Optimization of Transfer Factor Vitality Test and Establishment of a New Method Based on Micro Plate- Leukocyte Adherence Inhibition Test / 中国药学杂志

OBJECTIVE: To optimize the current T cell-E Rose test method, establish a new alternative method, and measure 57 batches of transfer factor preparations using the two methods. METHODS: Cell smear was prepared by modified cell smear-dry staining method. Cross-test was performed with fresh porcine thymus and rabbit thymus respectively with sheep erythrocytes. The micro plate-leukocyte adherence inhibition alternative method was established and verified for the linearity, repeatability, and specificity. RESULTS: The improved smear-dyeing method had clear and reliable microscopic effect, which was easy to read the preservation. Porcine thymus lymphocytes and rabbit thymus lymphocytes were able to form E rosette with sheep red blood cells, and there was no significant difference between the two methods. The analysis results of 57 batches of transfer factor injection showed that the leukocyte adherence inhibition rate had no significant difference with the results of rosette viability. CONCLUSION: The improved method can replace the original cell counting plate method, which can better respond to the actual value of the sample with long-term preservation, and it is suitable for reading and reviewing the analysis result of large quantities of samples at any time. The alternative micro plate-leukocyte adherence test method may reduce the human subjective factors, and is more accurate, rapid, reproducible, and specific than the original method, and suitable for high-throughput analysis.

Application of Leucocyte Adherence Inhibition Test in Determining Immunological Activity of Ribonucleic Acid / 中国药师

Objective:To establish a method for determining the immunological activity of ribonucleic acid. Methods: Leucocyte adherence inhibition test ( LAI) was applied, and the important parameters of LAI including the mouse strain, drug concentration, treatment time, content of buffer solution and cell density were researched. The immunological activity of RNAⅠ, Ⅱand Ⅲ was re-spectively determined by the method. Results:Stable and reliable parameters were obtained: the sample concentration was 10 mg· ml-1 , the treatment time was 2 hours, Ca2+ and Mg2+ were necessary for the buffer solution, and the cell density was about 4 × 107 cell·ml-1 . The strain of mouse showed no effect on the results. As a result, the determination method for immunological activity was established. Using the method, the immunological activity of RNA Ⅰ,Ⅱand Ⅲ was determined 3 times, and the results met the re-quirements with RSD below 20%. Conclusion:The method is suitable for determining the immunological activity of RNA.

Synergistic inhibition of Lactobacillus acidophilus and Clostridium butyricum against Shigella flexneri in vitro / 中国药科大学学报

@#An agar diffusion method was used to investigate the antimicrobial activities of fermentation broth of Lactobacillus acidophilus(L. acidophilus), and Clostridium butyricum(C. butyricum)against Shigella flexneri(S. flexneri)infection in vitro. It was found that cell-free culture supernatants(CFCSs)of L. acidophilus and C. butyricum possessed remarkable synergistic anti-S. flexneri activity in vitro and the antimicrobial activity of the mixed culture was 17. 2% and 22. 4% greater, than single strains alone, respectively. Meanwhile, there was a symbiotic relationship between L. acidophilus and C. butyricum. The result showed that the biomass accumulation of the mixed culture reached 4. 27 g/L(DCW)and increased by 6. 0% and 30. 6% compared to the L. acidophilus and C. butyricum, respectively. Moreover, L. acidophilus and C. butyricum clearly inhibited S. flexneri adhesion to Caco-2 cells by 75. 8% and 81. 2%, and the combination of these two probiotic strains demonstrated the highest inhibition rate, reaching 84. 2%, while the viability of Caco-2 cells treated with L. acidophilus, C. butyricum or their combination was increased by 11. 5%, 12. 5% and 22. 9%, respectively. The synergistic effect of L. acidophilus and C. butyricum provided better protection against Shigella flexneri infection, which represents a promising alternative therapy for shigellosis.

In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

Study on adhesion of Lactobacillus acidophilus to mice Peyer's patches and inhibition of pathogen invasion / 中华微生物学和免疫学杂志

Objective To investigate the adhesion mechanism of Lactobacillus acidophilus FN001外to Peyer's patches. Methods Adhesion of L. acidophilus FN001 to mice Peyer's patches was studied in vitro using a fluorescent quantization method. The nature of adhesion mediator was studied by the effects of physical, chemical and enzymatic pre-treatments of the bacteria on their adhesion and effect of sugars on in- hibition of adhesion. The presence of lectin-like proteins in the cell surface was determined by hemagglutina- tion. Effect of L. acidophilus FN001 on inhibition of adhesion of pathogens to Peyer's patches was also stud- ied. Results The adhesion of L. acidophilus FN001 was strongly inhibited in the presence of D-mannose and methyl-ct-D-mannoside. Pretreatment of L. acidophilus FN001 with pepsin and trypsin decreased the ad- hesive capacity indicating that cell surface proteins are involved in adhesion to Peyer's patches. L. acidophi- lus FN001 could agglutinate rabbit red cell in mannose specific manner and protease pretreatment could de-crease hemagglutinin, suggesting that L. acidophilus FN001 has mannose specific lectin (s). In adherence inhibition assay, L. acidophilus NF001 could significantly inhibit adhesion of E. coli ATCC25922 to Peyer's patches when L. acidophilus NF001 were applied to Peyer's patches first or at the same time with pathogen. Conclusion It was concluded that a mannose-specific protein mediated adhesion of L. acidophilus FN001 to the Peyer's patches, and L. acidophilus FN001 could inhibit adhesion of pathogen with similar lectins speci- ficity to Peyer's patches.

Studies of LAIT and morphology of immune organs in vasectomized rabbits / 中国病理生理杂志

23 rabbits were bilaterally vasectomized and investigated for 12 months with 17 normal male rabbits as control. It was found that (1) positive results of leucocyte adhesion inhibition test occured in 43.5% of the vasectomized rabbits and a significant difference of positive rate was found between 10 and 12 monthes after vasectomy, (2) pathomorphological findings in the immune organs of the vasectomized rabbits showed that the lymph tissues proliferated. The pathomorphological findings were found to be in line with the result of leucocyte adhesion inhibition test.

Experimental studies of autoimmune response to sperm antigens in vaseetomized rabbits / 中国病理生理杂志

Thirty-three rabbits were bilaterally vasectomized and observed for 1-12 months with 7 normal male rabbits control. It was found that (1) 67.9% of the vase ctomized rabbits revealed antibodies to sperm antigens with a titre ranging from 1:5 to 1:1280 as detected by indirect hemagglutination test; (2) 90.9% of the vasectomized rabbits showed antibodies to sperm by indirect immunofluorescence; (3) 43.5% of the vasectomized rabbits showed positive leucocyte adhesion inhibition test; (4) while pathomorphological findings in the section of immune organs of the vasectomized rabbits showed that the lymphatic tissues were proliferative, macrophages were active. Circulatory immune complex was not detected in any of the 33 vasectomized rabbits. Epididymides were swollen in 70% of the experimental animals in 5-7 months after vasectomy, subsidence of epididymal swelling occurred usually with the increase of antibody titres against sperm antigen.