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.
Subject(s)
Inflammation/complications , Inflammation/drug therapy , Shock, Septic/complications , Shock, Septic/drug therapy , Single-Domain Antibodies/therapeutic use , Tumor Necrosis Factor-alpha/immunology , Animals , Biomarkers/blood , Disease Models, Animal , Humans , Inflammation/blood , Inflammation/pathology , Interleukin-10/metabolism , Interleukin-6/metabolism , Lipopolysaccharides , Liver/metabolism , Male , Mice , Mice, Inbred BALB C , Nitrates/metabolism , Nitric Oxide Synthase Type II/metabolism , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , Shock, Septic/blood , Survival Analysis , Treatment OutcomeABSTRACT
An immunoenzymatic test (DIG-ELISA) was serologically evaluated for the serodiagnosis of onchocerciasis. Control and infected sera from the onchocerciasis endemic area of Mexico was collected and the donors assessed for onchocerciasis according to parasitological, clinical, and epidemiological data. The sera were submitted to the DIG-ELISA test using a crude extract prepared from O. volvulus adults woems which had been preserved in nodules in 67 percent glycerol. The test showed a 100 percent sensitivity with sera from 38 microfilariae carriers and 96 percent specificity with sera from 133 non-infected people living outside the endemic zone. In addition, seropositivity was 52.9 percent with samples from nononchocercotic people living indide the endemic area, while 82.5 and 90.5 percent of sera from patients with clinical symptoms and subcutaneous nodules, respectively, were positive. A high rate (30 percent) of cross-reactivity with serum samples from people infected with Wuchereria bancrofti or Brugia timori was obtained, which is in contrast with the low seropositivity rates (7.4 percent) obtained with sera from patients infected with other parasites. These results suggest that DIG-ELISA test may be a useful serological test for antibody detection in onchocerciasis, especially for epidemiological surveillance of disease, but because of the high cross-reactivity observed, its use must be limited to areas where this parasitic infection does not coexist with other human filiariasis. Finally, the arrangement of sera in groups according to the relative likelihood to have onchocerciasis seems to be a useful procedure for evaluation of serologicales tests
