ENA 78 Human

What is the molecular structure of human ENA-78 and how does it relate to its function?

Human ENA-78/CXCL5 is a CXC subfamily chemokine that exists in multiple forms. Full-length CXCL5/ENA-78 is 114 amino acids with a molecular weight of approximately 12 kDa. Following signal peptide removal, the bioactive form is 78 amino acids in length. The protein can undergo N-terminal cleavage by enzymes like Cathepsin G and Chymotrypsin to produce shorter variants including CXCL5/ENA-74 (74 aa) and CXCL5/ENA-70 (70 aa) . These truncated forms exhibit increased biological potency compared to the full-length protein. The structural characteristics of ENA-78 enable its binding to the CXCR2 receptor, which mediates its chemotactic effects on neutrophils .

How does ENA-78 differ from other CXC chemokines in cellular expression and function?

ENA-78 shares structural similarities with other CXC chemokines but exhibits distinctive expression patterns and functional properties. Unlike many chemokines with broad expression, ENA-78 is upregulated specifically at sites of inflammation and is expressed by multiple hematopoietic cell types, fibroblasts, endothelial cells, vascular smooth muscle cells, and adipocytes . Its production is primarily stimulated by pro-inflammatory cytokines, particularly interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα) . Functionally, while many chemokines have overlapping targets, ENA-78 demonstrates particular importance in neutrophil recruitment, angiogenesis promotion, and connective tissue remodeling . An important species distinction is that human ENA-78 lacks a true murine ortholog, with murine LIX initially thought to be the ortholog but later recognized as distinct based on genome-wide analysis .

What are the most reliable methods for measuring ENA-78 levels in different biological samples?

For accurate quantification of human ENA-78 in research settings, several validated methodologies exist:

ELISA-Based Methods:
The Quantikine Human ENA-78 Immunoassay provides a solid-phase ELISA approach with a 4.5-hour protocol, suited for cell culture supernatants, serum, and plasma samples . This method utilizes E. coli-expressed recombinant human ENA-78 and specific antibodies, demonstrating high precision with intra-assay CVs of 3.8-8.3% and inter-assay CVs of 6.7-9.8% depending on sample type .

Automated Immunoassay Platforms:
The Simple Plex platform (Ella instrument) offers a cartridge-based automated approach with high sensitivity and reproducibility. Performance metrics indicate intra-assay precision with CVs between 6.5-7.8% and inter-assay precision with CVs between 4.3-5.1% across concentration ranges of approximately 32-1511 pg/mL .

Multiplex Detection Systems:
For simultaneous analysis with other biomarkers, cytometric fluorescence detection systems like the Luminex platform can be employed. This approach has been validated in clinical research settings, including studies of cardiovascular biomarkers .

When selecting a method, researchers should consider sample type, required sensitivity, and whether parallel analysis of other inflammatory markers is needed.

How can researchers address variability in ENA-78 measurements across different sample types?

Addressing measurement variability requires understanding the unique characteristics of each sample type:

Matrix-Specific Considerations:

• Serum vs. Plasma: The search results show that while both can be used, precision data may vary. For example, serum, EDTA plasma, and heparin plasma show slightly different performance metrics in validated assays .

• Recovery and Linearity: ENA-78 demonstrates natural linearity from neat sample to 1:16 dilution in properly validated assays, minimizing the need for complex recovery calculations .

Standardization Approaches:

• Include matrix-matched calibrators and controls

• Perform appropriate sample dilutions based on expected concentration ranges

• Validate assay performance specifically for each sample type

• Consider that different anticoagulants (EDTA, heparin, citrate) may affect measurements

Technical Recommendations:

• Minimize freeze-thaw cycles for all sample types

• Standardize collection procedures, including processing time and storage conditions

• For cell culture supernatants, standardize culture conditions and collection timepoints

• Consider the potential influence of medications or concurrent inflammatory conditions when interpreting clinical samples

The precision data from the search results demonstrate that validated assays can achieve both intra- and inter-assay CVs below 10% across sample types when proper procedures are followed .

What is the role of ENA-78 in rheumatoid arthritis and how has it been validated experimentally?

ENA-78 plays a significant role in rheumatoid arthritis (RA) pathogenesis, as evidenced by both human studies and animal models:

Expression in Human RA:
Human studies have demonstrated increased expression of ENA-78 in inflamed synovial tissue and fluid of RA patients compared to osteoarthritis patients . This increased expression correlates with neutrophil infiltration, a hallmark of RA.

Experimental Validation in Animal Models:
The adjuvant-induced arthritis (AIA) rat model has been instrumental in understanding ENA-78's role in arthritis. Key experimental findings include:

• Increased levels of ENA-78-like protein in sera of AIA animals by day 7 post-adjuvant injection, which continued to rise as arthritis developed

• Elevated ENA-78-like protein in joint homogenates by day 18 during maximal arthritis

• Correlation between ENA-78-like protein levels in both serum and joints with progression of joint inflammation

Intervention Studies:
Critical evidence for ENA-78's causal role came from antibody intervention experiments. Administration of anti-human ENA-78 antibodies before disease onset significantly modified the severity of AIA, providing evidence for its functional importance in disease initiation . Interestingly, administration after clinical onset did not modify the disease, suggesting ENA-78 may be more critical in the initiation phase rather than maintenance of established disease .

These findings collectively validate ENA-78 as an important chemokine in the progression and maintenance of inflammatory arthritis, positioning it as both a potential biomarker and therapeutic target in RA research.

How does ENA-78 contribute to cardiovascular disease and blood pressure regulation?

ENA-78/CXCL5 has emerging significance in cardiovascular pathophysiology, with genetic evidence suggesting a role in blood pressure regulation:

Genetic Association Studies:
Research has identified polymorphisms in the CXCL5 gene associated with variable blood pressure. A study examining the −156 G > C and 398 G > A CXCL5 polymorphisms found significant associations with blood pressure measurements . These polymorphisms were in high linkage disequilibrium (r² ranging from 0.51 to 1.0 depending on ethnicity) .

Blood Pressure Correlations:
The research demonstrated that genetic variations in CXCL5 were associated with measurable differences in systolic, diastolic, and pulse pressure. This association remained significant even after controlling for demographic factors and inflammatory markers .

Potential Mechanisms:
The involvement of ENA-78 in cardiovascular regulation likely relates to its inflammatory and angiogenic properties. As a neutrophil chemoattractant, it can influence vascular inflammation, while its angiogenic effects may impact vascular remodeling . Additionally, ENA-78 is expressed by vascular smooth muscle cells and adipocytes, suggesting direct effects on vascular function and potential links to metabolic factors influencing blood pressure .

Inflammatory Biomarker Correlations:
The studies examined correlations between ENA-78 levels, other inflammatory markers (like CRP), and blood pressure measurements. In the study population (n=192), median ENA-78 levels were 362 pg/mL (range 32.2-3970 pg/mL), providing reference data for researchers investigating cardiovascular correlations .

These findings suggest that genetic variations affecting ENA-78 expression or function may influence cardiovascular phenotypes, establishing this chemokine as a relevant factor in cardiovascular research.

What experimental approaches are most effective for studying ENA-78 function in vitro?

When designing in vitro studies of ENA-78 function, researchers should consider the following validated approaches:

Recombinant Protein Studies:
Utilizing high-quality recombinant human ENA-78 is essential for functional studies. E. coli-expressed recombinant human ENA-78 has been validated for experimental use . When designing experiments, researchers should consider:

• Using appropriate concentrations based on physiological levels (reference ranges from human studies: 362 pg/mL median with range 32.2-3970 pg/mL)

• Testing multiple isoforms, including the full-length protein and truncated variants (ENA-78, ENA-74, ENA-70) which may exhibit different potencies

• Including appropriate positive controls (e.g., other well-characterized chemokines)

Neutrophil Migration Assays:
As a potent neutrophil chemoattractant, migration assays represent a core functional assessment:

• Transwell migration chambers with isolated human neutrophils

• Real-time cell migration tracking systems

• 3D matrix models to simulate tissue environments

Cell Signaling Studies:
To investigate signaling pathways, researchers should focus on:

• CXCR2 receptor binding and activation assays

• Downstream signaling cascades using phosphorylation-specific antibodies

• Receptor antagonist studies to confirm specificity

Gene Expression Manipulation:

• Overexpression systems in relevant cell types (epithelial cells, endothelial cells, fibroblasts)

• RNA interference or CRISPR-based gene editing to modulate expression

• Reporter systems to monitor transcriptional regulation in response to stimuli like IL-1 or TNFα

Angiogenesis Models:
Given ENA-78's angiogenic properties, endothelial tube formation assays and endothelial cell proliferation assays are particularly relevant.

These approaches can be combined for comprehensive functional characterization of ENA-78 in controlled experimental settings.

What are the most appropriate animal models for studying ENA-78 in disease states?

When selecting animal models for ENA-78 research, several important considerations must be addressed:

Species-Specific Considerations:
An important limitation in ENA-78 research is that human CXCL5/ENA-78 does not have a true murine ortholog, despite earlier assumptions that murine LIX was equivalent . This species difference necessitates careful model selection and interpretation.

Validated Disease Models:

• Inflammatory Arthritis Models:

  • Adjuvant-induced arthritis (AIA) in rats has been validated for ENA-78 research, with documented increases in ENA-78-like protein that correlate with disease progression

  • This model allows for intervention studies, including antibody neutralization approaches that have demonstrated efficacy when administered before disease onset

• Cardiovascular Models:

  • Genetic association studies suggest that models examining blood pressure regulation may be appropriate

  • Models should account for the complex relationship between inflammation, ENA-78 expression, and cardiovascular parameters

Genetic Approaches:

• Transgenic models expressing human CXCL5 may provide more translatable results than relying on endogenous rodent chemokines

• CXCR2 receptor-modified animals may be useful for studying downstream effects

Experimental Design Considerations:

• Include appropriate timing of interventions, as the search results indicate that anti-ENA-78 intervention was effective before but not after disease onset in arthritis models

• Incorporate both tissue and systemic measurements of ENA-78, as the search results demonstrated different temporal patterns between serum and joint tissue levels in arthritis models

• Consider sex differences and genetic background effects, as human studies have shown variable associations with blood pressure across different demographic groups

Researchers should carefully document the limitations of their chosen model regarding the translatability to human ENA-78 biology.

How should researchers interpret variability in ENA-78 measurements in clinical studies?

Interpreting variability in ENA-78 measurements requires consideration of multiple factors:

Assay-Related Variability:
Based on the provided precision data, researchers should expect inherent variability within the following ranges:

• Intra-assay CVs: 3.8-8.3% (ELISA) or 6.5-7.8% (Simple Plex)

• Inter-assay CVs: 6.7-9.8% (ELISA) or 4.3-5.1% (Simple Plex)

Variability exceeding these validated ranges may indicate methodological issues requiring attention.

Biological and Clinical Variables:
Several factors contribute to biological variability that should be considered in interpretation:

• Demographic Factors:

  • Age and sex distributions should be carefully documented and controlled (reference population: mean age 39±12 years, 65% women)

  • Racial/ethnic differences in both ENA-78 levels and genetic polymorphisms (variant allele frequencies of −156 C allele: 14% in Caucasians, 45% in Blacks, 11% in non-Black Hispanics)

• Inflammatory Status:

  • Correlation with other inflammatory markers (CRP median: 1.78 mg/L, range 0.1-16.9 mg/L)

  • White blood cell count (reference: mean 6.3±2.0 ×10⁹ cells/L)

• Comorbidities and Medications:

  • Exclusion criteria used in reference studies included known CVD, CVD-risk equivalents, and anti-hypertensive medications

  • Response to inflammatory stimuli like IL-1 or TNFα should be considered

Statistical Approaches:

• Multiple regression analysis should adjust for relevant covariates (age, sex, smoking status, BMI, CRP concentration, WBC count)

• Race-by-genotype interaction terms should be considered in genetic association studies

• Power calculations should be conducted (reference: 80% power with two-sided α of 0.05 to detect a 6-mmHg difference in SBP, 4-mmHg difference in DBP)

The interpretation of ENA-78 measurements should acknowledge these sources of variability and control for them through appropriate study design and statistical analysis.

What are the best practices for comparing ENA-78 data across different experimental platforms?

When comparing ENA-78 data generated using different experimental platforms, researchers should implement the following best practices:

Cross-Platform Standardization:

• Reference Material Calibration:

  • Utilize common reference standards across platforms

  • Consider E. coli-expressed recombinant human ENA-78 as a reference standard, which has been validated in multiple assay systems

• Bridge Testing:

  • Run a subset of identical samples on multiple platforms to establish conversion factors

  • Develop statistical models to harmonize data when direct comparison is needed

Assay-Specific Considerations:
Based on the search results, researchers should be aware of platform-specific performance characteristics:

Methodological Documentation:

• Comprehensively document assay conditions, including antibody clones, detection methods, and sample preparation

• Report absolute concentrations rather than relative values when possible

• Always specify the exact isoform of ENA-78 being measured (full-length vs. truncated variants)

Data Integration Approaches:

• Meta-analytic techniques that account for assay-specific variability

• Z-score normalization within each platform before comparison

• Machine learning approaches for complex multi-platform data integration

Reporting Standards:

• Always report the dynamic range of the assay used

• Include quality control data with all reported results

• Clearly delineate between different sample types (serum vs. plasma vs. cell culture supernatants)

By implementing these practices, researchers can more confidently compare ENA-78 data across different experimental platforms while minimizing technical artifacts.

How can ENA-78 genetic polymorphisms be effectively studied in relation to disease risk and treatment response?

Studying ENA-78 genetic polymorphisms requires sophisticated methodological approaches:

Genotyping Methodologies:
The search results describe validated techniques for CXCL5 polymorphism analysis, including:

• PCR followed by pyrosequencing for reliable detection of variants such as −156 G > C and 398 G > A

• Verification of genotype frequencies by allele counting and assessment of Hardy-Weinberg equilibrium by chi-square analyses

Allelic Expression Analysis:
For functional validation of polymorphisms, the following approaches are recommended:

• Parallel pyrosequencing reactions of DNA and mRNA to detect allele expression imbalance

• Analysis of pooled results from multiple PCR amplification products to minimize cycle variability

• Quantitative assessment using pyrosequencing allele quantification algorithms

Study Design Considerations:
Based on the cardiovascular genetics study:

• Power calculations: 80% power with a two-sided α of 0.05 was sufficient to detect clinically relevant differences in blood pressure (6-mmHg difference in SBP, 4-mmHg difference in DBP)

• Sample size considerations: The reference study included 192 subjects, with genotyping successful in 188-189 individuals

• Appropriate exclusion criteria: Known CVD, CVD-risk equivalents, pregnancy, malignancy, substance abuse, and medications affecting WBC counts

Statistical Analysis Framework:

• Univariate analyses to identify potential covariates (age, sex, smoking status, BMI, inflammatory markers)

• Multiple regression with step-type selection methods to determine joint effects of CXCL5 genotypes and clinical variables

• Inclusion of race as a covariate and consideration of race-by-genotype interaction terms

• Analysis of linkage disequilibrium between polymorphisms (reference: r² for −156 G > C and 398 G > A was 0.82 in Caucasians, 1.0 in Blacks, and 0.51 in Hispanics)

These methodologies provide a robust framework for investigating how ENA-78 genetic variations influence disease susceptibility and treatment outcomes.

What are the emerging therapeutic approaches targeting ENA-78 in inflammatory diseases?

Research on therapeutic targeting of ENA-78 is evolving with several promising approaches:

Antibody-Based Approaches:
Neutralizing antibodies against ENA-78 have shown efficacy in experimental models:

• In adjuvant-induced arthritis, anti-ENA-78 antibodies administered before disease onset successfully modified disease severity

• Timing appears critical, as administration after clinical onset was less effective, suggesting a window of opportunity for intervention

• Human-ready antibody development would need to consider specificity for different ENA-78 isoforms (78aa, 74aa, 70aa forms)

Receptor Antagonism Strategies:
Since ENA-78 signals through CXCR2, targeting this receptor represents an alternative approach:

• Small molecule CXCR2 antagonists could inhibit ENA-78 signaling

• Receptor antagonists have the advantage of blocking multiple CXC chemokines that share this receptor

• Careful evaluation of side effects is necessary given the importance of CXCR2 in host defense

Gene Expression Modulation:
Understanding the transcriptional regulation of ENA-78 opens possibilities for upstream intervention:

• Inhibitors of IL-1 or TNFα pathways may indirectly reduce ENA-78 expression

• Targeting specific transcription factors regulating CXCL5 expression

• RNA-based therapeutics to modulate ENA-78 expression

Context-Specific Considerations:
The search results reveal disease-specific aspects relevant to therapeutic development:

• Rheumatoid Arthritis: Targeting the early inflammatory phase may be most effective based on animal models

• Cardiovascular Applications: Genetic polymorphism data suggest personalized approaches based on CXCL5 genotype might be considered

• Cancer: Given ENA-78's contribution to cancer progression , targeting in the tumor microenvironment represents another therapeutic avenue

Biomarker-Guided Therapy:
With validated assays for ENA-78 quantification , therapy could be guided by:

• Baseline ENA-78 levels to identify patients most likely to benefit

• Monitoring ENA-78 reduction as a pharmacodynamic marker of treatment effect

• Integration with other inflammatory markers for comprehensive assessment

These emerging approaches highlight the therapeutic potential of targeting the ENA-78 pathway in various inflammatory conditions.

What are the key ethical considerations when conducting human studies involving ENA-78 biomarker analysis?

Human studies involving ENA-78 biomarker analysis must address several ethical considerations:

Informed Consent Requirements:
The search results reference IRB-approved studies where all subjects provided written informed consent to specimen and data use in genetic association and related studies . Key consent elements should include:

• Clear explanation of sample collection procedures

• Explicit permission for genetic analysis if CXCL5 polymorphisms will be studied

• Options for future use of biological specimens

• Disclosure of potential incidental findings

Inclusion/Exclusion Criteria Justification:
Studies need ethically justifiable criteria:

• The reference cardiovascular study excluded subjects with known CVD, CVD-risk equivalents, pregnancy, malignancy, substance abuse, and those using medications affecting WBC counts

• Exclusion criteria should be scientifically necessary rather than merely convenient

• Efforts to ensure demographic diversity should be documented, especially given the reported ethnic variations in CXCL5 polymorphism frequencies

Privacy and Data Protection:
With genetic and biomarker data being particularly sensitive:

• De-identification protocols for samples and data

• Secure storage of genetic information

• Protection against re-identification risks

• Clear policies on data sharing and publication

Risk-Benefit Assessment:

• Minimal risks from standard blood collection should be balanced against scientific and clinical value

• Potential benefits to scientific understanding of disease mechanisms involving ENA-78

• Consideration of return of results policies, especially for genetic findings with potential clinical significance

Regulatory Compliance:

• Multi-institutional review board approvals for collaborative studies

• Compliance with genetic information protection regulations

• Adherence to biobanking standards for sample storage and use

These ethical considerations provide a framework for responsible conduct of ENA-78 biomarker research in human subjects.

What are the most promising areas for future ENA-78 research based on current knowledge gaps?

Based on the search results and current understanding, several high-priority research areas emerge:

Structural and Functional Characterization:

• Comparative analysis of different ENA-78 isoforms (78aa, 74aa, 70aa) to better understand their differential potency

• Crystal structure determination of ENA-78-CXCR2 complexes to inform targeted drug design

• Investigation of post-translational modifications that may affect function

Genetic and Epigenetic Regulation:

• Expanded studies of CXCL5 polymorphisms across diverse populations, building on existing data showing ethnic variation in allele frequencies

• Epigenetic regulation of CXCL5 expression in different disease states

• Functional characterization of regulatory elements controlling tissue-specific expression

Disease Mechanism Elucidation:

• Further investigation of the role of ENA-78 in early vs. established phases of inflammatory arthritis, following the observation that antibody intervention was only effective before disease onset

• Exploration of the mechanisms linking ENA-78 polymorphisms to blood pressure regulation

• Investigation of ENA-78's role in additional inflammatory disorders not yet well-characterized

Novel Therapeutic Approaches:

• Development of selective inhibitors targeting specific ENA-78 isoforms

• Investigation of combination approaches targeting multiple chemokines or their receptors

• Biomarker-guided therapeutic strategies based on ENA-78 levels or genetic profile

Methodological Advancements:

• Development of more sensitive and specific assays for different ENA-78 isoforms

• Standardization of reference ranges across diverse populations

• Novel imaging approaches to visualize ENA-78 activity in vivo

Translational Research:

• Longitudinal studies correlating ENA-78 levels with disease progression

• Clinical trials of ENA-78-targeted therapies in inflammatory conditions

• Personalized medicine approaches based on CXCL5 genotype

These research directions address critical knowledge gaps while building on the established understanding of ENA-78 biology and pathophysiology.

How might advances in single-cell technologies enhance our understanding of ENA-78 biology?

Single-cell technologies offer transformative potential for ENA-78 research:

Cellular Source Identification:
While we know ENA-78 is expressed by multiple cell types including hematopoietic cells, fibroblasts, endothelial cells, vascular smooth muscle cells, and adipocytes , single-cell RNA sequencing (scRNA-seq) can provide unprecedented resolution:

• Identification of specific cellular subpopulations responsible for peak ENA-78 production in disease states

• Temporal dynamics of expression during disease progression

• Co-expression patterns with other inflammatory mediators

Receptor-Ligand Interactions:
Single-cell technologies can map the CXCL5-CXCR2 signaling axis with precision:

• Quantification of receptor expression on target cell populations

• Correlation between receptor density and functional responses

• Competitive interactions with other CXCR2 ligands at the single-cell level

Spatial Transcriptomics Applications:
Beyond identifying cellular sources, understanding the spatial distribution of ENA-78 production is critical:

• Mapping of ENA-78 expression relative to tissue architecture in inflamed synovium

• Gradient formation visualization in inflammatory environments

• Co-localization with other inflammatory mediators and responding cells

Single-Cell Proteomics Integration:
Advancing beyond transcriptional analysis:

• Correlation between mRNA and protein expression at single-cell resolution

• Post-translational modification patterns across cell types

• Secretion dynamics using approaches like single-cell secretion profiling

Multi-omic Integration:
Combining genetic, transcriptomic, and proteomic data:

• Effects of CXCL5 polymorphisms on single-cell expression patterns

• Epigenetic regulation at single-cell resolution

• Metabolic correlates of ENA-78 production and response

Translational Applications:

• Patient stratification based on cellular ENA-78 expression patterns

• Prediction of therapeutic response using single-cell signatures

• Monitoring of treatment effects at cellular resolution

These applications of single-cell technologies promise to revolutionize our understanding of ENA-78 biology by providing unprecedented resolution of its cellular sources, targets, and regulatory mechanisms in health and disease.