Recombinant Macaca fascicularis Tumor necrosis factor (TNF), partial

What is TNF-alpha and what is its role in the Macaca fascicularis immune system?

Tumor necrosis factor alpha (TNF-alpha), also known as cachectin and TNFSF2, serves as a pleiotropic molecule that plays a central role in inflammation and immune system regulation in cynomolgus macaques. TNF-alpha functions as the prototypic ligand of the TNF superfamily and is critical for orchestrating immune responses to pathogens. In Macaca fascicularis, TNF-alpha is involved in granuloma formation and maintenance during tuberculosis infection, with studies showing abundant TNF-alpha mRNA expression localized to granulomatous lesions . The cytokine works synergistically with IFN-gamma to coordinate immune responses, participating in the recruitment of immune cells to infection sites and helping establish protective granuloma structures .

How are recombinant macaque TNF preparations typically produced for research applications?

Recombinant TNF from macaques is typically produced in bacterial expression systems, particularly E. coli. In the case of rhesus macaque TNF-alpha (which shares high sequence homology with cynomolgus macaque TNF), the recombinant protein represents the Val77-Leu233 portion of the full protein sequence . The expression in E. coli systems allows for cost-effective production of significant quantities of the cytokine for research purposes . Following expression, the protein undergoes purification and is typically supplied as a lyophilized preparation filtered through 0.2 μm filtration systems to ensure sterility and purity .

What formulations of recombinant macaque TNF are available and how should they be reconstituted?

Recombinant macaque TNF is typically available in two primary formulations:

Both formulations require careful handling during reconstitution to maintain bioactivity . The carrier protein (typically BSA) enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at more dilute concentrations .

What are the optimal storage conditions for maintaining stability of recombinant macaque TNF?

For optimal preservation of recombinant macaque TNF activity, researchers should employ a manual defrost freezer and meticulously avoid repeated freeze-thaw cycles that can degrade protein structure and function . The reconstituted protein should be aliquoted before freezing to minimize the need for repeated thawing of the entire stock . While the product is typically shipped at ambient temperature, it should be stored according to manufacturer recommendations immediately upon receipt to maintain stability . For short-term storage during experimental procedures, the reconstituted protein should be kept on ice or at 4°C to minimize degradation.

How can researchers verify the bioactivity of recombinant macaque TNF preparations?

The biological activity of recombinant macaque TNF can be validated through functional cytotoxicity assays. The standard approach involves measuring the cytotoxic effect on L929 mouse fibroblast cells in the presence of actinomycin D, with the ED50 for this effect typically in the range of 15-60 pg/mL for rhesus macaque TNF-alpha . Researchers should establish a dose-response curve to confirm that their preparations fall within this expected range of activity. Additionally, protein integrity can be verified through techniques such as SDS-PAGE and Western blotting prior to experimental use.

What techniques are most effective for measuring TNF-alpha expression in Macaca fascicularis tissues?

In situ hybridization (ISH) has proven highly effective for measuring TNF-alpha mRNA expression levels directly in tissue samples from Macaca fascicularis . This technique allows researchers to:

• Visualize the spatial distribution of TNF-alpha expression within tissue structures

• Correlate expression with pathological features (e.g., granuloma formation)

• Compare expression levels between different tissue regions

• Assess co-expression with other cytokines or chemokines

In tuberculosis research, ISH has been successfully employed to examine TNF-alpha expression in over 300 granulomas from experimentally infected cynomolgus macaques, revealing that TNF-alpha mRNA+ cells were abundant and predominantly localized to granulomatous lesions .

How does TNF-alpha expression in Macaca fascicularis relate to granuloma characteristics in tuberculosis models?

Research in cynomolgus macaques experimentally infected with Mycobacterium tuberculosis has revealed critical patterns of TNF-alpha expression in relation to granuloma development and characteristics . TNF-alpha mRNA+ cells are abundant within both solid and caseous granulomas, with minimal expression in non-granulomatous regions of lung tissue . The expression pattern closely correlates with the presence of mycobacterial 16S rRNA, suggesting that the local presence of mycobacteria partially drives TNF-alpha upregulation . Notably, some granulomatous lesions show exceptionally high levels of both mycobacterial 16S rRNA and TNF-alpha mRNA, indicating a direct relationship between pathogen burden and cytokine expression .

How does TNF-alpha function in concert with other immune mediators in Macaca fascicularis infection models?

In cynomolgus macaque infection models, TNF-alpha operates as part of a coordinated network of cytokines and chemokines . Studies have demonstrated that TNF-alpha expression patterns closely mirror those of IFN-gamma, with both cytokines abundantly expressed within granulomatous lesions . Additionally, IFN-gamma-inducible chemokines (CXCL9/monokine induced by IFN-gamma, CXCL10/IFN-gamma-inducible protein, and CXCL11/IFN-gamma-inducible T-cell alpha-chemoattractant) show similar expression patterns within these structures . This coordinated expression suggests a synergistic role in recruiting CXCR3+ cells to infection sites, which were also found to be abundant in granulomatous lesions . The interaction between these immune mediators appears essential for effective granuloma formation and maintenance during mycobacterial infection.

What safety considerations should researchers be aware of when using recombinant cytokines in Macaca fascicularis studies?

While the search results don't provide specific safety data for recombinant TNF-alpha in cynomolgus macaques, insights can be gained from studies with other recombinant cytokines. Research with recombinant IL-21 in cynomolgus monkeys revealed several important safety considerations that may be relevant :

• Hematological effects: Cyclical anemia and thrombocytopenia were observed

• Hepatic responses: Leukocyte infiltrates in hepatic sinusoids and sporadic serum transaminase elevations (typically <3 times upper limit of normal)

• Acute-phase responses: Clinical pathology changes consistent with inflammatory stimulation

• Reversibility: All adverse effects were reversible upon cessation of treatment

• Antibody development: Anti-cytokine antibodies developed in a dose-dependent manner, affecting long-term studies

Researchers should conduct appropriate dose-finding studies and monitor for similar effects when working with recombinant TNF-alpha in macaque models .

How should researchers address the development of anti-cytokine antibodies in macaque studies?

The development of anti-cytokine antibodies represents a significant challenge in longitudinal studies using recombinant proteins in non-human primates. Based on experiences with recombinant IL-21 in cynomolgus macaques, researchers should implement several strategies :

• Regular monitoring: Implement scheduled screening for anti-cytokine antibodies throughout the study duration

• Dose optimization: Consider that antibody development may be dose-dependent, with higher doses potentially increasing immunogenicity

• Individual analysis: Account for significant individual variation in antibody responses among animals

• Pharmacodynamic assessment: Monitor for decreased biological responses over time, which may indicate neutralizing antibody development

• Study design adaptation: Consider the potential limitations for long-term studies, possibly implementing shorter experimental timelines

The appearance of anti-cytokine antibodies can significantly impact experimental outcomes by neutralizing the recombinant protein and altering its intended biological effects .

What are the key considerations for comparing TNF-alpha data across different Macaca fascicularis studies?

When comparing TNF-alpha data from different Macaca fascicularis studies, researchers should carefully consider several methodological and biological factors:

• Detection methods: Different techniques (e.g., ISH, ELISA, flow cytometry) may yield varying measurements of TNF-alpha levels

• Infection models: Differences in pathogen strains, inoculation routes, and doses can significantly impact TNF-alpha responses

• Sampling timepoints: The dynamic nature of cytokine responses means that sampling at different disease stages will yield different results

• Individual variation: Significant inter-animal variability exists in cytokine responses, as observed in studies with recombinant IL-21

• Genetic background: Differences in genetic backgrounds between macaque colonies could influence immune responses

• Environmental factors: Housing conditions, microbiome composition, and previous exposures may modify baseline and induced TNF-alpha levels

Standardization of protocols and comprehensive reporting of methodological details can help improve comparability between studies.

How can researchers optimize experimental design when using recombinant macaque TNF in functional studies?

For optimal experimental design using recombinant macaque TNF in functional studies, researchers should:

• Include carrier controls: When using carrier-containing TNF preparations, include appropriate controls with the carrier protein alone

• Establish dose-response relationships: Determine the effective dose range for specific biological readouts in your experimental system

• Include species-specific controls: When possible, compare with human TNF to assess species-specific differences in biological activity

• Implement time-course analyses: TNF-alpha often elicits time-dependent responses that may peak and resolve at different timepoints

• Consider receptor expression: Verify expression of TNF receptors on target cells to ensure they can respond to the cytokine

• Account for endogenous production: Design experiments to distinguish between effects of exogenous recombinant TNF and endogenous TNF induced in your experimental system

These considerations help ensure robust, interpretable results when using recombinant macaque TNF in research applications.

How should researchers interpret observed differences in TNF-alpha expression between individual macaques?

When interpreting differences in TNF-alpha expression between individual macaques, researchers should consider several biological and experimental factors:

• Genetic polymorphisms: Individual genetic differences may affect TNF production, receptor expression, or signaling pathways

• Prior immune exposures: Previous pathogen exposures can condition the immune system and alter cytokine responses

• Disease progression stages: Animals at different stages of infection may show varying TNF-alpha expression patterns

• Antibody development: As observed with recombinant IL-21, macaques can develop anti-cytokine antibodies that vary between individuals and affect biological responses

• Microbiome composition: Differences in gut microbiota can influence systemic immune responses, including TNF-alpha production

• Age and sex: These biological variables can impact immune function and cytokine expression

Statistical approaches should account for this individual variation, potentially employing repeated measures designs and mixed-effects models that can accommodate individual-level random effects.

What are the implications of TNF-alpha localization patterns for understanding disease pathogenesis?

The distinct localization patterns of TNF-alpha expression in tissues have important implications for understanding disease pathogenesis, particularly in infectious diseases . In tuberculosis models, the finding that TNF-alpha mRNA expression is predominantly localized to granulomas, with minimal expression in non-granulomatous regions, suggests that TNF-alpha production is tightly regulated and spatially restricted to sites of active immune response . This localization pattern, coupled with the observation that high levels of TNF-alpha expression correlate with high levels of mycobacterial 16S rRNA, indicates that TNF-alpha is produced in direct response to local pathogen presence .

The co-localization of TNF-alpha with IFN-gamma and IFN-gamma-inducible chemokines suggests a coordinated cytokine response that orchestrates the recruitment and activation of immune cells within granulomas . This spatial organization of cytokine production appears essential for containing infections and preventing dissemination, highlighting the importance of studying not just the levels of TNF-alpha but also its spatial distribution within affected tissues.