lymphoma cells over-express chemokine receptors that favor leukocyte infiltration and promote metastasis. Chemokine receptor 4 (CXCR4) antagonists disrupt the specific interaction of chemokines with its receptor and may elicit therapeutic potential.

Utilization of a topical autologous blood clot for treatment of pressure ulcers.

Management and treatment of pressure ulcers (PUs) are met with great difficulty due to various factors that cause vulnerability of the soft tissue such as location, limited mobility, increased friction and shearing forces, as well as other comorbidities that may delay or halt wound healing. The topical autologous blood clot therapy (TABCT) is a point-of-care treatment used as a blood clot to assist in recreating and repairing the extracellular matrix (ECM). The mechanism of action consists of reconstruction of the ECM by incorporating into the ulcer, providing protection from further external destruction, while assisting in advancement through the wound healing phases via interaction of necessary growth factors, mediators, and chemokines. This study aims to assess the efficacy of the TABCT in the treatment of PUs in comparison to standard of care (SOC) treatment. Twenty-four patients, 18 years or older, with PUs ranging from stage 1 to 4, were included in this study. TABCT was created by using the patient's own peripheral blood in a point of care setting. Efficacy in percent area reduction (PAR) on weeks 4 and 12 with TABCT over SOC was assessed. Treatment using TABCT in PUs resulted in 77.9% of the patients achieving a 50% PAR on week 4. The mean PAR on week 12 was 96.23% with 45% of the wounds treated with TABCT achieving complete wound closure. TABCT exhibited efficacy in PAR of PUs. In addition, TABCT use prompted granulation tissue formation over vital structures, such as bone, which is often present in later stage PUs. The potential of bringing an affordable, cost-effective, advanced biologic bedside treatment that is efficacious in resolution of these complex wounds has the potential to drastically reduce the burden of treatment on the health system.

Identification of biomarkers, pathways and potential therapeutic agents for salt-sensitive hypertension using RNA-seq.

BackgroundSalt-sensitive hypertension (SSH) is a common type of essential hypertension in China. In recent years, although an increasing number of researches have focused on SSH, few studies have been researched on patients with SSH. The objective of this study was to explore the genes and pathways linked with SSH using RNA-sequencing (RNA-seq).Materials and methodsWe used RNA-seq to analyze the transcriptome of peripheral blood mononuclear cells (PBMCs) of five SSH patients and five SRH patients. Next, we analyzed the differentially expressed genes (DEGs) using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Gene Set Enrichment (GSEA) enrichment analysis. Then, Cytoscape was used to construct the protein-protein interaction (PPI) network and the hub genes. Finally, CMAP analysis found that several small molecular compounds could reverse the altered DEGs.ResultsA total of 431 DEGs were found in the PBMC samples, including 294 up-regulated and 137 down-regulated genes. Functional enrichment analysis found significant enrichment in immune-related associations such as inflammation, chemokine, and cytokine-cytokine receptor interaction. The hub genes of the two modules were IL-6, IL-1A, CCL2, CCL3L3, and BUB1. In addition, we identified two small molecular compounds (iopromide and iloprost) that potentially interacted with DEGs.ConclusionThis study suggests some potential biomarkers for the diagnosis of SSH. It provides new insights into SSH diagnosis and possible future clinical treatment.