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Effect Of Colchicine On Cellular And Humoral Immune Responses In Mice

By: Shahzada Khurram Syed (2007-VA-444) | Dr. Aqeel Javeed.
Contributor(s): Prof. Dr. Muhammad Ashraf | Dr. Jawad Nazir | Dr. Shahbaz Yousaf.
Material type: materialTypeLabelBookPublisher: 2016Description: 96p.Subject(s): Pharmacology and Toxicology | Phd thesesDDC classification: 2650-T Dissertation note: Colchicine is a medication that treats gout. It is a natural product and secondary metabolite, originally extracted from plants Colchicum autumnale .It causes modulation of chemokine and prostanoid production and inhibition of neutrophil and endothelial cell adhesion molecules by which it interferes with the initiation and amplification of the joint inflammation. The present study is designed to evaluate the effects of colchicine on cellular and humoral immunity in mice. There were five groups for each assay i.e. group I (negative control), positive control and three colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg). The number of mice in each group was five to eight. All these groups were administered doses intraperitoneally. To determine the effect of colchicine on cell mediated immunity , delayed type hypersensitivity (DTH) assay, macrophage engulfment assay, cyclophosphamide induced neutropenic test and nitric oxide production was performed .DTH was performed by measuring skin thickness. DTH showed significant difference (P<0.001) of negative control to colchicine treated groups 40μg/kg, 80μg/kg and 160μg/kg. With increasing dose, there was decrease in skin thickness of the mice. Highest reduction of skin was found at 160μg/kg. Macrophage engulfment assay was performed to evaluate the effect of macrophage induced phagocytosis. There was significant ( P <0.001) difference of engulfment of SRBCs by macrophages with negative control to colchicine treated group II (40μg/kg), group III(80μg/kg) and group IV(160μg/kg) groups. There was significant difference of engulfment of macrophages at 45 and 90 minutes. Cyclophosphamide induced neutropenic test was performed to assess the effect of colchicine on total leukocyte count (TLC) and differential leukocyte count (DLC). There was SUMMARY 77 reduction of TLC to about 45.3% in control to 48.3%, 54.68% and 65.42% in group II (40μg/kg), group III (80 μg/ kg) and group IV (160μg/kg) respectively when these were compared with primary values of TLC. There was significant difference of reduction in the neutrophil count of negative control 1057 (±120) to 902 (±67) in group II (40μg/kg), 734(±69) in group III (80 μg/ kg) and 609 (±71) in group IV (160μg/kg) of doses of colchicine. This test showed that with the increasing dose of colchicine, there was significant (P<0.001) difference of TLC count and neutrophil count. Nitric oxide (NO) production by macrophages was performed for measuring different concentrations of nitric oxide produced. There was significant difference (P<0.001) in NO production by macrophages alone and LPS stimulated between negative control to group II (40 μg /kg), group III (80μg/kg), group IV (160μg/kg) of colchicine. With increasing dose, there was significant reduction in production of NO. There was significant P<0.0001 reduction in body weight andspleen weight difference of mice in different groups of colchicine treated 40μg/kg, 80μg/kg and 160μg/kg from negative control after treatment. There was difference of weight of Thymus of group II (40 μg/kg), group III (80μg/kg) and group IV (160μg/kg) but difference was statistically not significant. There were no histopathological changes observed in spleen and Thymus at 40μg/kg and 80μg/kg doses of colchicine. At 160μg/kg dose, increase in thickness of trabecular was seen .due to edema in the spleen. For evaluation of colchicine effect on humoral immunity, haemagglutination assay, mice lethality test and Jerne hemolytic plaque formation were performed. Haemagglutination assay (HA) was performed by using red blood cells injected intraperitoneally in mice to measure antibody titer. There was significant difference of (P >0.001) to colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg)with group I (negative control).With the increasing dose, there was reduction in the SUMMARY 78 HA titer. Mice lethality test was performed by testing immune response of the mice to the challenge infection of P.multocida. It was performed by comparing mortality ratio of mice after administration of drug. There was no death of mice in the negative control group in which there was administration of PBS and vaccine. At 40μg/kg dose of colchicine, there was 50% mortality ratio. At 80μg/kg dose of colchicine 75% mortality ratio was observed. Maximum mortality ratio was observed at the 160μg/kg colchicine dose i.e. 100%. Jerne plaque formation test was performed and plaques formed was enumerated and recorded as the number of plaque forming cells (PFCs) per million cells. There was significant difference (P<0.001) of reduction in number of plaques from negative control to all doses of colchicine 40 μg/kg, 80 μg/kg and 160μg/kg. Antibody formation was decreased with increasing the dose of colchicine. Therefore, it is concluded that colchicine suppresses the cellular and humoral responses in mice.
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Colchicine is a medication that treats gout. It is a natural product and secondary metabolite, originally extracted from plants Colchicum autumnale .It causes modulation of chemokine and prostanoid production and inhibition of neutrophil and endothelial cell adhesion molecules by which it interferes with the initiation and amplification of the joint inflammation.
The present study is designed to evaluate the effects of colchicine on cellular and humoral immunity in mice. There were five groups for each assay i.e. group I (negative control), positive control and three colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg). The number of mice in each group was five to eight. All these groups were administered doses intraperitoneally. To determine the effect of colchicine on cell mediated immunity , delayed type hypersensitivity (DTH) assay, macrophage engulfment assay, cyclophosphamide induced neutropenic test and nitric oxide production was performed .DTH was performed by measuring skin thickness. DTH showed significant difference (P<0.001) of negative control to colchicine treated groups 40μg/kg, 80μg/kg and 160μg/kg. With increasing dose, there was decrease in skin thickness of the mice. Highest reduction of skin was found at 160μg/kg. Macrophage engulfment assay was performed to evaluate the effect of macrophage induced phagocytosis. There was significant ( P <0.001) difference of engulfment of SRBCs by macrophages with negative control to colchicine treated group II (40μg/kg), group III(80μg/kg) and group IV(160μg/kg) groups. There was significant difference of engulfment of macrophages at 45 and 90 minutes.
Cyclophosphamide induced neutropenic test was performed to assess the effect of colchicine on total leukocyte count (TLC) and differential leukocyte count (DLC). There was
SUMMARY
77
reduction of TLC to about 45.3% in control to 48.3%, 54.68% and 65.42% in group II (40μg/kg), group III (80 μg/ kg) and group IV (160μg/kg) respectively when these were compared with primary values of TLC. There was significant difference of reduction in the neutrophil count of negative control 1057 (±120) to 902 (±67) in group II (40μg/kg), 734(±69) in group III (80 μg/ kg) and 609 (±71) in group IV (160μg/kg) of doses of colchicine. This test showed that with the increasing dose of colchicine, there was significant (P<0.001) difference of TLC count and neutrophil count.
Nitric oxide (NO) production by macrophages was performed for measuring different concentrations of nitric oxide produced. There was significant difference (P<0.001) in NO production by macrophages alone and LPS stimulated between negative control to group II (40 μg /kg), group III (80μg/kg), group IV (160μg/kg) of colchicine. With increasing dose, there was significant reduction in production of NO. There was significant P<0.0001 reduction in body weight andspleen weight difference of mice in different groups of colchicine treated 40μg/kg, 80μg/kg and 160μg/kg from negative control after treatment. There was difference of weight of Thymus of group II (40 μg/kg), group III (80μg/kg) and group IV (160μg/kg) but difference was statistically not significant. There were no histopathological changes observed in spleen and Thymus at 40μg/kg and 80μg/kg doses of colchicine. At 160μg/kg dose, increase in thickness of trabecular was seen .due to edema in the spleen. For evaluation of colchicine effect on humoral immunity, haemagglutination assay, mice lethality test and Jerne hemolytic plaque formation were performed. Haemagglutination assay (HA) was performed by using red blood cells injected intraperitoneally in mice to measure antibody titer. There was significant difference of (P >0.001) to colchicine treated group II (40μg/kg), group III (80μg/kg) and group IV (160μg/kg)with group I (negative control).With the increasing dose, there was reduction in the
SUMMARY
78
HA titer. Mice lethality test was performed by testing immune response of the mice to the challenge infection of P.multocida. It was performed by comparing mortality ratio of mice after administration of drug. There was no death of mice in the negative control group in which there was administration of PBS and vaccine. At 40μg/kg dose of colchicine, there was 50% mortality ratio. At 80μg/kg dose of colchicine 75% mortality ratio was observed. Maximum mortality ratio was observed at the 160μg/kg colchicine dose i.e. 100%.
Jerne plaque formation test was performed and plaques formed was enumerated and recorded as the number of plaque forming cells (PFCs) per million cells. There was significant difference (P<0.001) of reduction in number of plaques from negative control to all doses of colchicine 40 μg/kg, 80 μg/kg and 160μg/kg. Antibody formation was decreased with increasing the dose of colchicine. Therefore, it is concluded that colchicine suppresses the cellular and humoral responses in mice.

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