The Study of Immunological And Cytogenetic Effects of Polyvinyl Alcohol

This st udy has be en des igned t o investigate th e t oxicity effects of polyvinyl alcohol at different concentrations on peri pheral blood l ymphocytes by measuring each of blastogenic index, mitotic index and chromosomal aberration in addition to th e polymo rph nuclear leukoc ytes activity through determine both of t he phagocytosis percent and phagocytosis index .The results show that PVA have no toxic effects at t he co ncentrations 0.1,1,10,100,250 µg/ml be cause t he cytogenetic parameters undergo not significantly raised (at P < 0.05) a nd phagocytosis percent and phagocytosis index have been increased significantly (at P ≤ 0.05). The immune response has been found to be significantly too ( P ≤ 0.05) i n the presence of PVA wit h Candida alb icans antigen as adjuvant via increasing t he fo ot pad sw elling i n the imm unized mice wit h 50 µg/ml of C.albicans antigen.


Introduction
Adjuvants have been used for more than 70 years to enhance the immune response of the host animal to an antigen [1].Adjuvants are substances injected along with an antigen that are intended to enhance humeral and cell mediated immune response to the antigen [2, 3, and 4].Adjuvants generally permit the use of smaller antigen dose and may modulate the immune response to the antigen, more than 100 Adjuvant preparation have been described [5].The mechanisms by which adjuvants promote the immune response are depot effect, antigen presentation effect, an antigen distribution effect and CTL induction effect [6] experimentally we can provide a controlled release formulation comprising biodegradable polymer microspheres where in a vaccine suspended in a polymer matrix.Poly vinyl alcohol powder is white in color, have different viscosity character Amorphous density at 25 o C: 1.26 g/cm3, crystalline density at 25 o C: 1.35 g/cm 3 , molecular weight of repeat unit: 44.00 g/mol.PVA water soluble in the aqueous solution are colloidal and compatible with lower alcohol the PVA is insoluble in petroleum solvents, use in the plastic industry, surface coating ,film resistance to gasoline ,artificial sponges , printing inks, pharmaceutical ,and cosmetics products [7].The diameter of PVA fibers above respiable limits and most of them are not inhalable, have a lower density as mineral fibers ranging (10-16) µm in diameter and they don't fibrillate [8].

Experiment Materials and methods:
1-candida albicans antigen preparation : Fresh growth of C. albicans was suspended with 100 ml of extract solution (Na H2PO4 0.37, Na 2HPO4 1.42, NaCl 2.5, phenol 4) gm / L following to [9] and using the method [10] for determination the total protein.

2-biodegradable polymer micro spheres preparation :
Two highly water soluble polymer micro spheres were used (PVA and starch) in ratio 20:80 % the aqueous solution of (PVA and starch) added to the C. albicans antigen the aqueous phase mixed with an emulsifying medium (glycerol) the mixture (antigen and adjuvant) homogenized by placed together in beaker and the two were emulsified, through use of a syringe only by pulling the material back forth rapidly the homogenized micro droplet suspension slowly to absolute methanol with stirring the micro droplet thus causing micro spheres to precipitate ,slowly evaporating the solvent leaving behind micro spheres [11].

3-chromosomal analysis:
Five concentrations of PVA (0.1, 1, 10,100 and 250) µg/ ml were used to determine the genotoxicity effect of PVA on blood lymphocytes according to [12].

4-phagocytosis assay:
five ml of venous blood were obtained by heparenized syringe and then divided into five tubes 1ml/ each, 5 concentrations of PVA with phosphate buffer saline (0.1, 1, 10,100,250µg/ ml) were added to single tube .All tubes have been PDF created with pdfFactory Pro trial version www.pdffactory.comincubated for one hour with 100 µl of Candida suspension (1×10 3 cell/ml) at 37º C. The mixture centrifuged (3000RPM) for five minutes .One drop of the mixture (blood,PVA,and Candida suspension) was speared on slide ,air dried and fixed with absolute methanol and stained with Giemsa stain, ,washed with PBS and the percentage of phagocytosis was calculated using the following equation .

5-DTH and Arthur reaction:
Nine albino mice were immunized with biodegradable microspheres (adjuvant and C. albicans antigens) for three weeks.50 µl of C. albicans antigens was injected in the right foot bad while the left foot pad was injected with 50 µl of PBS.The swilling of foot pad was detected by using Vernea [13].

6-Toxicity assay:
To detect the effect of PVA on poly morph nuclear leukocytes 100 µl of (0.1, 1, 10,100,250µg/ml) of PVA were mixed with 100µl of whole blood incubated for one hour at 37º C.100 µl of mixture and 100 µl of trypan blue dye (0.2gm of stain with 100 ml normal slain) at 37º C for 3 minutes and the viability percentage was calculated by the following equation: Viability percentage % =

Results and Discussion 1-chromosomal analysis:
The

Arthur and delayed type hypersensitivity assay:
The immediate and delayed type hypersensitivity has been raised in the immunized mice with Candida antigens conjugated with 10 and 100 µg/ml of PVA (significantly at P≤0.05 level).Because the presence of PVA gave the antigen complexity and high molecular weight, this lead to slow releasing of an antigen.Foot pad swelling of immunized mice increase according to the concentration of the PVA as compare with the negative control (PBS only) and the positive control (C.albicans antigen only) table (3).

Toxicity assay:
To determine the hagocytosis assay on the PMN leukocytes the viability must be ranging between 96-100 % [15]

table (
As will as the studies of [14] on bacteria Escherichia coli and Salmonella typhimurium observed that PVA compounds were not genotoxic in a range of in vivo and in vitro studies.PVA fibres, as manufactured, are above the respirable limit, and most of them are not inhalable.Theonly study on lung cancer risk in workers exposed to PVA fibres did not show positive results, PVA itself is not genotoxic[14].
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Table ( 1) chromosomal analysis of peripheral blood lymphocytes exposed to Graduated concentrations of PVA Table (2) Phagocytic index and phagocytic percent of polymorph nuclear leukocytes exposed to graduated concentrations of PVA Chromosomal aberration Mitotic index Blastogenic index
. The PVA have low toxicity on the PMN table (4) because the PVA is non toxic as it self in the human, animals, and microorganisms [13].PDF created with pdfFactory Pro trial version www.pdffactory.com PDF created with pdfFactory Pro trial version www.pdffactory.com

Table ( 3) Arthus and DTH assay in mice treated with different concentrations of PVA and C. albicans antigen Type of treatment NO. of samples Arthus reaction Foot pad swilling in millimeter DTH assay Foot pad swilling in millimeter
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