Study on protective effect of specific igy antibody on cholera toxin-intoxicated suckling mice

Nội dung text: Study on protective effect of specific igy antibody on cholera toxin-intoxicated suckling mice

1. Journal of military pharmaco-medicine n o1-2018 STUDY ON PROTECTIVE EFFECT OF SPECIFIC IgY ANTIBODY ON CHOLERA TOXIN-INTOXICATED SUCKLING MICE Hoang Trung Kien*; Nguyen Anh Tuan* Le Thu Hong**; Nguyen Dang Dung* SUMMARY Objectives: To evaluate the protective effect of anti-choleratoxin IgY antibody on choleratoxin-intoxicated animal. Subjects and methods: Suckling mice (3 - 5 days old) were intoxicated with choleratoxin, then treated with anti-choleratoxin IgY. The protective effect of IgY antibody was evaluated by measuring survival time of choleratoxin-intoxicated suckling mice treated with anti-choleratoxin IgY and by histopathological analysis of epithelial cells of the mice's intestinal mucosa. Results: After 66 hours of choleratoxin infection, 65% of mice treated with anti-choleratoxin IgY were still alive (compared to 0% in control group); pathohistological images of the mice's intestinal mucosa showed less damages in those from mice treated with anti-choleratoxin IgY antibody compared to controls. Conclusions: IgY antibody against cholera toxin helps protect suckling mice from toxic effect of choleratoxin. * Keywords: Vibrio cholerae; IgY antibody; Sucking mice. INTRODUCTION expressing on epithelial cells of the intestinal mucosa. Once bound, the Cholera is a serious diarrhea disease cleavage between subunit A1 and A2 caused by gastrointestinal infection of Vibrio segments is facilitated and A1 portion cholerae. In 2016, 172,454 cholera cases moves into the cells. The A1 component were reported by WHO, including 1,304 stimulates the production of adenylate cases of deaths in 42 countries in the cyclase, leading to increased production world with fatality rate of 0.8%, and the of cyclic AMP inside the cells. This total number of cholera cases and deaths increased intracellular level of cyclic AMP has not decreased in the last five years, results in a disruption of the active but the fatality rate for cholera is till high. transport of electrolyte across the cell Following the gastrointestinal infection, membrane, which leads to fluid secretion the bacteria secretes choleratoxin (CT), into small intestine. The diarrhea occurs which consists of one A (active) and five when the volume of fluid entering the B (binding) subunits. The B subunits of colon from intestine is over the re- CT (CTB) can bind to GM1 ganglioside absorptive capability of the colon. * Vietnam Military Medical University ** 103 Military Hospital Corresponding author: Nguyen Dang Dung (dzungmd@yahoo.com) Date received: 20/10/2017 Date accepted: 18/12/2017 151
2. Journal of military pharmaco-medicine n o1-2018 The utilization of antibodies specific to SUBJECTS AND METHODS V. cholerae exerted preventive and 1. Subjects. therapeutic effects against cholera on - Suckling mice (3 - 5 days old), animal orally infected with live regardless of gender, provided by the V. cholerae . However, whether IgY Animal House of Military Medical University. antibody specific to CT can protect animal - C0852 CT freeze-dried powder provided from cholera-like disease caused by oral CT infection has not yet been completely by Sigma. elucidated. - IgY isolated from egg yolk laid of hens In this study, we developed an animal immunized with CT by method described model of CT infection and evaluate the previously [1, 3]. protective effect of specific IgY antibody - Reagents used for immunohistochemical on CT-intoxicated suckling mice. (IHC) staining. 2. Methods. * Establishing choleratoxin-intoxicated animal model: Table1: Choleratoxin intoxication model on suckling mice. Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) (n = 5) Mice given Mice given Mice given Mice given Mice given with Mice given with with 50 µL of with 50 µL of with 50 µL of with 50 µL of 50 µL of 50 µL of NaCl 0.9% choleratoxin choleratoxin choleratoxin choleratoxin choleratoxin 0.008 mg/mL 0.04 mg/mL 0.2 mg/mL 1 mg/mL 5 mg/mL The experimental animal was suckling mice (3 - 5 days old), separated from their mother 3 hours before the start of experiment. A total of 30 mice were devided into 6 groups, with 5 mice in each group. In group 1 (control group): mice were given with 50 µL of NaCl 0.9% through esophageal intubation using a plastic flexible needle; in the remaining groups (from group 2 to 6), mice were given with 50 µL of CT at concentrations of 0.008; 0,04; 0,2; 1 and 5 mg/mL in NaCl 0.9%, respectively. Thereafter, all the mice were inspected every 2 hours for diarrhea and death, of which number of mice died in each group was recorded at 2-hour time intervals until all mice in the CT-intoxicated groups died. 152
3. Journal of military pharmaco-medicine n o1-2018 * Investigation of the protective effect of specific IgY antibody against CT on CT- intoxicated suckling mice: Table 2: Group 1 2 3 4 Intervention (n = 20) (n = 20) (n = 20) (n = 20) Choleratoxin intoxication 50 µL 50 µL choleratoxin 50 µL choleratoxin 50 µL choleratoxin (0 hour) NaCl 0.9% 1 mg/mL 1 mg/mL 1 mg/mL Treatment 50 µL 50 µL 50 µL 50 µL (3 hours after IgY against Freund IgY against NaCl 0.9% NaCl 0.9% choleratoxin intoxication) adjuvant choleratoxin 4 groups of suckling mice (3 - 5 days number of mice died in each group old), with 20 mice in each group, were recorded at 2-hour time intervals until all adopted for this experiment. Suckling mice in group 2 (treated with NaCl 0.9%, mice were separated from their mother served as negative control-1 group) died. 3 hours before testing. * Pathohistological analysis: In group 1 (biological control group): Immunohistochemistry (IHC) technique mice were given with 50 µL of NaCl 0.9% was adopted to determine CT on through esophageal intubation; in the intestinal mucosal epithelium, and to groups 2, 3, and 4, mice were given with evaluate the intestinal epithelial injuries of 50 µL of CT at 1 mg/mL. Three hours the experimental CT-intoxicated suckling after CT injection, mice in the groups 1 mice. and 2 were given with 50 µL of NaCl Briefly, a mouse with anti-CT IgG antibody 0.9%; mice in group 3 were administered (the primary antibody) was incubated with with 50 µL of IgY isolated from egg yolk laid of hens immunized with Freund intestinal specimen's slide. After being adjuvant only (served as negative control- washed, the slide was incubated with the 2 group), meanwhile mice in group 4 were secondary antibody (a biotynated rabbit- administered with 50 µL of IgY antibody anti-mouse IgG antibody). The slide was isolated from egg yolk laid of hens washed, and then incubated with an immunized with a mixture of CT antigen avidin-peroxidase conjugate. Finally, and Freund adjuvant. diaminobenzidine substrate was added All the mice were then inspected every for slide's microscopic visualization and 2 hours for diarrhea and death, with analysis. 153
4. Journal of military pharmaco-medicine n o1-2018 RESULTS AND DISCUSSION 1. Establishment of choleratoxin intoxication model on suckling mice. Graph 1: Survival time of mice after choleratoxin intoxication. In an attemp to establish an animal mice, respectively. CT infection could only model for CT infection, we have chosen kill the experimental animals significantly suckling mice, since the animal was when using a CT solution at concentration of previously reported to be susceptible to 1 mg/mL and 5 mg/mL (group 5 and cholera caused by gastrointestinal infection group 6), with all mice died at 58 and 66 hours with live V. cholerae . In the present study, following CT intubation, respectively. No we investigated the susceptibility of the significant difference in survival rate and mice with 5 different doses of CT infection. time of CT-intoxicated mice in the two groups Mice in group 1 served as biological control (intoxicated with 1 mg/mL and 5 mg/mL of group, all of which were given NaCl 0.9% CT, respectively) was observed. only, to ensure that an the esophageal The survival rate of mice in group 1 intubation and injection of the solutions (non-CT infection group) and in groups 2, did not unexpectedly kill the mice. 3 and 4 (intoxicated with CT at Graph 1 presented the results of concentrations of 0.008, 0.04 and establishment of the CT-infection animal 0.2 mg/mL, respectively) at 66 hours model, with a single injection (through following CT infection ranged from 60 to esophageal intubation) of 50 µL of CT at 80%. The survival time of mice in group 1 0.008 mg/mL; 0.04 mg/mL; 0.2 mg/mL; compared to those in groups 5 and 6 was 1 mg/mL; 5 mg/mL for each suckling significantly different (p < 0.01). 154
5. Journal of military pharmaco-medicine n o1-2018 Analysis on pathohistological images non-CT-intoxicated mice (figure 1A) which of intestinal mucosa from CT-intoxicated showed negative choleratoxin staining by mice revealed obvious presence of immuno-histochemical staining method choleratoxin on intestinal mucosa (figure 2B), (figure 1B) ; this is in accordance with the and epithelial cells' damages (figure 2A) role of CT in pathogenesis of V. cholerae were not observed in specimens from infection. (A) (B) Figure 1: Normal intestinal mucosa of mice; (A): HE staining, X400; (B): immuno-histochemical staining, using anti-choleratoxin antibody, X400: choleratoxin (-). (A) (B) Figure 2: Intestinal mucosa of mice intoxicated with CT: (A) CT-intoxicated, no anti-CT IgY treatment; HE staining, X400; (B) CT-intoxicated, no anti-CT IgY treatment; immuno-histochemical staining with anti-choleratoxin antibody, 400X: choleratoxin (+). It was therefore revealed by the results that oral CT intubation can cause death to suckling mice (3 - 5 days old), and the death was likely due to CT-induced cholera-like disease. 155
6. Journal of military pharmaco-medicine n o1-2018 2. Protective effect of anti-choleratoxin likely due to the specificity of the IgY to IgY on choleratoxin-intoxicated mice. CT; in other words, it was very likely that the CT-specific IgY bound to the CT molecule and therefore neutralized it, thus exerting the therapeutic effect as observed. Graph 2: Survival time of choleratoxin- Figure 3: Intestinal mucosa of CT- intoxicated mice treated with intoxicated mice treated with anti-CT IgY; anti-choleratoxin IgY antibody. immuno-histochemical staining with The results presented in graph 2 showed anti-choleratoxin antibody, that survival time of mice in group 2 X400: choleratoxin (+). (CT-intoxicated mice treated with NaCl Importantly, pathohistological images 0.9%) and in group 3 (CT-intoxicated mice of intestinal mucosa from CT-intubated treated with IgY against Freund adjuvant, mice treated with anti-CT IgY showed little i.e IgY isolated from egg's yolk laid of hens epithelial damages compared to that of immunized with Freund adjuvant only) control ( figure 3 ). was similar; additionally, the survival rates of mice in both groups at 66 hours Hirai K (2010) demonstrated that IgY following CT infection were 0%. These against choleratoxin B (CT-B) subunit results indicated that a "normal" IgY (i.e exerted therapeutic effect on suckling non-specifically CT-immunized IgY) had mice infected with live V. cholerae . Together no therapeutic effect on CT infection. with our findings in this study, it was Meanwhile, in group 4 (CY-intoxicated suggested that anti-CT IgY used in our mice, treated with anti-CT IgY), survival time study bound to CT-B subunit on the CT of mice was significantly higher compared molecule, therefore inhibited the binding to those in groups 2 and 3 (p < 0.001). of CT (or CT-B subunit) to its GM1 receptor The results indicated that IgY specific on intestinal mucosa's epithelial cell. Further to CT administered gastrointestinally investigation is needed to elucidate the possessed a therapeutic effect on CT- mechanism of action of anti-CT IgY in intoxicated suckling mice. This effect was protecting mice intoxicated with CT. 156
7. Journal of military pharmaco-medicine n o1-2018 CONCLUSIONS burn lesions. Journal of Military Pharmaco- Medicine. 2011, 8, pp.44-49. Single injection of 0.05 mL choleratoxin 3. Kazuyuki Hirai, Hideyuki Arimitsu, Koji (at concentration of 1 mg/mL) through Umeda et al. Passive oral immunization by esophageal intubation caused cholera-like egg yolk immunoglobulin (IgY) to Vibrio disease in suckling mice (3 - 5 days old). cholerae effectively prevents cholera. Acta In choleratoxin-intoxicated suckling mice, Medica Okayama. 2010, 64 (3), pp.163-170. a single administration of anti-choleratoxin 4. World Health Organization. Cholera, IgY gastrointestinally after infection can 2015. Weekly Epidemiological Record. 2016, protect choleratoxin-intoxicated suckling 38 (91), pp.433-440. mice from the toxic effect. 5. World Health Organization. Cholera, REFERENCES 2014. Weekly Epidemiological Record. 2015, 40 (90), pp.517-528. 1. Hoang Trung Kien; Do Khac Dai; Nguyen Ngoc Tuan et al. Production of 6. World Health Organization. Cholera, antibody to Edwardsiella ictaluri the causative 2013. Weekly Epidemiological Record. 2014, pathogen of liver pus disease in catfish by 31 (89), pp.345-356. chicken IgY technology. Journal Information of 7. World Health Organization. Cholera, Pharmaco-medicine. 2010, 3, pp.71-76. 2012. Weekly Epidemiological Record. 2013, 2. Tim Sunnary, Do Minh Trung, Le Thu 31 (88), pp.321-336. Hong, Lo Van Dong. Effect of IgY antibody 8. World Health Organization. Cholera, against Pseudomonas aeruginosa in vivo on 2011. Weekly Epidemiological Record. 2012, Pseudomonas aeruginosa -infected experimental No 31-32, 87, pp.289-304. 157