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Abstract
Materials and Methods
Twenty rabbits were utilized. A d/luciferase (adenovirus construct) were injected into nucleus pulposus of lumbar vertebrae. Group 1 received intradiscal injection of A d/luciferase only, Group 2 received subcutaneous and intradiscal injection simultaneously, Group 3 received subcutaneous injection then intradiscal injection with 2 weeks interval. Blood sam-ples were obtained serially after injection. A nimals were sacrificed at 7 weeks. A ntibody to adenovirus in peripheral blood was measured with ELISA and transgene expression was measured with standard luciferase kits.
Results
All rabbits in the Group 2 and 3 exhibited increased production of neutralizing antibody. There were clearly two sub-groups in Group 1, three rabbits exhibited production of antibody but remaining three rabbits showed little or no production of antibody. All rabbits showed robust increase in transgene expression regardless of titer of neutralizing antibody.
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Fig. 1.
Qualitative analysis of intradiscal transgene expression of A) 3 weeks, B) 6 weeks after direct application of Ad/lacZ into lumbar intervertebral discs of adult New Zealand white rabbits. There were strong transgene expresson indicated by the arrow (dark staining, originally blue color) in the nucleus pulposus without evidence of infiltration of inflamatory cell and neovascularization (X-Gal staining with counter staining with hematoxylin-eosin, Original magnification × 100).
Fig. 2.
Sequential production of specific antibody for adenoviral proteins in peripheral blood as function of time. Transverse bar chart on the right denote intradiscal transgene expression in each individual rabbit. A Three rabbits injected with Ad/luciferase produced little or no neutralizing antibody while remaining three rabbits showed production of antibody (likely due to leakage of adenovirus from injected disc). B Six rabbits in Group 2 after simultaneous injection of Ad/luciferase into subcutaneous tissue and intervertebral disc produced neutralizing antibody in significant amount. C Six rabbits in Group 3 were immunized two weeks prior to intradiscal gene transduction with injection of Ad/luciferase into subcutaneous tissues. All rabbits showed significant amount of transgene (luciferase) expression.
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