Recombinant Human C-C motif chemokine 5 protein (CCL5)
Description
Production Methods
Recombinant CCL5 is primarily expressed in E. coli, with recent advances improving yield and bioactivity:
Key Innovations:
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SUMO fusion tags: Increase solubility, achieving ~25 mg/L yield in SHuffle cells without inclusion body purification
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Low-pH dialysis: Maintains structural integrity during refolding
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Endotoxin reduction: <0.1 EU/µg, critical for cell-based assays
Comparative Production Data:
| Method | Yield | Bioactivity Confirmed By | Reference |
|---|---|---|---|
| SUMO-tagged (SHuffle) | 25 mg/L | Calcium flux, chemotaxis | |
| MBP fusion | 66.8 mg/L* | Partial receptor binding | |
| His-tagged | 2.5 mg/L | Ovarian cancer proliferation |
Functional Mechanisms
CCL5 activates multiple signaling pathways through G-protein coupled receptors (GPCRs):
Signaling Pathways:
Disease Implications:
Validated Assays:
| Assay Type | Readout | ED50/EC50 | Source |
|---|---|---|---|
| Chemotaxis (T cells) | Transwell migration | 5–15 ng/mL | |
| Calcium flux | Fluo-8AM fluorescence | 10–100 nM | |
| HIV-1 inhibition | p24 antigen reduction | IC50 = 1–10 nM |
Therapeutic Targeting:
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CCR5 antagonists: Maraviroc (MVC) and TAK-779 block CCL5-induced signaling
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Phase separation inhibitors: Disrupt heparan sulfate-CCL5 condensates to modulate inflammation
Challenges and Future Directions
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Structural heterogeneity: Oligomerization states impact functional studies
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Disease-specific roles: Context-dependent pro/anti-tumor effects require tissue-specific models
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Delivery optimization: Nanoparticle formulations to enhance in vivo stability
Current research focuses on engineering CCL5 variants with improved pharmacokinetics and receptor selectivity, leveraging insights from cryo-EM studies of CCR5 complexes .
Product Specs
Our Recombinant Human CCL5 protein is a versatile tool for advancing immunological research. Produced in E. coli, this full-length mature protein encompasses amino acids 24-91 and is tag-free for optimal compatibility with your experiments. Supplied as a lyophilized powder, the product can be conveniently reconstituted according to your specific requirements.
Our commitment to quality ensures a purity of >98% for the Recombinant Human CCL5 protein, as verified by SDS-PAGE and HPLC analysis. Endotoxin levels are meticulously controlled below 1.0 EU/µg using the LAL method. Furthermore, the protein's biological activity is demonstrated through a chemotaxis bioassay utilizing human peripheral blood monocytes, with an effective concentration range of 1.0-10 ng/ml.
CCL5 plays a pivotal role in the recruitment and activation of immune cells, with research highlighting its involvement in diverse immune responses and inflammatory processes[1]. Consequently, our Recombinant Human CCL5 protein serves as a valuable tool in elucidating the molecular mechanisms underlying immune function and developing novel therapeutics for immune-related disorders[2].
References:
1. Appay V, Rowland-Jones SL. RANTES: a versatile and controversial chemokine. Trends Immunol. 2001;22(2): 83-87.
2. Proost P, Struyf S, Van Damme J. Natural post-translational modifications of chemokines. Biochem Soc Trans. 2006;34(Pt 6): 997-1001.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
CCL5 acts as a chemoattractant for blood monocytes, memory T-helper cells, and eosinophils. It induces histamine release from basophils and activates eosinophils. CCL5 may activate several chemokine receptors, including CCR1, CCR3, CCR4, and CCR5. It is one of the primary HIV-suppressive factors produced by CD8+ T-cells. Recombinant RANTES protein exhibits a dose-dependent inhibition of various strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). The processed form RANTES(3-68) acts as a natural chemotaxis inhibitor and is a more potent inhibitor of HIV-1 infection. The second processed form RANTES(4-68) exhibits reduced chemotactic and HIV-suppressive activity compared to RANTES(1-68) and RANTES(3-68) and is generated by an unidentified enzyme associated with monocytes and neutrophils. CCL5 may also act as an agonist of the G protein-coupled receptor GPR75, stimulating inositol trisphosphate production and calcium mobilization through its activation. In conjunction with GPR75, CCL5 may contribute to neuron survival by activating a downstream signaling pathway involving the PI3, Akt, and MAP kinases. Through its activation of GPR75, CCL5 may also play a role in insulin secretion by islet cells.
- These CCL5 derivatives can now be tested against several inflammation-related pathologies where the CCL5:CCR5 axis plays a significant role. PMID: 29382912
- The CCL5 In1.1T/C polymorphism may modulate the risk of pulmonary early-onset tuberculosis. PMID: 29608337
- Using the CPRC prostate cancer model, we demonstrate that endothelial cells secrete significant amounts of CCL5 and induce autophagy by suppressing AR expression in prostate cancer cell lines. Consequently, elevated autophagy accelerates focal adhesions proteins disassembly and promotes prostate cancer invasion. Inhibition of both CCL5/CCR5 signaling and autophagy significantly reduces metastasis in vivo. PMID: 30200999
- CCL5, secreted by endothelial cells, acts in a paracrine manner on triple-negative breast cancer (TNBC) cells to enhance their migration, invasion, and metastasis. CCL5, in turn, accelerates TNBC cell secretion of PAI-1 and promotes TNBC cell metastasis, thus forming a positive feedback loop. Moreover, this enhanced metastatic ability is reversible and dependent on CCL5 signaling via the chemokine receptor, CCR5. PMID: 28899878
- High concentrations of plasma CCL5 may promote EMT of breast cancer cells. Plasma CCL5 could be a promising candidate to predict chemotherapy response in NCT of LABC. PMID: 29758926
- The polymorphism of CCR1 rs3733096 and CCL5 rs3817656 are associated with spontaneous clearance of hepatitis C virus in the Chinese Han population. PMID: 29703961
- Our findings suggest that the CCL5 level is influenced collectively not only by the genotypes of -403G>A SNP and bacillary load but also by the treatment. Thus, CCL5 may be considered for the development of a diagnostic marker and also as an indicator of recovery. PMID: 29664036
- Serum levels in active vitiligo are significantly elevated compared to those in stable vitiligo patients. PMID: 29115683
- These findings collectively indicate that TGF-beta regulates CCL5 expression in a stage-dependent manner during breast cancer progression. PMID: 29107385
- KLF5-regulating cancer-associated fibroblasts affect gastric cancer cells progression by CCL5 secretion and activation of CCR5. PMID: 28934010
- Data show that plasminogen activator inhibitor-1 (PAI-1) and chemokine CCL5 (CCL5) overexpression promoted cell proliferation and migration in breast cancer cells. PMID: 29601121
- Among infants with lower CCL5 levels, the Haemophilus-dominant microbiota profile was associated with a higher risk of intensive care use and hospital length-of-stay >/=3 days compared to the Moraxella-dominant profile. Conversely, among those with higher CCL5 levels, there were no significant associations between the microbiota profiles and these severity outcomes. PMID: 28306146
- The current study suggests that TLR3 signaling induces CCL5 expression via NF-kappaB and IRF3 in bile duct cells, and this pathway may be involved in the pathogenesis of BA. PMID: 29070776
- Study demonstrates that increased CCL5 expression was restricted to human mesenchymal glioblastoma (GBM) and suggests that CCL5 functions in an autocrine growth-promoting circuit, and establishes a new receptor responsible for CCL5 function in mesenchymal glioblastoma cells. PMID: 28380429
- Study showed that bone stromal cells promoted prostate cancer progression through the secretion of CCL5. In vitro co-culture of bone stromal cells with prostate cancer cells induced the expression of CCL5, which promoted prostate cancer cell migration. CCL5 was found to have a key role in the progression of prostate cancer in the bone metastasis microenvironment. PMID: 29288523
- Identifies the essential role of the chemoattractive cytokine CCL5 for liver disease progression and especially hepatocellular carcinoma development. PMID: 28011329
- Breast cancer cell CCL5 mediates bone marrow independent angiogenesis via paracrine signaling. PMID: 27863423
- The present study has demonstrated a novel pathway involving CCl5/CCR1/beta-catenin/Slug, via which human Mesenchymal stem cells promotes colorectal cancer development. PMID: 28542126
- The present study suggests that TT genotype of CCL5 In1.1T/C (rs2280789) polymorphism play an important role to increased CCL5 expression in T cell which may enhanced Th1 immunity and help in protection against tuberculosis. PMID: 28336310
- CSF levels of RANTES were remarkably high only in active multiple sclerosis patients. RANTES levels were associated with transcranial magnetic stimulation measures of cortical synaptic excitability, but not with long-term potentiation (LTP)-like plasticity. PMID: 26733422
- We document for the first time that CCL5 induces tumor lymphangiogenesis by the induction of VEGF-C in human cancer cells. PMID: 27166194
- Our findings indicate that the -403 G/A RANTES (CCL5) promoter gene polymorphism is connected with psoriasis vulgaris disease severity. PMID: 27859608
- Baseline serum CCL5 levels and decrease of the serum VEGF-A levels may serve as potential predictive markers for survival or treatment-specific toxicities in metastatic colorectal cancer patients receiving regorafenib. PMID: 27166185
- This meta-analysis suggests that RANTES -403G/A and -28C/G polymorphisms confer possible protection against HIV-1 infection, whereas In1.1T/C polymorphism may increase risk of HIV-1 infection, especially in Asians. PMID: 26690919
- CCL5 and CXCL11 expression were also induced in response to the activation of the PKC pathway, and gene silencing experiments indicated that their inducible expression was dependent on RIPK4 and IRF6. Moreover, gene reporter assays suggested that RIPK4 induces CCL5 and CXCL11 expression by stimulating the transactivation of their promoters by IRF6. PMID: 27014863
- Data suggest that inhibition of CCL5 in the adipose microenvironment may represent an approach for the therapy of highly malignant Triple Negative Breast Cancer (TNBC). PMID: 27027351
- RNA-binding protein HuR (HuR) expression negatively correlated with chemokine (CC motif) ligand 5 (CCL5) expression and macrophage appearance in a cohort of breast tumors. PMID: 28731284
- We have utilized a broad-scaled affinity proteomics approach to identify three proteins (CCL5, HPGDS, and NPSR1) with altered plasma levels in asthmatic children compared to healthy controls, representing the first evaluation of HPGDS and NPSR1 in plasma. PMID: 27145233
- Data provide evidence that CCL5 enhances the proliferation and the invasive capacity of human breast cancer cell lines mediated by CCR5 activation. PMID: 27335323
- Cancer-FOXP3 serves as a prognostic biomarker and a crucial determinant of immunosuppressive microenvironment via recruiting Treg cells by directly trans-activating CCL5. Therefore, cancer-FOXP3 could be used to select patients with better response to CCL5/CCR5 blockade immunotherapy. PMID: 27991933
- Data suggest that STAT2 plays a role in the psoriasis pathogenesis by regulating the expression of CXCL11 and CCL5, and thereby attracting IFNgamma-producing immune cells to the skin. PMID: 28472186
- Mean RANTES concentrations in nasal fluid in patients with perennial allergic rhinitis and nonallergic and allergic chronic rhinosinusitis with nasal polyps patients were significantly higher in comparison to control subjects. PMID: 28587510
- All fatty necrotic and osteolytic jawbone (FDOJ) samples showed high expression of RANTES and fibroblast growth factor (FGF)-2. PMID: 28685531
- This study showed that RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation. PMID: 26873938
- These data indicate that ECFCs, not SPCs, are the major players in MMD pathogenesis and that the chemokine CCL5 mediates the interactions. It can be hypothesized that in MMD patients, defective ECFCs direct aberrant SPC recruitment to critical vascular locations through the action of CCL5. PMID: 28072843
- miR-200c represses IL-6, IL-8 and CCL-5 and improves osteogenic differentiation. PMID: 27529418
- This study shows that melanoma peptides vaccination and intratumoral administration of IFNgamma increases production of CCL5 in patient tumors. PMID: 27522581
- Findings indicate the importance of chemokine (CC motif) ligand 5 (CCL5) genetic variability and CCL5-CCR5 (CC chemokine receptor 5) axis on the susceptibility to HCV. PMID: 27304910
- Combined experimentally determined binding affinities (KD) of several orthologs of CCL5 with HNP1 with in silico studies to identify the most likely heterodimeric CCL5-HNP1 complex which was subsequently used as a starting structure to rationally design peptidic inhibitors. PMID: 26871718
- CCL5 plays a pivotal regulatory role in hepatic fibrosis during nonalcoholic fatty liver disease. PMID: 27639593
- Intermolecular interactions of RANTES with its receptor CCR5 have been reported based on NMR spectroscopy measurements. PMID: 28052516
- Our findings proposed that CCL5 -403G>A polymorphism may be a risk factor for susceptibility TO pulmonary tuberculosis. PMID: 27668525
- IL-17A could enhance the expression of RANTES, but not IL-16, in cultured primary OFs in cooperation with CD40L. PMID: 27832278
- We also found that the activation of H4R caused the release of IL-13 and RANTES on human mast cells. These data demonstrate that the H4R activates divergent signaling pathways to induce cytokine and chemokine production in human mast cells. PMID: 27400655
- The chemokine RANTES level could become a useful marker of severity of coronary artery disease. PMID: 27226191
- Findings show the significant upregulated expression of chemokine CCL5 (RANTES) in plasma, compared to CSF and contused brain tissue following severe traumatic brain injury (TBI). PMID: 28340601
- There were no associations of CCL5 gene promoter polymorphism with the risk of diabetic microvascular complications (DMI); However, the 59029A polymorphism in CCR5 might affect individual susceptibility for DMI [Meta-Analysis]. PMID: 27042273
- RANTES Gene Polymorphisms are Associated with HIV-1 Infections. PMID: 27821902
- Through the self-production of CCL5, ovarian cancer stem-like cells are induced to differentiate into endothelial cells and participate in tumor angiogenesis. PMID: 27033454
- Monocytes and lymphocytes cooperate to enhance migration towards CXCR3 chemokines and CCL5 in COPD. PMID: 26965295
Q&A
What is CCL5 and what are its primary biological functions?
CCL5, also known as RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted), is a chemokine that plays crucial roles in immune regulation and inflammatory responses. CCL5 is a key component in the tumor microenvironment and has been documented to play significant roles in tumor invasion and metastasis processes . It functions primarily by recruiting various leukocytes, including T cells, macrophages, and eosinophils, to inflammatory sites. In pathological contexts, elevated CCL5 expression has been observed in residual tumors and recurrent tumors, suggesting its involvement in cancer progression and recurrence mechanisms .
How is recombinant human CCL5 typically produced for research applications?
Recombinant human CCL5 protein is commonly expressed in prokaryotic systems, with E. coli being the predominant expression host . The typical production process involves:
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Amplification of the human CCL5 gene
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Insertion into expression vectors such as pET-30a
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Transformation into E. coli BL21 strains
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Induction of protein expression using isopropyl-β-D-1-tiogalactopiranoside (IPTG) at specific concentrations (typically 0.1 mmol/L) and temperatures (optimally at 25°C)
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Purification of the expressed protein using affinity chromatography, leveraging histidine tags engineered into the recombinant protein
This production methodology yields soluble His-tagged CCL5 protein suitable for various downstream research applications.
What are the common methods for detecting and quantifying CCL5 in experimental samples?
Several established methodologies are available for CCL5 detection and quantification:
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Enzyme-Linked Immunosorbent Assay (ELISA): Provides quantitative measurement of CCL5 in biological fluids and tissue lysates. This method has been successfully used to measure CCL5 levels in primary tumors, residual tumors, and recurrent tumors .
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Quantitative PCR (qPCR): Enables quantification of CCL5 mRNA expression using TaqMan probes (e.g., Mm01302427_m1 for mouse CCL5). This approach is particularly useful for analyzing transcriptional regulation of CCL5 in various experimental conditions .
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Western Blotting: Used for detecting CCL5 protein expression and assessing downstream signaling pathways such as NF-κB activation (through p-NFκB p65 detection) .
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RNA-seq Analysis: Allows comprehensive examination of CCL5 expression patterns across different tissues and disease states, as demonstrated in cancer studies utilizing TCGA and GTEx datasets .
How can researchers effectively study the role of CCL5 in cancer progression and recurrence?
Studying CCL5's role in cancer requires multifaceted approaches:
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Overexpression and Knockdown Models: Engineer cancer cell lines to overexpress CCL5 or use RNA interference to suppress its expression. These modified cells can then be used in orthotopic xenograft models to assess tumor growth, regression, and recurrence dynamics .
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Recurrence Assays: Utilize inducible oncogene systems (e.g., doxycycline-regulated Her2) in combination with CCL5 expression manipulation to study cancer recurrence mechanisms. This approach has demonstrated that CCL5-expressing tumors recur significantly earlier than control tumors (HR = 2.14, p = 0.023) .
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Single-cell Analysis: Use platforms such as the Tumor Immunity Single Cell Center (TISCH) to quantify CCL5 expression in various cell types within the tumor microenvironment, visualizing results through heatmaps and scatter plots .
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Correlation Analysis with Clinical Outcomes: Employ Kaplan-Meier curves and Cox regression analyses to establish associations between CCL5 expression and patient prognosis across different cancer types .
What methodological approaches are recommended for analyzing CCL5's interaction with immune cells in the tumor microenvironment?
To investigate CCL5-immune cell interactions:
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Immune Infiltration Analysis: Utilize the Tumor Immunity Estimation Resource (TIMER) database to quantify immune cell infiltration levels in relation to CCL5 expression. This allows researchers to perform Spearman correlation analyses between CCL5 mRNA expression and 21 different immune cell types .
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Hallmark Gene Set Enrichment Analysis: Apply this methodology to identify pathways linked to CCL5 expression that serve as cancer flag markers, providing insights into mechanisms of CCL5-mediated immune modulation .
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Immunotherapy Response Assessment: Analyze datasets from immune checkpoint blockade (ICB) clinical trials to examine the prognostic function of CCL5 in immunotherapy response, particularly in relation to common biomarkers such as tumor mutational burden (TMB) and microsatellite instability .
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Macrophage Recruitment Studies: Design experiments to investigate how CCL5 promotes tumor recurrence through the recruitment of CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors .
What are the optimal conditions for expressing and purifying recombinant human CCL5 protein?
Optimal expression and purification conditions include:
| Parameter | Recommended Condition | Rationale |
|---|---|---|
| Expression Host | E. coli BL21 | Provides high yield and proper folding |
| Expression Vector | pET-30a | Enables efficient expression control |
| IPTG Concentration | 0.1 mmol/L | Balances expression yield and protein solubility |
| Induction Temperature | 25°C | Minimizes inclusion body formation |
| Purification Method | Affinity chromatography | Leverages His-tag for selective isolation |
Researchers should optimize these parameters based on their specific experimental requirements, as modifications may be necessary depending on the intended downstream applications .
How can researchers effectively study CCL5-receptor interactions, particularly focusing on CCR5?
To investigate CCL5-CCR5 interactions:
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Molecular Docking Studies: Retrieve CCL5 protein structures from the PDB database and perform protein-small molecule docking using software such as MOE. This approach allows simulation of CCL5's binding mechanism with potential therapeutic molecules .
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Genetic Variant Analysis: Study natural variants like CCR5-Δ32 to understand receptor-ligand interactions. The CCR5-Δ32 variant has been extensively studied in HIV research and provides insights into the structural determinants of CCL5-CCR5 binding .
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Functional Assays: Design experiments to assess downstream signaling pathways activated by CCL5-CCR5 interaction, such as NF-κB pathway activation, which can be detected through phosphorylation of NF-κB p65 .
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Antagonist Studies: Utilize CCR5 antagonists like maraviroc to block CCL5-CCR5 interactions and assess functional consequences in disease models, as demonstrated in stroke recovery studies .
What statistical approaches are recommended for analyzing CCL5 expression data in relation to clinical outcomes?
For robust statistical analysis:
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Cox Regression Analysis: Apply this methodology to datasets from different patient cohorts to establish hazard ratios associated with CCL5 expression. Studies have shown significant hazard ratios greater than 1 for high CCL5 expression in kidney renal clear cell carcinoma (KIRC) and esophageal carcinoma (ESCA), indicating worse prognosis .
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Concordance Index (C-index): Calculate C-index values to assess the predictive accuracy of CCL5-based prognostic models. Values around 0.6 suggest moderate predictive accuracy .
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Hardy-Weinberg Equilibrium Testing: When studying CCL5-related genetic variants in population studies, apply HWE testing to identify potential associations with disease susceptibility, as demonstrated in HIV research with CCR5-Δ32 .
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Explained Fraction Calculations: Use mathematical approximations based on mutual information theory to estimate the relative influence of CCL5 and other factors on disease progression, as employed in HIV-AIDS studies .
How can CCL5 be studied in the context of infectious diseases beyond HIV?
While CCL5-CCR5 interactions are well-documented in HIV research, approaches for studying other infectious diseases include:
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Historic Epidemic Analysis: Examine the frequency of CCL5-related genetic variants in populations with historic exposure to epidemic diseases. For example, the elevated frequency of CCR5-Δ32 in Eyam (15% compared to 10% in neighboring villages) suggests potential selection during the Great Plague .
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Population Genetics Approaches: Analyze the geographical distribution of CCL5-related variants to identify potential selective pressures from historic infectious disease outbreaks .
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Comparative Pathogenesis Studies: Design experiments comparing CCL5 expression and function across different infectious disease models to identify common and disease-specific mechanisms.
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Receptor Antagonist Trials: Assess the therapeutic potential of blocking CCL5-receptor interactions in various infectious disease models, similar to approaches used in HIV and stroke research .
What are the technical challenges in translating CCL5 research findings to clinical applications?
Translational challenges include:
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Target Specificity: CCL5 interacts with multiple receptors (CCR1, CCR3, CCR5), making selective targeting challenging. Researchers must carefully design interventions that modulate specific receptor interactions without disrupting beneficial CCL5 functions.
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Context-Dependent Effects: CCL5 exhibits both pro-tumorigenic and anti-tumorigenic effects depending on the cancer type and microenvironment context, as evidenced by varying prognostic significance across cancer types .
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Ethical Considerations: Genetic manipulation of CCL5-related genes requires careful ethical consideration, particularly given the precedent of CCR5 gene editing in human embryos and subsequent controversies .
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Data Privacy: Large-scale genomic studies of CCL5-related variants must address privacy concerns while still enabling open science. Approaches like GWATCH have been developed to protect patients' personal data behind firewalls while making computed population-based results accessible .
What emerging technologies are likely to advance CCL5 research in the next decade?
Promising technological advances include:
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CRISPR-Cas9 Gene Editing: This technology offers unprecedented precision for manipulating CCL5 and its receptors in various model systems, enabling detailed functional studies and potential therapeutic applications .
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Single-Cell Multi-omics: Integration of transcriptomics, proteomics, and epigenomics at single-cell resolution will provide deeper insights into CCL5's cell type-specific functions in complex tissues and disease states .
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Advanced Computational Modeling: Improved molecular docking and simulation techniques will enhance understanding of CCL5-receptor interactions and facilitate drug discovery efforts .
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Biobank-Scale Association Studies: Large-scale analysis of biobank data linked to electronic health records will enable more robust assessments of CCL5-related variants and their impact on diverse health outcomes .
How might the understanding of CCL5's role in cancer evolve with integrated multi-omics approaches?
Multi-omics integration will likely reveal:
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Pathway-Level Insights: Better characterization of how CCL5 signaling interfaces with other cancer-relevant pathways, moving beyond single-gene analysis to network-level understanding .
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Biomarker Panels: Development of prognostic and predictive models that incorporate CCL5 expression alongside other molecular features, improving upon the moderate predictive accuracy (C-index ~0.6) of CCL5 alone .
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Microenvironment Dynamics: Deeper understanding of how CCL5 mediates communication between cancer cells and immune cells, particularly in the context of therapy resistance and cancer recurrence .
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Personalized Therapeutic Approaches: Identification of patient subgroups most likely to benefit from CCL5-targeted interventions based on comprehensive molecular profiling .
