A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75(NTR.).

The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.

Immunohistochemical detection of nerve growth factor and its receptors in the rat periodontal ligament during tooth movement.

OBJECTIVES: Nerve growth factor (NGF) and its receptors, p75 and tyrosine receptor kinase A (Trk A), have been shown to increase following trauma. The aims of this study were to examine changes in the detection of NGF and its receptors during orthodontic tooth movement in the rat, and the effects of anti-NGF on these changes. DESIGN: Orthodontic separators were placed between the right maxillary first and second molars of Sprague-Dawley rats which were equally divided into two groups. Animals from the second group were injected with anti-NGF. The left sides served as controls, and animals were sacrificed at 0, 3, 7 and 14 days. RESULTS: Results of immunohistochemical localisation for p75, Trk A, calcitonin gene-related peptide (CGRP) and NGF showed staining intensity increased at day 3, with a peak at day 7 and decreasing intensity at day 14. Anti-NGF injected animals showed reduced staining at all observation periods. CONCLUSION: Data suggest that orthodontic injury induces NGF production, leading to sprouting and invasion by CGRP-positive nerve fibers and that injection of anti-NGF reduces NGF tissue levels and prevents innervation by CGRP-positive fibers.

Graft of pre-injured sural nerve promotes regeneration of corticospinal tract and functional recovery in rats with chronic spinal cord injury.

A possible treatment approach for chronic spinal cord injuries has been tested. We report that minced, autologous, pre-injured peripheral nerve administered as a single injection into an injury-induced cyst, resulting from a contusion injury of the thoracic spinal cord, stimulates recovery of hindlimb locomotor function in rats, as measured by the Basso, Beattie, Bresnahan Locomotor Rating Scale. This response was further enhanced by the addition of exogenous neurotrophic factors. Histological analysis showed axons of the corticospinal tract exhibited significant regeneration past the injury site, when quantified both by number and length. Results indicate that the use of a pre-injured peripheral nerve graft stimulates chronically injured descending nerves to overcome a local inhibitory environment. The resulting sprouting and growth past the injury site is associated with a significant improvement in locomotor function.

Efficacy of N-acetylcysteine and hydration versus placebo and hydration in decreasing contrast-induced renal dysfunction in patients undergoing coronary angiography with or without concomitant percutaneous coronary intervention.

OBJECTIVE: Contrast-induced renal dysfunction is an iatrogenic complication that occurs more frequently in patients with preexisting renal dysfunction. A prospective, double-blind, randomized, placebo, controlled trial was completed to assess the efficacy of N-acetylcysteine in decreasing the incidence of contrast-induced renal dysfunction in patients with an acute coronary syndrome and renal insufficiency who underwent coronary angiography with or without percutaneous coronary intervention. METHODS: With similar intravenous hydration protocols, 20 patients received N-acetylcysteine (treatment group) and 20 patients received placebo (control group) in a twice per day dosing regimen with one dose before and three doses after contrast media exposure. RESULTS: The two groups were similar at baseline on demographic and clinical characteristics, and preexisting renal insufficiency. Contrast-induced renal dysfunction, defined as an increase in serum creatinine greater than 44 micromol/L (.5 mg/dL) and/or 25% above baseline within 48 hours, occurred in 7.5% of the cohort, with 2.5% in the treatment group, and 5% in the control group, for an absolute difference of 2.5%. There was no difference in serum creatinine or creatinine clearance at 24 hours or at 48 hours between the treatment and control groups. CONCLUSION: These results suggest that this cohort gained no added protection to renal function with the use of N-acetylcysteine.

GDNF gene delivery via the p75(NTR) receptor rescues injured motor neurons.

The retrograde axonal transport mechanism of motor neurons has been exploited to deliver the gene encoding Glial cell line-derived neurotrophic factor (GDNF) into the central nervous system to provide trophic support following injury. A nonviral gene delivery system, consisting of a monoclonal antibody (MC192) that binds the neurotrophic receptor, p75(NTR), coupled to poly-L-lysine, was constructed and used to deliver the gene via a receptor-mediated mechanism. The MC192-poly-l-lysine/pGDNF complex was injected into the hind limb of newborn rats to allow gene expression within motor neurons prior to sciatic nerve transection. In adult rats, the gene delivery complex was administrated in gel foam placed on a transected hypoglossal nerve. We show that the delivered construct is internalized following binding to p75(NTR) and is transported into the brain and spinal cord, bypassing the blood-brain barrier. The presence of the GDNF transgene and its transcript could be detected for up to 8 weeks in spinal cord and brain stem. Expression of the GDNF protein rescued 38% of the targeted motor neurons 1 week postinjury in newborn rats while the survival rate in control group was below 12%. In adult rats, neuronal death induced by axotomy was almost completely reversed by the introduction of the transgene (95 +/- 3%). Thus, the significant functional outcomes of this novel gene delivery system are demonstrated both in postnatal and adult motor neurons.

Use of engineered peripheral nerve autografts for spinal cord repair.

We developed a clinically compatible protocol for the production of engineered tissue for grafting into the injured spinal cord. We used autologous tissue derived from pre-ligated peripheral nerves, which avoids supply, immunocompatibility and ethical hinderances, combined with non-viral transfection, which is a versatile and non-immunogenic gene transfer method. In-vitro transfection of glial cells or primary tissue from pre-ligated rat peripheral nerve with the neurotrophic gene brain-derived neurotrophic factor significantly enhanced its expression, when quantified or labelled by immunofluorescence. Engineered tissue expressed brain-derived neurotrophic factor after being grafted into the spinal cord of rats that had received spinal contusion injury 3 weeks before. Anatomical and functional assays of repair, conducted on a small cohort, showed that the treatment may promote axonal regeneration and improve motor performance.

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action.

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.