Nerve Growth Factor Receptor Expression Research Articles

Ocular Application of Nerve Growth Factor Protects Degenerating Retinal Ganglion Cells in a Rat Model of Glaucoma

Elevated intraocular pressure is a crucial pathologic event for the development of glaucoma (GL). We have reported that nerve growth factor (NGF) reaches retinal cells and the optic nerve (ON) when applied to the eye. Whether ocular application of NGF prevents or reduces damage to retinal ganglion cell (RGC) is not known. GL was induced in adult rats by the injection of hypertonic saline into the episcleral vein of the right eye and the left eye used as control. Rats were then treated daily with ocular application of NGF or vehicle solution for 7 weeks. Retinal and ON tissues were then used for structural, immunohistochemical, and biochemical studies. The injection of hypertonic saline into the episcleral vein led to progressive degeneration of RGCs, with the loss of nearly 40% of these cells after 7 weeks of treatment. This cellular loss is associated with the downregulation of NGF and NGF-receptor expression in the retina and ON of the glaucomatous eye and ocular treatment with NGF significantly reduced the deficit induced by GL. These findings indicate that NGF can exert protective action on RGC degeneration occurring in glaucomatous retina. We suggest that ocular NGF treatment might be a suitable pharmacologic approach to investigate protective mechanisms of degenerating RGCs.

NGFR (p75) Expression in Cutaneous Scars; Further Evidence for a Potential Pitfall in Evaluation of Reexcision Scars of Cutaneous Neoplasms, in Particular Desmoplastic Melanoma

Desmoplastic melanoma is a rare variant of malignant melanoma composed of spindle cells in a collagenous matrix. The antibody against NGFR (low affinity nerve growth factor receptor, also known as p75) stains cells of desmoplastic melanoma with high sensitivity; however, the specificity of this marker is not well established. Although there are established histologic criteria for recognition of desmoplastic melanoma, the evaluation of residual disease in cutaneous reexcision scars can be challenging. If residual spindle cells in scar are sufficiently atypical and NGFR positive, their presence could be interpreted as residual desmoplastic melanoma. In this study, we reevaluated the use of antibody against NGFR to detect residual disease in reexcision specimens of melanocytic neoplasms as the previously published works are contradictory. Our data indicate that anti-NGFR antibody stains many cells in the scar, some of which seem to be myofibroblasts, nerve twigs, and Schwann cells. Our findings further suggest that NGFR is not a suitable marker to evaluate reexcision scars for desmoplastic melanoma, especially as a sole marker, as its specificity is low.

Altered expression of nerve growth factor and its receptors in the kidneys of diabetic rats

Type 1 diabetes (DB) is a multifactorial metabolic disorder characterized by loss of insulin-producing pancreatic ß-cells, and metabolic and functional deficits in a number of cells, including kidney cells. Nerve growth factor (NGF) is a signaling molecule that is up-regulated in cells affected by diabetes-linked disorders. However, whether DB alters the expression of NGF in the kidney is not known. DB was induced in adult male rats with a single injection of streptozotocin (STZ), and NGF protein levels were analyzed in a time-course study in serum and kidney. The expression of NGF receptors in the kidneys of healthy and DB rats was evaluated by immunohistochemistry. NGF levels as well as apoptotic features in the kidneys of healthy rats injected with purified NGF were also assessed. This study revealed that DB elevates NGF levels in serum and NGF expression in the kidney and that subcutaneous administration of NGF causes a marked uptake of NGF in kidney cells. The elevated presence of NGF in kidney cells is not associated with proapoptotic factor expression. The present data suggest that NGF presence in the kidney might play a survival, and most probably protective, role in kidney cells.

Transient lysosomal activation is essential for p75 nerve growth factor receptor expression in myelinated Schwann cells during Wallerian degeneration

Myelinated Schwann cells in the peripheral nervous system express the p75 nerve growth factor receptor (p75NGFR) as a consequence of Schwann cell dedifferentiation during Wallerian degeneration. p75NGFR has been implicated in the remyelination of regenerating nerves. Although many studies have shown various mechanisms underlying Schwann cell dedifferentiation, the molecular mechanism contributing to the re-expression of p75NGFR in differentiated Schwann cells is largely unknown. In the present study, we found that lysosomes were transiently activated in Schwann cells after nerve injury and that the inhibition of lysosomal activation by chloroquine or lysosomal acidification inhibitors prevented p75NGFR expression at the mRNA transcriptional level in an ex vivo Wallerian degeneration model. Lysosomal acidification inhibitors suppressed demyelination, but not axonal degeneration, thereby suggesting that demyelination mediated by lysosomes may be an important signal for inducing p75NGFR expression. Tumor necrosis factor-α (TNF-α) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-α in vivo did not significantly suppress the induction of both lysosomes and p75NGFR. Thus, these findings suggest that lysosomal activation is tightly correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration.

Modulation of Nerve Growth Factor receptors in human monocytes and their influence in pulmonary inflammatory diseases

Neurotrophins (NTs) are a family of growth/survival factors with well-established functions in the nervous system. In the last decade, novel biological actions, from oncogenicity to inflammation, have been attributed to these factors. In particular, Nerve Growth Factor (NGF) acts through two different classes of receptors: the high affinity transducing TrKA receptor, associated to proliferation/survival, and the low affinity p75 receptor that, depending on its cross talk with TrKA, induces either apoptosis or survival. Recently, NGF and its receptors have been detected in healthy pulmonary tissues and seem to be involved in pulmonary inflammatory diseases. Since the expression of these NGF receptors in circulating monocytes is controversial, our initial aim was to investigate the role of these receptors both in pulmonary tissues and in peripheral blood monocytes of patients with Chronic Obstructive Pulmonary Disease (COPD). We therefore analyzed 38 healthy control subjects divided in two groups based on their smoking status. Serendipitously, the data obtained in these two control groups may have a value of their own, with potential implications in preventive medicine. We show here that in healthy subjects, smoking induces an early increase in p75 expression in monocytes, while TrKA seems unaffected. Furthermore, our control subjects could be divided in three subsets according to the constitutive TrKA expression in monocytes: TrKA-negatives, -intermediates (up to 50%) or -high (> 50%), independently on their smoking status. Since TrKA activation promotes inflammation, we hypothesize that subjects with high-TrKA monocytes could be more prone to pulmonary inflammatory diseases, such as COPD. In fact, all 28 COPD patients in our series belonged to the high-TrKA subset, while all long term heavy smokers with no evidence of pneumologic diseases belonged to TrKA-negative subset. In conclusion, our data support the hypothesis that, since TrKA expression promotes survival, elevated levels of TrKA-positive monocytes may render subjects more prone to long term inflammatory diseases (e.g., COPD). Moreover, in patients constitutively expressing high levels of TrKA in monocytes, the smoking-dependent increase in p75 may in turn further extend monocytes survival, contributing to a chronic inflammation. Furthermore, the early increase in p75 expression in monocytes following smoking may support the hypothesis that p75 determination might represent a novel marker for passive smoking. Acknowledgments. This study was totally supported by Fondazione Cassa di Risparmio di Terni

Vascular Endothelial Growth Factor and Its Receptors Are Expressed in Human Nerve Grafts

Vascularization and angiogenicity of human nonvascularized nerve grafts in the second stage of facial reanimation were studied. Immunohistochemistry for endothelial markers (CD-31) and vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 was performed on distal end biopsies from 35 cross-facial nerve grafts. In grafted nonvascularized nerve, density of vascular structures (also clearly immunopositive for VEGF and both receptors) showed a mean of 166 vessels (range 78 to 267) per unit area, corresponding to control values. In addition, VEGF was expressed in axons and perineural structures. In control samples, VEGF expression was low and occurred in the myelin sheath. In nerve grafts, expression of Flt-1 and Flk-1 (less intense) was seen in axons and perineural structures. A higher density of vessels was associated with lower VEGF expression (not significant). In short, expression of VEGF and its receptors is described in human nerve grafts and compared with basic histology and p75 nerve growth factor receptor expression of the nerve graft and functional outcome of patients.

Mouse Natural Killer (NK) Cells Express the Nerve Growth Factor Receptor TrkA, which Is Dynamically Regulated

BackgroundNerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.Methodology/Principal FindingsWe used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.Conclusions/SignificanceWe demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.

P75 nerve growth factor receptor staining is superior to S100 in identifying spindle cell and desmoplastic melanoma

Spindle cell melanoma (SCM) including desmoplastic melanoma (DM) is a rare variant of malignant melanoma that may present diagnostic difficulties particularly when staining with S100 is negative, weak, focal, or a combination of these. Conventional melanocytic markers in SCM are usually negative. We sought to compare the staining of p75 nerve growth factor receptor (NGF-R) and S100 in SCMs. We evaluated the staining of p75 NGF-R and S100 in 13 cases of SCMs: 3 SCMs without desmoplasia, 5 pure DMs, and 5 combined DMs with a conventional component. Staining with p75 NGF-R was positive in 13 of 13 (100%) cases of SCMs. In 3 cases the intensity of staining and the percentage of cells staining with this marker were greater than those with S100. One case of SCM was negative for S100 but demonstrated strong expression of p75 NGF-R. One case was focally and weakly positive with S100 but expressed strong positive staining with p75 NGF-R. Absence of staining with p75 NGF-R was noted in the conventional round cell component of two of 5 (40%) combined DMs whereas the same areas were strongly positive for human melanoma black (HMB)-45 and Melan-A. In 5 of 5 (100%) cases of combined DMs the desmoplastic component stained positive with p75 NGF-R, demonstrating an inverse relationship with the staining of conventional melanocytic markers. Small study size was a limitation. p75 NGF-R exhibits superior staining characteristics and greater sensitivity in identifying SCM and DMs than S100. P75 NGF-R may be a useful diagnostic and ancillary stain in addition to S100.

Abstract 1431: Nerve growth factor-expressing stromal cells in the microenvironment of hepatic colorectal carcinoma metastasis: Clinical occurrence and functional implications

Abstract Nerve growth factor (NGF) is increased during hepatic regeneration and carcinogenesis, but its role during hepatic metastasis is unknown. A tissue-array collection of metastases from 24 patients who had undergone hepatic excision of colorectal adenocarcinoma metastases was used to investigate NGF and neurotrophin receptor expression by cancer and stromal cells. NGF immunostaining of cancer cells only occurred in 2 out of 24 patients with hepatic metastases, while around 80% of patients had metastases with NGF-expressing stromal cells. Conversely, high affinity TrkA neurotrophin receptor immunoreactivity was mainly concentrated in cancer cells, with low expression occurring in tumor stroma. However, NGF immunostaining of tumor stroma and cancer cell immunostaining with anti-ki67 antibodies did not correlate, suggesting that NGF was not associated to metastatic cell proliferation. Anti-alpha-smooth muscle actin (ASMA) antobodies revealed that majority of metastasis-associated NGF-expressing cells had a myofibroblast phenotype. Interestingly, NGF immunoreactivity was unequivocally localized to desmin-expressing hepatic stellate cells (HSC), a prototypic myofibroblast precursor, and perimetastatic hepatocytes, located at the invasion front of metastases. NGF-expressing hepatocytes had phenotypic features suggesting epithelial-to-mesenchymal transition (snail, vimentin and ASMA overexpression with E-cadherin expression decrease). Consistent with clinical findings, a similar immunostaining pattern was reproduced in the experimental hepatic colonization of C26 and 51b colorectal cells. Moreover, NGF increased by two-fold in the hepatic blood from metastasis-bearing mice and also significantly increased by 2-3-fold in the supernatant of primary cultured HSC given tumor cell-conditioned medium (CM), and hepatocytes given tumor-activated HSC-CM, but not tumor cell-CM. Both NGF secretion and epithelial-to-mesenchymal transition in hepatocytes were TGFbeta-dependent. Recombinant NGF dose-dependently increased chemotactic migration, but not proliferation and adhesion of neurotrophin receptor-expressing cancer cells in vitro. HSC migration-stimulating activity of VEGF and tumor-activated hepatocytes were also NGF-mediated as shown with anti-NGF antibodies. Our results demonstrate that both hepatocyte- and HSC-derived myofibroblasts secrete NGF in the hepatic metastasis microenvironment of human and experimental colorectal carcinoma. The mechanism is TGFbeta-dependent. The migration-stimulating effect of NGF on both cancer and stromal cells suggests its contribution to hepatic metastasis development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1431.

Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells

Adenoviral vector-mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC) when transient gene expression is preferred. Previous studies have demonstrated that fiber-retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we investigated the potential of bi-directional promoter-controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs. We have engineered Ad5F35-DeltaLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (DeltaLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase promoter and minimal core promoter from human cytomegalovirus. The expression pattern of DeltaLNGFR and GFP following Ad5F35-DeltaLNGFR-BiDp gene transfer in various cell types, including candidate HSCs, was compared to Ad5F35-DeltaLNGFR-IRES vector encoding phosphoglycerate kinase promoter-controlled bicistronic expression cassette for DeltaLNGFR and GFP. Using Ad5F35-DeltaLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34(+) cells. However, the ability of Ad5F35-DeltaLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between repopulating progenitor cells. In nonobese diabetic severe combined immune deficient mice bone marrow transplantation assay, sorted CD34(+)/DeltaLNGFR(+)/GFP(+) cells following infection with Ad5F35-DeltaLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34(+)/DeltaLNGFR(+)/GFP(-) cells exhibited both myeloid and lymphoid reconstitution. This study indicates that bi-directional promoter-controlled Ad5F35 vector, such as Ad5F35-DeltaLNGFR-BiDp, can be particularly useful for manipulation of myeloid progenitor cells and potentially in myeloid lineage leukemic cells as well.

Functional significance of nerve growth factor and its receptor (TrkA) in inflammatory arthritis

We read the article by Barthel and colleagues [1] in this is sue of Arthritis Research & Th erapy with great interest. Th ey reported that neurotrophin receptors/ligands and specifi cally nerve growth factor (NGF) and NGF receptor (NGF-R) (TrkA and p75) are expressed in synovial fl uid (SF) cells and synovial tissue (ST) of patients with rheuma toid arthritis (RA) or spondyloarthritis (SpA). Th e authors also looked for the cellular source of NGF and demonstrated that the T cells and monocytes/macrophages derived from SF of patients with RA/SpA are enriched with NGF. Furthermore, they reported that STderived fi broblast-like synoviocytes (FLSs) did not produce NGF in vitro. In a recent publication, we observed similarly that NGF levels in SF were signifi cantly higher in patients with psoriatic arthritis (PsA) (365.5 ± 85.2 pg/ mL) or RA (120 ± 35 pg/mL) than in patients with osteoarthritis (OA) (30 ± 6 pg/mL) [2]. However, in regard to the source of NGF, we observed that FLSs produced a signifi cant amount of NGF. Here, we would like to share our observations about the NGF/TrkA system in human FLSs and its function. Synovial biopsies from patients with meniscal injury without any other joint diseases or PsA, OA, or RA were collected. FLSs were isolated and examined for NGF/ NGF-R expression in accordance with our standardized protocols [2]. Using a highly sensitive enzyme-linked immuno sorbent assay (ELISA) for human NGF (NGFEmax assay; Promega Corporation, Madison, WI, USA) and Hi-D fl uorescence-activated cell sorting analyses, we observed that under basal conditions FLSs from healthy individuals express low levels of NGF/TrkA. However, there was a marked upregulation of NGF and TrkA in these FLSs following stimulation with tumor necrosis factor-alpha and interleukin-1-beta (Table 1). A critical observation was that FLSs from patients with PsA or RA produced spontaneously higher levels of NGF and had increased expression of TrkA compared with FLSs of patients with OA (Table 1). Furthermore, we observed that NGF signifi cantly stimulated the proliferation of FLSs derived from PsA synovial tissue (Figure 1). Barthel and colleagues [1] cultured FLSs from only one patient with RA and one patient with SpA and noticed that FLSs did not produce NGF, but the authors do mention that they can’t rule out the production of NGF by FLS. We cultured FLSs from 15 subjects (Table 1), and it is also possible that the NGF ELISA kit that we used is more sensitive. A fully formed pannus is characterized by proliferation of FLSs, infl ammatory infi ltrates, and a marked angiogenesis. NGF and its receptor system are known to

P16-50. Immune reconstitution and antiviral control of SIV following adoptive transfer of anti-CD3/28 expanded CD4+ T cells: Induction of antiviral control

Results Antiviral control was characterized with the extended presence multifunctional antigen specific CD4 and CD8+ T cells detected even 1 year post transfer while viral loads remained mostly undetectable. Monitoring of trafficking and turnover of adoptively transferred anti-CD3/28 expanded CFSE labeled CD4+ T cells demonstrated extensive recirculation and dissemination of these cells with relatively slow homeostatic proliferation over time and long time engraftment in LTNP monkeys. In efforts to examine the long-term contribution of adoptively transferred anti-CD3/28 expanded CD4+ T cells, another set of monkeys was treated with transduced with a retroviral vector directing intracellular expression of the HSV-tk suicide gene and the extracellular expression of the nerve growth factor receptor (NGFR). Transduced cells enriched based on NGFR expression were expanded with anti-CD3/ 28 coated beads and transfused in SIV infected monkeys under short term ART. Two weeks following the last adoptive transfer, the transferred cells were selectively eliminated by ganciclovir administration, resulting in various levels of viral rebound after initial control. The data lends support to a long term therapeutic effect of adoptively transferred in vitro expanded CD4+ T cells even in the absence of transduction of antiviral moiety, these cells appear refractory to SIV production, for at least 2 weeks post expansion.

Mechanical ventilation with high tidal volume or frequency is associated with increased expression of nerve growth factor and its receptor in rabbit lungs

Nerve growth factor (NGF), a neurotrophin, is induced in lung cells by proinflammatory cytokines, and has a role in bronchial hyperreactivity and lung tissue repair. Ventilation induced lung injury, on the other hand, is known to increase the levels of proinflammatory cytokines in the lungs. We investigated whether, and to what extent, various degrees of lung injury induced by short-term ventilation affect NGF levels in the lung tissue of adolescent rabbits. The rabbits were randomized to different modes of ventilation: (1) CON: normal ventilation for 30 min; (2) NVT: normal ventilation for 6 hr; (3) HFQ: ventilation for 6 hr at double frequency, but normal tidal volume (VT); and (4) HVT: 6 hr ventilation at double VT but normal frequency. NGF protein was detected in bronchoalveolar lavage fluid (BALF) and lung tissue in all animals. Ventilation for 6 hr significantly increased NGF levels, in both BALF and lung tissue, in the HFQ and HVT groups as compared to control (P < 0.05). The maximum increase in BALF NGF was seen in the HVT group (P = 0.02 vs. CON and NVT groups, and P = 0.05 vs. HFQ). A parallel increase in interleukin 1-beta (IL1-beta) was observed. Expression of the high-affinity NGF-receptor, tropomyosin-related kinase A (TrkA), was also upregulated in these two groups. Injurious modes of mechanical ventilation upregulate NGF and its receptor TrkA in rabbit lungs, and IL1-beta may be a mediator for this response. We speculate that this increase in NGF level may translate into the development of bronchial hyperreactivity.

Increased Expression of Nerve Growth Factor Receptor and Neural Endopeptidase in the Lesional Skin of Melasma

Characteristic clinical features of melasma are symmetry of hyperpigmentation and distribution related to trigeminal nerves, which suggest that the neural involvement could play a part in the pathogenic mechanisms of pigmentation. To evaluate whether some neuropeptides and neurotrophins and their receptors were associated with the pathogenesis of melasma. To investigate the involvement of neuronal system and neuropeptides in melasma, we examined the expression of nerve growth factor receptor (NGFR) and neural endopeptidase (NEP) in melasma lesional and nonlesional skin. Skin biopsies were obtained from lesional and nonlesional facial skin of six Korean women with melasma. Confocal laser scanning microscopic examination and western blot were performed. Melasma lesions showed markedly greater expression of NGFR and NEP than nonlesional skin. We suggest that neuroactive molecules, including NGF, is one of the critical factors for the pathogenesis of melasma, which may directly affect the microenvironment around melanocytes through a NGFR immunoreactivity (NGFR-IR) nerve fiber pathway, and higher levels of NEP in melasma has an important role in regulation of melanogenesis.

Effect of acetyl- l-carnitine on ovarian cancer cells' proliferation, nerve growth factor receptor (Trk-A and p75) expression, and the cytotoxic potential of paclitaxel and carboplatin

Objectives The incidence of chemotherapy induced peripheral neuropathy (CIPN) is 15–25% with platinum and taxanes. CIPN can be permanent and often requires dose reduction or change in chemotherapy. Acetyl- l-carnitine (ALCAR), an ester of l-carnitine, is used to treat CIPN in humans and in animal models. The goals of this study are: 1) examine the effects of ALCAR on ovarian cancer cells, 2) determine if ALCAR affects the cytotoxicity of standard chemotherapy on ovarian cancer cells. Methods OVCAR-3 and SKOV-3 ovarian cancer lines were incubated in ALCAR containing media. Viability, proliferation, and expression of the nerve growth factor receptors (NGFR) Trk-A and p-75 were determined by flow cytometry. Cytotoxicity assays examining ALCAR's effect on paclitaxel and carboplatin were done by flow cytometry and infrared plate-reader. Results Flow cytometry showed no change in percent live ( p = 0.87) or proliferation ( p = 0.95) of OVCAR-3 cells when comparing controls with up to 100 μM ALCAR. However, there was a slight but significant decrease in the proliferation of SKOV-3 cells incubated at higher ALCAR concentrations ( p = < 0.01). Flow cytometry showed no difference in the viability of OVCAR-3 cells when comparing ALCAR: +/− paclitaxel ( p = 1), +/− carboplatin ( p = 0.8), or both ( p = 0.4). Proliferation assays indicated that paclitaxel's cytotoxicity on OVCAR-3 and SKOV-3 cells was unchanged at higher ALCAR concentrations ( p = < 0.01–0.4). ALCAR did not affect the expression of NGFR on OVCAR-3 or SKOV-3 cells. Conclusion ALCAR does not affect the cytotoxicity of paclitaxel or carboplatin. There was no increase in proliferation, or NGFR of OVCAR-3 or SKOV-3 cells exposed to ALCAR.

Localization of the low‐affinity nerve growth factor receptor p75 in human limbal epithelial cells

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n= 5) and foetal (n= 3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n= 10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n= 4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.

Glaucoma alters the expression of NGF and NGF receptors in visual cortex and geniculate nucleus of rats: Effect of eye NGF application

We investigated the effect of glaucoma (GL) on nerve growth factor (NGF) presence in two brain visual areas. Rats with elevated intraocular pressure (EIOP), induced by hypertonic saline injection in the episcleral vein, were treated with eye topical application of saline or NGF. Rats were subsequently sacrificed, and brain tissues were used for immunohistochemical, biochemical, and molecular analyses. We found that GL alters the basal level of NGF and NGF receptors in brain visual centers and that NGF eye application normalized these deficits. These findings demonstrate that the reduced presence of NGF can arise due to degenerative events in retinal and brain visual areas.

Differential modulation of nerve growth factor receptor (p75) and cholinergic gene expression in purified p75-expressing and non-expressing basal forebrain neurons by BMP9

The synthesis of acetylcholine and its release from basal forebrain cholinergic neurons (BFCN) that innervate the cerebral cortex and hippocampus are considered essential processes for normal learning, memory and attention. We have developed a purification and cell culture method of BFCN in order to examine the regulation of their cholinergic phenotype. Cells isolated from the septal region of late embryonic mice were purified by fluorescence-activated cell sorting based on their expression of the nerve growth factor receptor (p75), a surface marker for mature BFCN. Consistent with previous reports, p75-postive (p75+) cells were enriched in choline acetyltransferase (ChAT) and the high-affinity choline transporter (ChT), as measured by reverse transcriptase PCR. In culture, these cells maintained their gene expression of p75, ChAT and ChT, while p75-negative (p75−) cells had a low expression of these genes. Incubation of the cells with BMP9 not only increased p75 and ChAT gene expression in p75− cells, but also augmented the expression of these genes in p75+ cells. Conversely, BMP9 decreased ChT gene expression in p75+ cells and had no such effect in p75− cells. Immunostaining confirmed that p75 protein expression was modulated by BMP9 in a similar way as p75 mRNA, and also revealed that only a subset of p75− cells respond to BMP9 in this manner. These data suggest that mature BFCN in culture may express their cholinergic phenotype in the absence of exogenous trophic input, but that BMP9 can further modulate this phenotype. Moreover, BMP9 induces the cholinergic phenotype in a set of basal forebrain non-cholinergic neurons or precursor cells.

Identification and early characterization of genetically modified NGF-producing neural stem cells grafted into the injured adult rat brain

Objective: To investigate whether genetically modified mouse neural stem cells (NSC) expressing recombinant human nerve growth factor (rhNGF) and transplanted in chemically injured rat brain, can survive and eventually acquire phenotypic characteristics of early nerve cells.Methods: Stably high expression of rhNGF in NSC was obtained by a new lentivirus-mediated expression system. To test the effectiveness of hNGF secreted by rhNGF-NSC, hereby we performed either a bioassay for neurite outgrowth in PC12 rat cells or immunoblot analysis for TrkA, the high-affinity NGF receptor, from engineered NSC. rhNGF and mock-NSC were grafted into adult injured rats striatum and 3 days later, animals were killed, and brains were removed and examined by immunohistochemical analysis.Results: The results showed that rhNGF-producing NSC cultured for extended period of time release bioactive hNGF in the culture media which promotes PC12 neuronal differentiation and correlates with the up-regulation of TrkA. rhNGF-NSC transplanted into the injured brain can survive, produce hNGF and induce the expression of NGF receptors, p75NTR and TrkA.Discussion:In vitro and in vivo experiments confirmed the ability of rhNGF-NSC to secrete bioactive hNGF. Our data provide by means of genetically modified rhNGF-producing NSC, a useful experimental tool to test the potential clinical effectiveness of trophic factors relevant to central nervous system (CNS).

Sleeping Beauty Transposon‐mediated Engineering of Human Primary T Cells for Therapy of CD19+ Lymphoid Malignancies

We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.