title: Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock.
abstract: BACKGROUND
  In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab')₂ fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy. Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured.
  
  
  RESULTS
  Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab')₂ antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose₁₀₀ (LD₁₀₀) LPS administration. TNF blockade with either VNAR domains or F(ab')₂ fragments were associated with lower mortality (60% and 75%, respectively) compared to LD₁₀₀. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS. In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab')₂ fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group.
  
  
  CONCLUSIONS
  Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.
authors.affiliation: Department of Immunology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico.
authors.forename: Rafael
authors.initials: R
authors.lastname: Bojalil
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Mata-González
authors.forename: Fausto
authors.initials: F
authors.lastname: Sánchez-Muñoz
authors.forename: Yepci
authors.initials: Y
authors.lastname: Yee
authors.forename: Iván
authors.initials: I
authors.lastname: Argueta
authors.forename: Lucía
authors.initials: L
authors.lastname: Bolaños
authors.forename: Luis Manuel
authors.initials: LM
authors.lastname: Amezcua-Guerra
authors.forename: Tanya Amanda
authors.initials: TA
authors.lastname: Camacho-Villegas
authors.forename: Edna
authors.initials: E
authors.lastname: Sánchez-Castrejón
authors.forename: Walter Jakob
authors.initials: WJ
authors.lastname: García-Ubbelohde
authors.forename: Alexei Fedorovish
authors.initials: AF
authors.lastname: Licea-Navarro
authors.forename: Ricardo
authors.initials: R
authors.lastname: Márquez-Velasco
authors.forename: Jorge Fernando
authors.initials: JF
authors.lastname: Paniagua-Solís
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000097:blood; D004195:Disease Models, Animal; D006801:Humans; D007249:Inflammation / Q000097:blood / Q000150:complications* / Q000188:drug therapy* / Q000473:pathology; D016753:Interleukin-10 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009566:Nitrates / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012772:Shock, Septic / Q000097:blood / Q000150:complications* / Q000188:drug therapy*; D061905:Single-Domain Antibodies / Q000627:therapeutic use*; D016019:Survival Analysis; D016896:Treatment Outcome; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D015415:Biomarkers; D015850:Interleukin-6; D008070:Lipopolysaccharides; D009566:Nitrates; D012333:RNA, Messenger; D061905:Single-Domain Antibodies; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D052247:Nitric Oxide Synthase Type II
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