ABSTRACT
A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r(2) = 0.95, P < 0.0001) and for diphtheria (r(2) = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.
Subject(s)
Diphtheria Antitoxin/blood , Diphtheria-Tetanus Vaccine/immunology , Diphtheria-Tetanus-Pertussis Vaccine/immunology , Tetanus Antitoxin/blood , Animals , Diphtheria Antitoxin/immunology , Female , Guinea Pigs , Male , Mice , Neutralization Tests/methods , Reference Standards , Reproducibility of Results , Tetanus Antitoxin/immunologyABSTRACT
A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.
Subject(s)
Animals , Male , Female , Guinea Pigs , Mice , Diphtheria Antitoxin/analysis , Diphtheria-Tetanus Vaccine/immunology , Diphtheria-Tetanus-Pertussis Vaccine/immunology , Tetanus Antitoxin/analysis , Diphtheria Antitoxin/immunology , Neutralization Tests/methods , Reference Standards , Reproducibility of Results , Tetanus Antitoxin/immunologyABSTRACT
The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.
Subject(s)
Bronchodilator Agents/pharmacology , Cyclic GMP/pharmacology , Nitric Oxide/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Trachea/physiology , Animals , Ca(2+) Mg(2+)-ATPase/drug effects , Dose-Response Relationship, Drug , Drug Interactions , In Vitro Techniques , Male , Nitroprusside/pharmacology , Protein Kinases/physiology , Rats , Rats, Wistar , Sodium-Potassium-Exchanging ATPase/drug effects , Vasodilator Agents/pharmacologyABSTRACT
The proximal and distal stumps of severed mouse sciatic nerves were inserted into opposite ends of polyethylene tubes. The tubes were implanted either empty or filled with collagen alone or in combination with interleukin-1 (IL1). Six weeks later, neurons in the L3-L5 dorsal root ganglia were back-filled with HRP. The number of HRP reactive sensory neurons detected in the IL1-treated animals was significantly greater than that seen in the other experimental groups. Thus, exogenous IL1 may partially mimic the effects obtained with in vivo administration of nerve growth factor in protecting sensory neurons from lesion-induced death.
Subject(s)
Interleukin-1/pharmacology , Nerve Regeneration/drug effects , Neurons, Afferent/drug effects , Sciatic Nerve/physiology , Animals , Male , Mice , Mice, Inbred C57BL , Sciatic Nerve/surgeryABSTRACT
The proximal and distal stumps of severed mouse sciatic nerves were inserted into opposite ends of polyethylene tubes. The tubes were implanted either empty ofr filled with collagen alone or in combination with interleukin-1 (IL1). Six weeks later, neurons in the L3-L5 dorsal root ganglia were back-filled with HRP. The number of HRP reactive sensory neurons detected in the IL1-treated animals was significantly greater than that seen in the other experimental groups. Thus, exogenous IL1 may partially mimic the effects obtained with in vivo administration of nerve growth factor in protecting sensory neurons from lesion-induced death
