Abstract
In this study, specific and nonspecific activity of calf thymus DNA and Dextran-Sulfate (DS)-containing synthetic carbonic adsorbents (0.7–7 mg of each ligands per 1 cm3 of activated carbonic beads, 0.3–0.6 mm diameter, bulk density γ = 0.1–0.2 g/cm3) have been compared in stir-bath and micro-column in vitro tests. DS coating as well as DNA coating does not demonstrate deep influence on the unspecific adsorptive activity of carbonic matrix toward creatinine, vitamin B12, and unconjugated bilirubin. No essential difference has been found in the specific activity of DNA and DS containing adsorbents toward anti-ds- and anti-ss-DNA-antibodies, as well as antibodies against DNA-protein complexes (anti-DNP-antibodies): in both cases the percentage of decrease of appropriate antibody concentration varied between 35 and 51% for single-pass microcolumn experiments with moderate enhancement of extraction efficacy (up to 60–75%) due to additional recirculation (2 h) or preliminary plasma dilution in 2–5 times. In the micro-column experiments with the proinflammatory cytokines DNA or DS-coating did not diminish TNF-α, IL-1β, and IL-6 adsorption from 3% BSA solution, but even improves to some extent its removal compared with uncoated matrix.
Introduction
Deoxyribonucleic acid-coated carbonic hemosorbents, consisting of porous carbonic matrix coated and thin layers of native calf thymus DNA, are known as one of the first clinical immunoadsorbents for whole blood purification (Snezhkova and Nikolaev, [Citation1994]; Snezhkova et al., [Citation1987]). The combination of unspecific adsorptive properties of activated carbonic matrix with the specific adsorption of DNA-coupling substances demonstrates a number of positive clinical effects of the usage of such hemoimmunoadsorbents in the treatment of exacerbations of severe psoriasis and, especially, psoriatic arthropathy, bronchial asthma, lupus nephritis, etc. (Amosova et al., [Citation1997]; Beloglasov et al., [Citation1998]; Snezhkova et al., [Citation1992]; Zakharash et al., [Citation2003]). Recently, the new type of high-porous carbonic adsorbents having specific internal surface up to 5000 m2 per 1 g was synthesized in the form of light beads of the regular spherical shape with pronounced mass-fractal organization of its internal dimension. These adsorptive materials were successfully used for extraction of the most typical protein-bound toxins from blood plasma of patients with hepatic and renal insufficiency (Nikolaev et al., [Citation2003]; Sarnatskaya et al., [Citation2002], [Citation2003]). At the same time, different synthetic polymers have been studied for removal of cationic anti-DNA antibodies from blood plasma (Letourneur and Jozefowich, [Citation1992]; Migonney et al., [Citation1997]; Suzuki et al., [Citation1994]). The plasmaperfusion through dextran-sulfate containing column was found effective for removal of high affinity anti-DNA-antibodies and is included now to the schedules of some immunodependent diseases treatment (Funauchi et al., [Citation1996]; Kutsuki et al., [Citation1998]). Recently, a well-known DNA-coupling substances HMG-1 nonhystonic chromosomal protein was rediscovered as an important regulator of cytokines cascade, responsible particularly for the late endotoxin lethality (Wang et al., [Citation1999]). It means that DNA or DS-coated high-porosity carbonic adsorbents should be regarded as a possible component of extracorporeal treatment of septic shock and multi-organ failure, which includes severe hepatic and renal failure as well as so-called “cytokine storm” (Michel and Glauser, [Citation2000]). Still, the influence of the DNA or DS-coating on the sorption of main proinflammatory cytotoxins TNF-α, IL-1β, IL-6, essentially responsible for clinical picture of bacterial shock, remains unknown. The goal of this study is to investigate the specific and unspecific activities of DNA or DS-coated high-porosity carbons as well as its adsorptive activity towards proinflammatory cytokines.
Materials and Methods
The combined method of ligands grafting, included UV-irradiation was used for immobilization of native calf thymus DNA and dextran-sulfate onto mass-fractal spherical carbonic beads (dia 0.3–0.6 mm) with the different level of porosity, produced by regulated pyrolysis of synthetic resins. Bulk density of carbonic matrix varied from 0.06 to 0.2 g/cm3, pore volume by benzene—from 1.85 to 3.04 cm3/g and the amount of immobilized ligand—from 0.7 to 7.0 mg per 1 cm3 of beads. In cytokines micro-column experiments, conventional medical charcoal for hemoperfusion (Adsorba 300C), manufactured by Gambro (Sweden), was also tested. Unspecific adsorptive properties of DNA and DS-containing carbonic materials were studied in the stir-bath experiments, using creatinine solution in phosphate buffer (concentration—0.3 mg/mL), the water solution of vitamin B12 (concentration—1 mg/mL), and the unconjugated bilirubin (concentration—0.2 mg/mL in 3% solution of human serum albumin). Modified ELISA method (Zakharash et al., [Citation2003]), including laboratory preparation and individual calibration of ELISA plates, together with nonlinear mathematical approximation of obtained data, was used for more accurate measurement of anti-ss-DNA, anti-ds-DNA, and anti-DNP antibodies’ concentration in the pool of plasma of patients with Sjogren's syndrome, before and after single-pass and additional recirculating perfusion of this plasma through the adsorbent-containing micro-column (column volume—2.5 cm3; column length—45 mm; perfusion rate—0.2 mL/min; plasma volume—12 mL). The plasma dilution in 2 and 5 folds (with an appropriate enhancement of perfusion rate) was also tested to obtain more deep plasma purification (Zakharash et al., [Citation2003]). Proinflammatory cytokines TNF-a, IL-1b, IL-6 (concentration 1000 pg/mL) adsorption from 3% BSA was studied with the same type of micro-column experiments using standard ELISA kits from Diaclon, France.
Results
A. Unspecific Activity
In stir-bath experiments (), small influence of intensive DNA or DS coating was found on nonspecific adsorptive activity of carbonic matrix toward free-diluted (creatinine, m.m.—114 a.u.m.; vitamin B12, m.m.—1240 a.u.m.) and protein-bound (bilirubin, m.m.—585 a.u.m.) markers, especially for high porosity (VS = 3.04 cm3/g) adsorbent sample.
Table 1. The influence of massive (7 mg/cm3) coating of mass-fractal carbonic matrices with DNA or DS on standard adsorption of free-diluted (creatinine, vitamin B12) and protein-bound (bilirubun) substances (n = 6)
B. Specific Activity
Following , no practical differences exist between the specific affinity of equally coated (0.7 mg per cm3 DNA or DS) “mass-fractal” carbonic adsorbents toward 3 main groups of plasma's DNA-coupling substances excluding moderate superiority of DS-coating in the case of anti-ss-DNA and anti-DNP-antibodies adsorption. For both ligands, prolonged recirculation or 2–5 folds plasma dilution tends to improve the purification process quality. Unspecific adsorption of anti-DNA-substances with uncoated carbonic matrix normally does not exceeded 10% of its initial concentration, correlating with overall adsorption of total protein from plasma.
Table 2. Differences in concentration (Δ, % of initial level) of main groups of DNA-coupling substances in whole and diluted blood plasma after its perfusion through microcolumn containing mass-fractal adsorbents (γ = 0.1 g/cm3) covered with DNA or DS (0.7 mg of ligand per 1 g carbonic matrix) (n = 6)
C. Proinflammatory Cytokines Adsorption
shows that Adsorba 300C activated charcoals exhibiting slight affinity to TNF-α was ineffective for IL-1β and IL-6 removal. In contrast, “mass-fractal” carbonic adsorbent removed 93–97% of all 3 cytokines in single-pass micro-column experiments. DNA-coating as well as DS-coating (1 mg of each ligand per 1 sm3 of carbonic beads) do not decrease the adsorption of the tested cytokines, but improve the removal in comparison with uncoated matrix.
Table 3. Residual concentrations of proinflammatory cytokines (initial is 1000 pg/mL of 3% BSA solution) after its contact with uncoated, DNA or DS-coated carbonic adsorbents (C.A.) or Adsorba 300C charcoal in single-pass microcolumn experiments
Discussion
As was mentioned above, DNA or DS-coating is necessary for removal from blood plasma such practically important DNA-trophic immunoreactants as anti-DNA-antibodies and HMG-1 protein. At the same time, adsorptive capacity of carbonic matrix toward free diluted and, especially, protein-bound waste metabolites should be maximally preserved despite the coating. From this point of view, massive coating of highly activated carbonic beads with DNA and DS has minimal effects on the adsorbent capacity (). This could be explained by the “semicoating mechanism” where wide inlet openings of transport pores on the external surface of carbonic beads remain uncovered with polymer film (Chang, [Citation1978]). This effect isn’t very important for small, free, diluted toxic molecules like creatinine, demonstrating sufficient removal rate via conventional hemodialysis, but is more essential for large molecules, and, especially, for protein-bound toxins with the high association constant (108 mol–1 for unconjugated bilirubin, for example), which practically can’t pass through a dialyzing membrane. According to , DNA and DS-coated high-porous carbonic adsorbents demonstrate approximately equal affinity towards antibodies to native (ds) and denaturated (ss) DNA and DNA-protein complexes (DNP), which are of the same order of magnitude as was shown for DNA-containing hemoimmunoadsorbents on the base of conventional synthetic carbonic matrix (Snezhkova and Nikolaev, [Citation1994]; Snezhkova et al., [Citation1987]). Theoretically, DS-coating is a matter of choice because it is free from any biocontamination risks. High-porosity mass-fractal carbonic adsorbents are rather more effective for the removal of TNF-α, IL-1β, and IL-6 than conventional activated charcoal from an Adsorba 300C device, widely used now clinically for extracorporeal blood purification. At the same time, DNA or DS coating of mass-fractal carbonic matrix doesn’t lead to decrease, but even stimulates to some extent adsorption of TNF-α, IL-1β, and IL-6 cytokines, which have been never mentioned before as DNA-trophic substances.
Conclusions
High-porosity synthetic carbonic adsorbents with mass-fractal internal structure can be used as support for native DNA and Dextran-sulfate immobilization.
Native DNA and Dextran-sulfate covering don’t inhibit deeply unspecific adsorptive potential of mass-fractal carbonic matrix toward free-diluted and protein-bound substances.
Both combined adsorbents exhibit sufficient and approximately equal potential to remove the main groups of DNA-coupling antibodies from blood plasma.
DS or DNA coating does not attenuate, but even enhances the adsorptive potential of mass-fractal carbonic matrix toward the main proinflammatory cytokines.
DNA and DS-containing mass-fractal carbonic adsorbent look to be new, prospective biomaterial for blood and plasma purification, particularly for removal of some humoral mediators of septic shock and protein-bound toxic metabolites, typical for hepatic and renal insufficiency.
Acknowledgments
This work was supported in part by Research Grant INTAS-2001-0346.
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