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Lona Jawaheer, Pankaj K Agarwal, Deepa Anijeet, Alan P Rotchford, Alastair Gracie, Lik Thai Lim, Alan Cameron, Kanna Ramaesh; Absorption of pro-inflammatory cytokines interleukin 6 and tumor necrosis factor α by amniotic membrane. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4710.
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To study the interaction of amniotic membrane and pro-inflammatory cytokines interleukin 6 (IL6) and tumor necrosis factor alpha (TNFα), in both fresh and preserved states.
Amniotic membrane grafts were obtained from placenta following caesarean section delivery from healthy mothers who had been consented prior to the operation. Sixteen square-inch pieces of amniotic membrane were prepared and frozen at -20oC in a 50/50 mixture of Glycerol/Roswell Park Memorial Institute medium supplemented with 10% fetal calf serum, penicillin-streptomycin and L-Glutamine. Eight pieces of amniotic membrane were stored for 1 week and eight for 3 months. After the respective storage times, they were defrosted and incubated in a multiwell plate for 24 hours in 5% CO2 at 37 degrees Celsius with 1000pg/mL, 500pg/mL, 250pg/mL and 125pg/mL of recombinant human TNFα or IL6. Corresponding wells without tissue were set up as controls. After 24 hours, we measured the residual concentration of the cytokines in the supernatant solutions by using an Enzyme-Linked Immunosorbent Assay.
There was a significant reduction in the concentration of both IL6 and TNFα with fresh and preserved samples of amniotic membrane, when compared to controls. For IL6, there was a mean decrease of 70.6% in the amniotic membrane group compared with 56.2% in the control group (p<0.05). For TNFα, there was a mean decrease of 78.4% in the amniotic membrane group compared with 36.8% in the control group (p<0.05). There was no statistically significant difference in this reduction between fresh and preserved samples of amniotic membrane.
Both fresh and preserved amniotic membrane grafts absorb IL6 and TNFα, which are known to be potent pro-inflammatory cytokines involved in ocular surface disease. Other studies have previously shown that amniotic membrane reduces inflammation through the sequestration of inflammatory cells and through reduction in the expression of pro-inflammatory proteins. The anti-inflammatory effect of amniotic membrane is probably multi-factorial and further studies are required to investigate the mechanisms thereof.
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