Comparison of biochemical and cytotoxic functions of hepatocytes from goat, pig and human fetuses.

BACKGROUND AND AIM: To overcome the problem of shortage of donor organs, xenotransplantation of cells offers an alternative to orthotopic transplantation. Of the higher animals, the pig is considered as a suitable donor because of the similarity in size and function of pig organs to human organs. However, successful transplantation of pig organs/cells for human therapy is limited by hyperacute rejection, improper functioning of xenografts and the risk of transmission of endogenous retroviruses to the recipient. Thus, there is a pressing need to explore an alternate mammalian source to bridge the gap between the donor and the recipient waiting for transplantation. This has warranted us to explore the application of goat hepatocytes as a treatment modality in acute liver failure. METHODS: In the present investigation, isolated goat hepatocytes were assessed for their viability, membrane integrity, synthetic and cytotoxic functions, and compared with the hepatocytes of pig and human fetuses (28-36 weeks). RESULTS: The isolated hepatocytes from goat, pig and human fetuses were comparable in their viability, membrane integrity and synthetic functions. However, the cytotoxic functions assessed using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated a significant reduction in the viability of the pig hepatocytes (38%) as compared with the goat and human fetal hepatocytes, which retained their viability (98%) on incubation with normal human serum. CONCLUSION: These observations are significant as they suggest that goat hepatocytes probably can be explored as a source for cell therapy in the treatment of acute liver failure.

Binding characteristics of bovine serum albumin-aflatoxin B1 to polystyrene microtiter plates: importance of hapten to carrier protein molar ratio.

Binding characteristics of bovine serum albumin-aflatoxin B1 conjugates with high (1:54), medium (2:25) and low (1:9) hapten to carrier molar ratios, to the polystyrene microtiter plates are influenced by the stoichiometry of the hapten (Aflatoxin B1) to the carrier protein (bovine serum albumin). Conjugates with optimal hapten to carrier molar ratios (1:25) showed a better binding capacity to the polystyrene microtiter plate as compared to the conjugates with the high molar ratios in a non-competitive ELISA for aflatoxin B1. Denaturation of the conjugate with molar ratio of 1:54 in order to enhance its binding capacity, however, did not result in any significant improvement.

Quantitation of epsilon-amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. A simple spectrophotometric method for the estimation of hapten to carrier protein ratio.

A simple, sensitive and direct spectrophotometric method is presented for the determination of epsilon-amino groups of L-lysine present in carrier proteins, using the free amino acids L-lysine and L-glutamic acid as reference standards, and 2,4,6-trinitrobenzene 1-sulfonic acid (TNBS) reagent. The amount of epsilon-amino group present in the carrier protein after coupling with hapten is directly quantitated using the standard curve generated by the difference in absorbance observed with L-lysine and L-glutamic acid after their reaction with TNBS reagent. Spectral analysis of L-lysine and L-glutamic acid (27.36 nmol) derivatives of TNBS at 335 nm showed that TNP-L-lysine had twice the absorbance of TNP-L-glutamic acid, since TNBS reagent interacts equally with the alpha-amino and epsilon-amino groups present in L-lysine and the alpha-amino group of L-glutamic acid, respectively. The relationship between absorbance and concentration of epsilon-amino groups (up to 16 micrograms/ml) was found to be linear. The number of epsilon-amino groups of lysine present in carrier proteins such as BSA, HSA, thyroglobulin and the enzyme, horseradish peroxidase, were analyzed by the present method and were found to be similar to the reported values. Various carrier protein-hapten conjugates (protein-mycotoxin/vitamin/steroid hormone conjugates) were made and analyzed by the method developed in order to determine their mole to mole ratio.

Evaluation of adjuvanticity of promising new synthetic MDP analogues.

A series of nor-MDP analogues were evaluated for adjuvanticity in rodents using beta hCG-TT conjugate as the antigen. Of these, one compound, N-acetylnor-muramyl-L-N-methylalanyl-D-isoglutamine octylamide (nor-MDP octylamide (N-Me-Ala] was found to be effective. This compound, formulated with beta hCG-TT in water-in-oil emulsion and administered to rodents, significantly enhanced the anti-hCG response. The anti-hCG titers induced were three-fold higher than that of control formulation. Moreover, inclusion of this compound in the first injection only gave adequate levels of antibodies to the hormone which persisted longer in the blood circulation. Effectiveness of antibodies in neutralizing hCG was tested in vitro by the mouse Leydig cell bioassay. Biological vs. immunological binding capacities (B/I ratio) were compared. The results suggest that nor-MDP octylamide (N-Me-Ala) will be useful as an adjuvant for human vaccines.