Human Beta-nerve Growth Factor Research Articles

4-PBA Enhances Autophagy by Inhibiting Endoplasmic Reticulum Stress in Recombinant Human Beta Nerve Growth Factor–Induced PC12 cells After Mechanical Injury via PI3K/AKT/mTOR Signaling Pathway

To investigate mechanism of endoplasmic reticulum (ER) stress-mediated autophagy in spinal cord injury (SCI). An invitro model of spinal cord injury (SCI) was established by recombinant human beta nerve growth factor (NGF)-induced PC12 cells. Immunofluorescence was used to detect properties of PC12 cells induced by NGF. Western blot assay was used to detect expressions of the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)I/II, the ER stress-related protein (HSPA5/GRP78), as well as the PI3K/AKT/mTOR signaling pathway-related proteins after mechanical injury at different time points. Then the sample assigned into sham, SCI, LY294002, SCI+LY294002, 4-PBA (4-phenylbutyric acid), and SCI+4-PBA groups. The expressions of the LC3I/II and PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blot assay. NGF-induced PC12 cells have neurophysiological characteristics. After administration of the PI3K-specific inhibitor LY294002, phosphorylation levels of AKT and mTOR decreased, and the ratio of LC3II/I was higher in the inhibitor-treated injury group than the simple-injury group. After administration of the ER stress inhibitor 4-PBA, the results were similar to LY294002 group's results compared with SCI group. Our study showed that NGF-induced PC12 cells can induce autophagy and ER stress after mechanical injury. ER stress inhibitor 4-PBA obtained similar effects to PI3K inhibitor LY294002, enhanced autophagy via PI3K/AKT/mTOR signaling pathway.

Mesenchymal stem cells modified with nerve growth factor improve recovery of the inferior alveolar nerve after mandibular distraction osteogenesis in rabbits

Distraction osteogenesis is widely used in the treatment of bony deformities and defects. However, injury to the inferior alveolar nerve is a concern. Our aim was to investigate the feasibility of using lentiviral-mediated human nerve growth factor beta (hNGFβ) of the inferior alveolar nerve in mandibular distraction osteogenesis in rabbits. To achieve this, mesenchymal stem cells (MSC) from the bone marrow of rabbit mandibles were isolated and genetically engineered using recombinant lentiviral vector containing hNGFβ. Twenty New Zealand white rabbits underwent mandibular distraction osteogenesis, and 5 million MSC transduced with hNGFβ-vector or control vector were transplanted around the nerve in the gap where the bone had been fractured during the operation (n=10 in each group). After gradual distraction, samples of the nerve were harvested for histological and histomorphometric analysis. We found that the genetically engineered MSC transduced by the lentiviral vector were able to secrete hNGFβ at physiologically relevant concentrations as measured by ELISA. Histological examination of the nerve showed more regenerating nerve fibres and less myelin debris in the group in which hNGFβ-modified MSC had been implanted than in the control group. Histomorphometric analysis of the nerve showed increased density of myelinated fibres in the group in which hNGFβ-modified MSC had been implanted than in the control group. The data suggest that implantation of hNGFβ-modified MSC can accelerate the morphological recovery of the inferior alveolar nerve during mandibular distraction osteogenesis in rabbits. The use of lentiviral-mediated gene treatment to deliver hNGFβ through MSC may be a promising way of minimising injury to the nerve.

Effects of subcutaneous transfection of human beta-nerve growth factor gene on function of sensory nerve in a rat model of diabetic neuropathic pain

Objective To evalute the effects of subcutaneous transfection of human beta-nerve growth factor (Ad-hNGFβ) gene on the function of sensory nerve in a rat model of diabetic neuropathic pain (DNP).Methods Healthy male Sprague-Dawley rats,weighing 180-220 g,were used in this study.Ten rats were randomly collected as normal control without DNP (group C).DNP model was established by intraperitoneal injection of streptozocin (STZ) 75 mg/kg.Seventy-five rats with DNP were randomly divided into 3 groups (n =25 each):DNP group; Ad-hNGF group and Ad-LacZ group.Groups Ad-NGF and Ad-LacZ received subcutaneous transfection of 1.12 × 1011 PFU Ad-hNGFβ 10 ul and 1.12 × 1011 PFU Ad-LacZ 10 ul respectively after pain thresholds were measured on 21 days after STZ injection.The mechanical and thermal pain thresholds were measured before STZ injection (baseline) and at 21,35 and 49 days after STZ injection.The conduction velocity and latency of the sensory nerve and the expression of substance P (SP) in dorsal root ganglion (by immmuno-histpchemistry) were determined after measurement of pain threshold on 49 days after STZ injection.Results Compared with group C,the mechanical and thermal pain thresholds were significantly increased at each time point after STZ injection in groups DNP,Ad-NGF and Ad-Lacz,the conduction velocity of the sensory nerve was significantly decreased,the latency of the sensory nerve was prolonged,and the expression of SP in dorsal root ganglion was down-regulated in groups DNP and Ad-LacZ,the conduction velocity of the sensory nerve was significantly decreased (P ＜ 0.05),and no significant change was found in the latency of the sensory nerve and expression of SP in dorsal root ganglion in group Ad-NGF (P ＞ 0.05).Compared with group DNP,the thermal pain threshold was significantly increased at 49 days after STZ injection,the conduction velocity of the sensory nerve was decreased,the latency of the sensory nerve was shortened,and the expression of SP in dorsal root ganglion was down-regulated (P ＜ 0.05 or 0.01),while no significant change was found in the mechanical pain threshold in group Ad-NGF,and no significant change was found in the parameters mentioned above in group Ad-LacZ (P ＞ 0.05).Conclusion Subcutaneous transfection of Ad-hNGFβ can improve the function of sensory nerve in a rat model of DNP through up-regulat-ing of the SP expression in dorsal root ganglion. Key words: Nerve growth factor; Transfection ; Diabetes mellitus; Neuralgia; Neural conduction

Effects of subcutaneous transfection of human beta-nerve growth factor gene on expression of substance P in dorsal root ganglion in a rat model of diabetic neuropathic pain

Objective To investigate the effects of subcutaneous transfection of human beta-nerve growth factor (Ad-hNGFβ) gene on the expression of substance P (SP) in the dorsal root ganglion in a rat model of diabetic neuropathic pain (DNP).Methods Male SD rats weighing 180-220 g were used in this study.Ten rats were randomly collected as normal control without DNP (group C).DNP model was made by intraperitoneal injection of streptozocin (STZ) 75 mg/kg.Seventy-five rats with DNP were randomly divided into 3 groups ( n =25 each):DNP group; Ad-hNGF group and Ad-LacZ group.Groups Ad-NGF and Ad-LacZ received subcutaneous transfection of 1.12 × 1010 PFU Ad-hNGFβ 10 μl and 1.12 × 1010 PFU Ad-LacZ 10 μl respectively after pain threshold was measured on 21d after STZ injection.The mechanical and thermal pain threshold was measured before STZ injection (baseline) and at 21,35 and 49 d after STZ injection.The expression of SP in the dorsal root ganglion was determined after the measurement of pain threshold on 49 d after STZ injection.Results Compared with group C,the mechanical and thermal pain threshold was significantly decreased at each time point after STZ injection in groups DNP,Ad-NGF and Ad-LacZ,and the expression of SP in the dorsal root ganglion was signilicantly downregulated in groups DNP and Ad-LacZ (P ＜ 0.05).Compared with group DNP,the thermal pain threshold was significantly increased on 49 d afar STZ injection,and the expression of SP in the dorsal root ganglion was significantly up-regulated in group Ad-NGF ( P ＜ 0.01 ),and no significant change was found in the mechanical and thermal pain threshold and the expression of SP in the dorsal root ganglion at each time point in group Ad-LacZ ( P ＞ 0.05).Conclusion Subcutaneous transfection of Ad-hNGFβ can attenuate DNP to some extent through upregulation of the expression of SP in rat dorsal root ganglion. Key words: Nerve growth factor; Transfcction; Diabetes mellitus; Neuralgia; Substance P; Ganglia, spinal

A Pilot Study Evaluating the Effect of Recombinant Human Bone Morphogenetic Protein‐2 and Recombinant Human Beta‐Nerve Growth Factor on the Healing of Class III Furcation Defects in Dogs

The quantity of regenerated bone induced by recombinant human bone morphogenetic protein-2 (rhBMP2) is encouraging, but sometimes the quality is inferior. Recombinant human beta-nerve growth factor (rh beta-NGF) plays a major role in bone remodeling. This study evaluates the quality and quantity of regenerated bone in periodontal regeneration following topical application of the two growth factors to Class III furcation defects. Thirty-six inflamed Class III furcation defects were created in six beagle dogs at sites of mandibular premolars 2, 3, and 4, and then biodegradable hydrogel incorporating rhBMP2 and rh beta-NGF was topically applied to the defects. The groupings were as follows: G1, untreated (control group A); G2, carrier alone (control group B); G3, 0.4% rhBMP2 + carrier; G4, 2% rh beta-NGF + carrier; G5, 0.4% rhBMP2 + 2% rh beta-NGF + carrier; and G6, 0.2% rhBMP2 + 1% rh beta-NGF + carrier. Eight weeks after application, the quality and quantity of regenerated tissue were evaluated by scanning electron microscopy observation, calcium/phosphorus ratio analysis, and histologic evaluation. The regenerated bone in G5 exhibited the highest calcium/phosphorus ratio among all groups and showed a denser structure with more calcified substances on the collagen fiber surface than that in the other groups. Histomorphometric analysis revealed that 0.4% rhBMP2 + 2% rh beta-NGF promoted the highest percentage of periodontal regeneration among all groups. The results of this pilot study suggest that a topical application of rhBMP2 and rh beta-NGF may improve the quality and quantity of regenerated bone in artificially created Class III furcation defects of beagle dogs.

Bladder Regeneration by Collagen Scaffolds With Collagen Binding Human Basic Fibroblast Growth Factor

Studies show that basic fibroblast growth factor can promote bladder regeneration. However, the lack of targeting delivery approaches limits its clinical application. We investigated a collagen based targeting system for bladder regeneration. A collagen binding domain was added to the native basic fibroblast growth factor N-terminal to allow it to bind to collagen. Sprague-Dawley rats underwent partial cystectomy. Collagen scaffolds loaded with collagen binding domain basic fibroblast growth factor, native basic fibroblast growth factor or phosphate buffered saline were grafted to the remaining host bladders, respectively. At days 30 and 90 reconstructed bladders were evaluated by histological analysis and urodynamics. This targeting basic fibroblast growth factor delivery system induced satisfying bladder histological structures. It promoted more vascularization and smooth muscle cell ingrowth. Urodynamics revealed well accommodated bladder tissue with volume capacity and compliance. Results show that the targeting delivery system consisting of collagen binding domain basic fibroblast growth factor and collagen membranes induced better bladder regeneration at the injury site. Thus, this targeting delivery system may be an effective strategy for bladder regeneration with potential clinical applications.

Recombinant expression of human nerve growth factor beta in rabbit bone marrow mesenchymal stem cells

Nerve growth factor (NGF) is required for the differentiation and maintenance of sympathetic and sensory neurons. In the present study, the recombinant expression of human nerve growth factor beta (hNGF-β) gene in rabbit bone marrow mesenchymal stem cells (rMSCs) was undertaken. Recombinant vector containing hNGF-β was constructed and transferred into rMSCs, the expressions of the exogenous in rMSCs were determined by reverse transcriptase PCR (RT-PCR), ELISA and Western blot, whereas the biological activity of recombinant hNGF-β was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. The results showed that the hNGF-β gene expressed successfully in the rMSCs, a polypeptide with a molecular weight of 13.2 kDa was detected. The maximal expression level of recombinant hNGF-β in rMSCs reached 126.8012 pg/10(6) cells, the mean concentration was 96.4473 pg/10(6) cells. The recombinant hNGF-β in the rMSCs showed full biological activity when compared to commercial recombinant hNGF-β.

Application of nerve growth factor by gel increases formation of bone in mandibular distraction osteogenesis in rabbits

The long period of bony consolidation is a concern in mandibular distraction osteogenesis (DO). We have previously shown that repeated local injections of human nerve growth factor beta (NGFβ) can appreciably improve bony consolidation in a rabbit model of DO. The present study was designed to test the effect of a single injection of human NGFβ delivered by collagen/nano-hydroxyapatite/kappa-carrageenan gels to sites of new bony formation in DO. Rabbits underwent mandibular DO at a rate of 0.75 mm/12 h for 6 days. At the end of the distraction period, the following injections were given percutaneously into the callus ( n = 6 in each of the four groups): human NGFβ in the gel; human NGFβ in saline; the gels alone; and saline alone. Fourteen days after the end of distraction, mechanical testing, histological and histomorphometric variables of the new bone were evaluated. Histologically, the NGFβ group had more advanced consolidation than the other three groups. Both maximal load and bone volume/total volume in this group were significantly higher than in the other three ( P < 0.05). In conclusion, the delivery of human NGFβ in the gels results in better acceleration of new bone formation than when it is given in saline, and may be a possible way to shorten the duration of craniofacial DO.

Cell Death and Learning Impairment in Mice Caused by in Vitro Modified Pro-NGF Can Be Related to Its Increased Oxidative Modifications in Alzheimer Disease

Pro-nerve growth factor (pro-NGF) is expressed at increased levels in Alzheimer's disease (AD)-affected brains and is able to induce cell death in cultures; however, the reasons for these phenomena remain elusive. Here we show that pro-NGF in human AD-affected hippocampus and entorhinal cortex is modified by advanced glycation and lipoxidation end-products in a stage-dependent manner. These modifications block pro-NGF processing to mature NGF, thus making the proneurotrophin especially effective in inducing apoptosis of PC12 cells in culture through the p75 neurotrophin receptor. The processing of advanced glycation and lipoxidation end-products in vitro modified recombinant human pro-NGF is severely impaired, as evidenced by Western blot and by examining its physiological functionality in cell cultures. We also report that modified recombinant human pro-NGF, as well as pro-NGF isolated from human brain affected by AD, cause impairment of learning tasks when administered intracerebroventricularly in mice, which correlates with AD-associated learning impairment. Taken together, the data we present here offer a novel pathway of ethiopathogenesis in AD caused by advanced glycation and lipoxidation end-products modification of pro-NGF.

Haploinsufficiency of the nerve growth factor beta gene in a 1p13 deleted female child with an insensitivity to pain

Pain insensitivity is mediated at the genetic level by the disruption of specific genes associated with neuronal development. Mammalian in vivo and in vitro studies have shown the nerve growth factor (NGF) gene to play an integral role in nerve maintenance and function. Pain insensitivity in humans can be attributed to hereditary sensory and autonomic neuropathies (HSAN) of which there are five classes (HSAN I - HSAN V). The human nerve growth factor beta gene (NGFB) located on chromosome 1p13.2 has been found to cause HSAN V within individuals homozygous for a point mutation in NGFB. Although heterozygotes can display a milder phenotype, this has only been observed in adults. We report a karyotypically normal 5-year-old female with developmental delay, mild facial dysmorphism, and unsteady gait. Pain and thermal insensitivity were noted as were recurrent mouth ulcers, facial flushing, recurrent episodes of increased body temperature and unexplained sweating, indicative of a sensory neuropathy with mild autonomic involvement. Array comparative genomic hybridization (aCGH) analysis revealed a de-novo deletion within chromosome 1p13 of the child involving the NGFB gene. Sequence analysis of the homologous NGFB gene identified no mutation, implying that sensory neuropathy was caused by haploinsufficiency of the NGFB gene.

Effects of transfection of human beta-nerve growth factor gene on substance P and calcitonin gene-related peptide content of the spinal cord in a rat model of neuropathic pain

Objective To investigate the effects of adenovirus containing human beta-nerve growth factor (Ad-hNGFβ) gene on substance P (SP) and calcitonin gene-related peptide (CGRP) content of the spinal cord in a rat model of neuropathic pain by chronic constrictive injury (CCI). Methods Forty-eight male SD rats weighing 200-250 g were randomly divided into 3 groups (n=16 each) : group Ⅰ sham operation; group Ⅱ CCI and group Ⅲ CCI + Ad-hNGFβ gene IT. The animals were anesthetized with intraperitoneal choral hydrate 300-350 mg/kg. The right sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1-2 nun intervals as described by Bennet and Xie[5]. In sham operation group, right sciatic nerve was exposed but not ligated. In group Ⅰ and Ⅱ artificial cerebrnspinal fluid was injected IT instead of Ad-hNGFβ gene. The behavior score and the paw-withdrawal latency (PWL) to radiant heat and mechanical stimulus were measured one day before operation and every 4 days within the 28 days after gene transfection. Four animals were killed at 4, 7, 14 and 28 day after IT gene transfection in each group and lumbar segment (L4-6 ) of the spinal cord was removed for determination of SP and CGRP content by immunohistochemistry. Results The behavior scores were significantly higher and PWL to radiant heat and mechanical stimulus were significantly lower in group Ⅱ and Ⅲ than in group Ⅰ. There was no significant difference in the behavior score and PWL to mechanical stimulus between group Ⅱ and Ⅲ while the PWL to radiant heat was significantly higher in group Ⅲ than in group Ⅱ. After operation SP and CGRP content were significantly higher in group Ⅱ and group Ⅲ than in group Ⅰ , and significangly lower in group Ⅲ than in group Ⅱ 7-28 days after operation. Conclusion The recomhinant Ad-hNGFβ gene transfection can attenuate heat hyperalgesia by reducing SP and CGRP content of the spinal cord in a rat model of neuropathic pain induced by CCI. Key words: Nerve growth factor; Injections, spinal; Transfection; Neuropathic pain; Substance P; Calcitonin gene-related peptide

Effects of locally applied nerve growth factor to the inferior alveolar nerve histology in a rabbit model of mandibular distraction osteogenesis

Distraction osteogenesis (DO) is widely used in deformities and defects of the craniofacial bone. Accelerating inferior alveolar nerve (IAN) recovery would aid the process. Nerve growth factor (NGF) plays a vital role in peripheral nerve regeneration. In this study, the ability of locally applied human NGF beta (hNGFβ) to enhance the morphological recovery of the IAN in a rabbit model of mandibular DO was studied. Rabbits underwent bilateral DO with a rate of 0.5 mm per 12 h. Two doses of 40 μg hNGFβ in buffer were injected into callus at the beginning the of consolidation time. The contralateral side received injections of placebo. Rabbits were killed at 14 and 28 days. IAN specimens were subjected to histological and histomorphometric analysis. In both 14 and 28 days consolidation experiments, nerve histological analysis showed less degeneration and more regeneration in nerve fibers on the hNGFβ treated side than the control side. Histomorphometric analysis showed that the myelinated fiber density on the hNGFβ treated side was significantly higher than on the control side ( p < 0.01). The data indicate that locally applied hNGFβ can accelerate the morphological recovery of the IAN and may play a role in reducing nerve injury in mandibular DO clinically.

Expression of Recombinant Human Nerve Growth Factor Beta in Milk of Goats by Recombinant Replication-Defective Adenovirus

For producing a large quantity of human nerve growth factor beta (hNGF-beta), the recombinant adenovirus containing an expression cassette of hNGF-beta complementary DNA gene was constructed and then instilled directly into goats' mammary glands. The recombinant hNGF-beta was detected in the milk. The maximal expression level of recombinant hNGF-beta in the milk reached 196.8 mg/l. The biological activity of the recombinant hNGF-beta, compared with the commercial hNGF-beta, was tested using PC12 cells. The results demonstrate that instilling recombinant adenovirus directly into the mammary gland of goat is an efficient approach to producing a large quantity of hNGF-beta.

High-level expression of recombinant human nerve growth factor beta in milk of nontransgenic rabbits

The technology for the large-scale production of therapeutic recombinant proteins remains a challenge in the biopharmaceutical industry. In this study, we reported a nontransgenic approach to producing a large quantity of human nerve growth factor beta (hNGF-beta) in rabbit milk by employing a recombinant adenoviral expression system. After directly instilling hNGF-beta recombinant adenoviruses into rabbit mammary glands, a polypeptide with a molecular weight of 13.2 kDa was detected in rabbit milk. The maximal expression level of hNGF-beta reached 346 mug/ml. The biological activity of recombinant hNGF-beta was confirmed using PC12 cells and cultures of dorsal root ganglion neurons from chicken embryos. Our data suggest that instilling recombinant adenovirus directly into the mammary gland of mammals is an efficient approach to producing a large quantity of hNGF-beta.

Proliferation and regeneration effects of constructed human beta-nerve growth factor gene on transfected cat corneal endothelial cells in vitro

To explore the mechanisms of proliferation and regeneration effects of a human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF) on the transfected cat corneal endothelial cells in vitro. To provide a new method for long term cultivation of human corneal endothelial cells in vitro and to establish theoretical basis of gene therapy for corneal endothelial defects. It was a experimental study. The human pcDNA4-beta-NGF expression vector was constructed and transfected into cultured cat corneal endothelial cells by Effectene lipofectine transfection technique. The expression of the reporter gene pcDNA4-beta-LacZ expression was used to determine the transfection efficiency 48 hours after the transfection. RT-PCR and immunohistochemistry techniques were used to check the transient expression status at mRNA and protein levels in cat corneal endothelial cells. Mitotic index and methyl thiazolyl tetrazolium (MTT) value were measured and cell numbers at different stages of cell cycles were determined by flow cytometer 96 hours after transfection. An in vitro quantitative cat corneal endothelial cell traumatic model was established which was used for observing the effect of human beta-NGF expression product on the DNA synthesis of cat endothelial cells and healing process of traumatized endothelial cells. A human nerve growth factor (beta-NGF) expression vector (pcDNA4-beta-NGF)was successfully constructed and confirmed by sequence analysis. Single layered pure cat corneal endothelial cells were obtained by a modified sliced tissue culture technique and confirmed by morphological analysis, neurone specific enolase immunohistochemistry study and transmission electronic microscope. Effectene lipofectine mediated transfection efficiency of pcDNA4-beta-NGF into cat corneal endothelial cells in vitro was 11.3%. The human beta-NGF could be highly expressed in the transfected corneal endothelial cells at mRNA and protein levels. Mitotic index, MTT value and G1 stage cell numbers, as well as traumatically defected endothelial cells numbers during the healing process of human beta-NGF transfected corneal endothelial cells were statistically differed from the pre-transfected cells and control groups. Effectene lipofectine transfection technique could be effectively used for transfecting pcDNA4-beta-NGF into cat corneal endothelial cells in vitro with good efficacy and the gene could stably express to improve the proliferation and regeneration of the cat corneal endothelial cells. This method could be managed as an experimental basis to be applied in the experimental study for transfecting the human beta-NGF gene into human corneal endothelial cells. Therefore a new method for resolving the problem of impossible regeneration of corneal endothelial cells could become possible.

Pro-NGF Isolated from the Human Brain Affected by Alzheimer's Disease Induces Neuronal Apoptosis Mediated by p75NTR

The pro-form of nerve growth factor (pro-NGF) has been shown to be a high affinity ligand for p75NTR and to induce apoptosis through this receptor. It has been reported that pro-NGF, rather than mature NGF, is the predominant form of this neurotrophin in human brain. In the present work we studied the potential involvement of pro-NGF purified from human brains affected by Alzheimer's disease (AD), where it is especially abundant, in the neuronal apoptosis observed in this disease. Western blot analysis of human brain tissue showed the existence of several pro-NGF forms. Some of these pro-NGF forms were significantly increased in AD brain cortex in a disease stage-dependent manner. Pro-NGF, purified by chromatography from human AD brains, induced apoptotic cell death in sympathetic neurons and in a p75NTR stably transfected cell line. Blocking p75NTR in cell culture abolished neuronal apoptosis caused by pro-NGF. p75NTR-transfected cells underwent apoptosis in the presence of pro-NGF while control wild-type cells did not. Taken together, these results indicate that pro-NGF purified from AD human brains can induce apoptosis in neuronal cell cultures through its interaction with the p75NTR receptor.

Overproduction of Bacterial Protein Disulfide Isomerase (DsbC) and Its Modulator (DsbD) Markedly Enhances Periplasmic Production of Human Nerve Growth Factor in Escherichia coli

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.

Human nerve growth factor beta (hNGF-β): mammary gland specific expression and production in transgenic rabbits

Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-β) cDNA were generated. Expression of hNGF-β mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-β was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-β depicted a molecular weight of 13 261 Da per subunit. The biological activity of the hNGF-β was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-β demonstrated full biological activity when compared to commercial recombinant hNGF-β.

Meetings and Notices

Distraction osteogenesis is widely used in the treatment of bony deformities and defects. However, injury to the inferior alveolar nerve is a concern. Our aim was to investigate the feasibility of using lentiviral-mediated human nerve growth factor beta (hNGFβ) of the inferior alveolar nerve in mandibular distraction osteogenesis in rabbits. To achieve this, mesenchymal stem cells (MSC) from the bone marrow of rabbit mandibles were isolated and genetically engineered using recombinant lentiviral vector containing hNGFβ. Twenty New Zealand white rabbits underwent mandibular distraction osteogenesis, and 5 million MSC transduced with hNGFβ-vector or control vector were transplanted around the nerve in the gap where the bone had been fractured during the operation (n = 10 in each group). After gradual distraction, samples of the nerve were harvested for histological and histomorphometric analysis. We found that the genetically engineered MSC transduced by the lentiviral vector were able to secrete hNGFβ at physiologically relevant concentrations as measured by ELISA. Histological examination of the nerve showed more regenerating nerve fibres and less myelin debris in the group in which hNGFβ-modified MSC had been implanted than in the control group. Histomorphometric analysis of the nerve showed increased density of myelinated fibres in the group in which hNGFβ-modified MSC had been implanted than in the control group. The data suggest that implantation of hNGFβ-modified MSC can accelerate the morphological recovery of the inferior alveolar nerve during mandibular distraction osteogenesis in rabbits. The use of lentiviral-mediated gene treatment to deliver hNGFβ through MSC may be a promising way of minimising injury to the nerve.

Effect of Nerve Growth Factor on the Release of Inflammatory Mediators by Mature Human Basophils

Nerve growth factor (NGF) is a neurotrophic cytokine known to regulate the survival and function of peripheral and central neuronal cells. Recently, the spectrum of action could be extended to non-neuronal cell types such as rat mast cells and human B lymphocytes. The present study shows that NGF affects the function of mature human basophils isolated from the peripheral blood of healthy donors. Both murine NGF 7S and recombinant human NGF beta enhance histamine release and strongly modulate the formation of lipid mediators by basophils in response to various stimuli. This priming effect of NGF on basophils occurs rapidly within 10 to 15 minutes of preincubation, is dose-dependent, and requires similarly low concentrations (1 to 40 pmol/L) of human NGF beta as the induction of neurite outgrowth in ganglion cells. Cell fractionation studies indicate that NGF acts directly on human basophils without an involvement of other cell types, suggesting the presence of high-affinity NGF receptors on basophils. NGF by itself (up to 4 nmol/L of human NGF beta) does not induce the release of inflammatory mediators directly. The effect of human NGF on basophil mediator release is similar to that of the hematopoietic growth factors interleukin-3, interleukin-5, and granulocyte-macrophage colony-stimulating factor. The present study further demonstrates that NGF acts as a pleiotropic cytokine at the interface between the nervous and the immune system, and that NGF may be involved in inflammatory processes and hypersensitivity reactions.