Affinity Nerve Growth Factor Receptor Research Articles

CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers.

At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.

Soluble low affinity nerve growth factor receptor (sLNGFR) may regulate pain in knee osteoarthritis.

Nerve growth factor β (β-NGF) is a protein which is important to the development of neurons particularly those involved in the transmission of pain and is central to the experience of pain in osteoarthritis (OA). Direct NGF antagonism has been shown to reduce OA pain but is associated with rapidly progressive OA. The aim of the study is to investigate the ability of soluble neurotrophin receptors in the NGF pathway to modulate pain in OA. Synovial fluid (SF) was obtained from the knee joints of 43 subjects who underwent total knee arthroplasty. Visual analogue scale (VAS) pain scores were obtained prior to surgery. Customised-automated-ELISAs and commercial-ELISAs and LEGENDplex™ were used to measure soluble low-affinity nerve growth factor (LNGFR), soluble tropomyosin receptor kinase (TrkA), proNGF, β-NGF, other neurotrophins (NT) and cytokines including inflammatory marker TNF-α. The VAS score positively correlated with β-NGF (r=0.34) and there was positive association trend with neurotrophin-3 (NT-3), BDNF and negative association trend with ProNGF. sLNGFR positively correlated with VAS (r=0.33). The β-NGF/soluble TrkA ratio showed a strong positive correlation with VAS (r=0.80). In contrast, there was no correlation between pain and the β-NGF/sLNGFR ratio (r=-0.08). TNF-α positively correlated with β-NGF (r=0.83), NT-3 (r=0.66), and brain-derived neurotrophic factor (BDNF) (r=0.50) and negatively with ProNGF (r= -0.74) and positively correlated with both soluble TrkA (r=0.62), sLNGFR (r=0.26). This study suggests that endogenous or cleaved sLNGFR, but not soluble TrkA may participate in OA pain modulation thus supporting further research into soluble LNGFR as a therapeutic target in OA.

Cerebellar Calcium-Binding Protein and Neurotrophin Receptor Defects in Down Syndrome and Alzheimer's Disease.

Cerebellar hypoplasia is a major characteristic of the Down syndrome (DS) brain. However, the consequences of trisomy upon cerebellar Purkinje cells (PC) and interneurons in DS are unclear. The present study performed a quantitative and qualitative analysis of cerebellar neurons immunostained with antibodies against calbindin D-28k (Calb), parvalbumin (Parv), and calretinin (Calr), phosphorylated and non-phosphorylated intermediate neurofilaments (SMI-34 and SMI-32), and high (TrkA) and low (p75NTR) affinity nerve growth factor (NGF) receptors as well as tau and amyloid in DS (n = 12), Alzheimer's disease (AD) (n = 10), and healthy non-dementia control (HC) (n = 8) cases. Our findings revealed higher Aβ42 plaque load in DS compared to AD and HC but no differences in APP/Aβ plaque load between HC, AD, and DS. The cerebellar cortex neither displayed Aβ40 containing plaques nor pathologic phosphorylated tau in any of the cases examined. The number and optical density (OD) measurements of Calb immunoreactive (-ir) PC soma and dendrites were similar between groups, while the number of PCs positive for Parv and SMI-32 were significantly reduced in AD and DS compared to HC. By contrast, the number of SMI-34-ir PC dystrophic axonal swellings, termed torpedoes, was significantly greater in AD compared to DS. No differences in SMI-32- and Parv-ir PC OD measurements were observed between groups. Conversely, total number of Parv- (stellate/basket) and Calr (Lugaro, brush, and Golgi)-positive interneurons were significantly reduced in DS compared to AD and HC. A strong negative correlation was found between counts for Parv-ir interneurons, Calr-ir Golgi and brush cells, and Aβ42 plaque load. Number of TrkA and p75NTR positive PCs were reduced in AD compared to HC. These findings suggest that disturbances in calcium binding proteins play a critical role in cerebellar neuronal dysfunction in adults with DS.

CUMS Promotes the Development of Premature Ovarian Insufficiency Mediated by Nerve Growth Factor and Its Receptor in Rats.

This study aimed to investigate whether chronic unpredictable mild stress (CUMS) affects follicular development in ovaries through the nerve growth factor (NGF)/high affinity nerve growth factor receptor, the Tropomyosin-related kinase A (TrkA) receptor, mediated signaling pathway and to reveal the relationship between chronic stress and premature ovarian insufficiency (POI) development. In this experiment, a CUMS rat model was constructed. It was found that serum estradiol (E2), anti-Mullerian hormone (AMH), and gonadotropin-releasing hormone (GnRH) levels decreased, while follicle-stimulating hormone (FSH) levels increased. The expression of NGF, TrkA, p75, and FSHR in ovarian tissue decreased significantly. The expression levels of TrkA and p75 protein in ovarian stroma and small follicles were observed by an immunofluorescence assay. In addition, the numbers of small follicles were significantly reduced. The expression of TrkA, p75, and FSHR in CUMS ovarian tissue was upregulated by exogenous NGF in vitro. Furthermore, after treatment with NGF combined with FSH, E2 secretion in ovarian tissue culture supernatant of CUMS rats also increased significantly. Therefore, CUMS downregulates NGF and TrkA and promotes the occurrence of POI in rats. Exogenous NGF and FSH can upregulate the NGF receptor, E2, and AMH in vitro, and improve the rat ovarian function. Future studies may associate these results with female population.

Human medicines European public assessment report (EPAR): Zalmoxis, allogeneic T cells genetically modified with a retroviral vector encoding for a truncated form of the human low affinity nerve growth factor receptor (ΔLNGFR) and the herpes simplex I virus thymidine kinase (HSV-TK Mut2), Hematopoietic Stem Cell Transplantation,Graft vs Host Disease, Date of authorisation: 18/08/2016, Revision: 1, Status: Withdrawn

Human medicines European public assessment report (EPAR): Zalmoxis, allogeneic T cells genetically modified with a retroviral vector encoding for a truncated form of the human low affinity nerve growth factor receptor (ΔLNGFR) and the herpes simplex I virus thymidine kinase (HSV-TK Mut2), Hematopoietic Stem Cell Transplantation,Graft vs Host Disease, Date of authorisation: 18/08/2016, Revision: 1, Status: Withdrawn

Orphan designation: Herpes simplex 1 virus-thymidine kinase and truncated low affinity nerve growth factor receptor transfected donor lymphocytes, Adjunctive treatment in haematopoietic cell transplantation

Orphan designation: Herpes simplex 1 virus-thymidine kinase and truncated low affinity nerve growth factor receptor transfected donor lymphocytes, Adjunctive treatment in haematopoietic cell transplantation

Relevance of Neurotrophin Receptors CD271 and TrkC for Prognosis, Migration, and Proliferation in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and often has a poor prognosis. The present study investigated the role of the low affinity nerve growth factor receptor CD271 as a putative therapy target in HNSCC. Neurotrophins that bind to CD271 also have a high affinity for the tropomyosin receptor kinase family (Trk), consisting of TrkA, TrkB, and TrkC, which must also be considered in addition to CD271. A retrospective study and functional in vitro cell line tests (migration assay and cell sorting) were conducted in order to evaluate the relevance of CD271 expression alone and with regard to Trk expression. CD271 and Trks were heterogeneously expressed in human HNSCC. The vast majority of tumors exhibited CD271 and TrkA, whereas only half of the tumors expressed TrkB and TrkC. High expression of CD271-positive cells predicted a bad clinical outcome of patients with HNSCC and was associated with distant metastases. However, the human carcinomas that also expressed TrkC had a reduced correlation with distant metastases and better survival rates. In vitro, CD271 expression marked a subpopulation with higher proliferation rates, but proliferation was lower in tumor cells that co-expressed CD271 and TrkC. The CD271 inhibitor LM11A 31 suppressed cell motility in vitro. However, neither TrkA nor TrkB expression were linked to prognosis or cell proliferation. We conclude that CD271 is a promising candidate that provides prognostic information for HNSCC and could be a putative target for HNSCC treatment.

Umbilical Cord Mesenchymal Stem Cell Transplantation Prevents Chemotherapy-Induced Ovarian Failure via the NGF/TrkA Pathway in Rats.

Chemotherapy leads to a loss of fertility and reproductive endocrine function, thereby increasing the risk of premature ovarian failure (POF). Studies have suggested that the transplantation of mesenchymal stem cells could inhibit apoptosis in ovarian granulosa cells and improve follicular development. In the present study, the effects of human umbilical cord mesenchymal stem cell (UCMSC) transplantation on ovarian function after ovarian damage caused by chemotherapy and the mechanism underlying these effects were investigated. POF model rats were obtained by the intraperitoneal injection of cyclophosphamide, and cultured UCMSCs were transplanted by tail vein injection. Serum estrogen, follicle-stimulating hormone, gonadotropin releasing hormone, and anti-Mullerian hormone levels were detected by ELISA. Folliculogenesis was evaluated by histopathological examination. The expression levels of nerve growth factor (NGF), high affinity nerve growth factor receptor (TrkA), follicle-stimulating hormone receptor (FSHR), and caspase-3 were evaluated by western blotting and RT-qPCR. The natural reproductive capacity was assessed by pregnant rate and numbers of embryos. The results indicated that UCMSC transplantation recovered disturbed hormone secretion and folliculogenesis in POF rats. NGF and TrkA levels increased, while FSHR and caspase-3 decreased. The pregnancy rate of POF rats was improved. Therefore, UCMSCs could reduce ovarian failure due to premature senescence caused by chemotherapy, and the NGF/TrkA signaling pathway was involved in the amelioration of POF.

Locating human splenic capillary sheaths in virtual reality

Stromal capillary sheath cells in human spleens strongly express CD271, the low affinity nerve growth factor receptor p75. Serial sections of a representative adult human spleen were double-stained for CD271 versus smooth muscle alpha actin (SMA) plus CD34 to visualise capillary sheaths, the arterial tree and endothelial cells by transmitted light. Preliminary three-dimensional (3D) reconstructions of single regions were inspected in virtual reality (VR). This method showed that a large number of CD271+ sheaths occur in a post-arteriolar position often surrounding capillaries located close to divisions of arterioles. The length and diameter of capillary sheaths are rather heterogeneous. Long sheaths were observed to accompany one or two generations of capillary branches. We hypothesise that human splenic capillary sheaths may attract recirculating B-lymphocytes from the open circulation of the red pulp to start their migration into white pulp follicles along branches of the arterial tree. In addition, they may provide sites of interaction among sheath macrophages and B-lymphocytes. Our innovative approach allows stringent quality control by inserting the original immunostained serial sections into the 3D model for viewing and annotation in VR. Longer series of sections will allow to unequivocally localise most of the capillary sheaths in a given volume.

Abstract PR17: Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations

Abstract Partial responsiveness of tumor cells due to drug adaptation (or tolerance) during the early phase of treatment with targeted therapeutics seems to be essential for creating a population of viable tumor cells from which resistant clones eventually arise. Thus, understanding transient drug adaptation is likely to be important for both improving the initial effectiveness of treatment and delaying or controlling acquired resistance. Despite the wealth of information available about the molecular events and feedback mechanisms leading to drug tolerance or adaptation, most of our knowledge in this area comes from studying drug response in bulk tumor cell populations. Furthermore, the phenotypic consequences of drug adaptation have been studied most frequently at a few fixed time-points, when drug-adapted cells exhibit a high population-average activity in multiple pro-growth or pro-survival signaling cascades. Therefore, it remains unclear how uniform or heterogeneous the early drug adaptation is across individual cells within a tumor cell population, and how the fate of drug-adapted cells is determined by a diversity of early drug-induced adaptive signaling responses. Uncovering the evolution of biochemical and phenotypic heterogeneity in drug-adapted cell populations is key to designing optimal combinations of drugs to overcome resistance and to achieve durable therapy. In this study, we monitor the responses of BRAFV600E melanoma cells to RAF/MEK inhibitors at the single-cell level in real time using time-lapse live-cell imaging, and then analyze the resulting cell states using transcriptional, biochemical and phenotypic profiling. We found that exposure of tumor cells to RAF/MEK inhibitors elicits heterogeneous and time-variable responses in which some cells die, some arrest and a fraction of slowly-cycling cells adapts to drug, adopting a reversible drug-resistance phenotype characterized by up-regulation of markers of neural crest, a melanocyte precursor, including NGFR (the low affinity nerve growth factor receptor, also known as p75NTR or CD271). The slowly-cycling NFGRHigh state induced by RAF/MEK inhibitors is only transiently stable: after 1-2 weeks of outgrowth in drug-free medium, such cells reset to their initial state as measured by the restoration of RAF/MEK inhibitor sensitivity, accelerated rate of cell division and reduced expression of NGFR. Transcriptional and biochemical profiling of cell lines and human tumors implicates a role for the c-Jun/ECM/FAK/Src cascade in driving the de-differentiated (NGFRHigh) resistance program. We identify multiple drugs targeting this cascade as well as BET bromodomain inhibitors that block the slowly-cycling NGFRHigh state in cell lines and in a BRAFV600E melanoma xenograft model and increase sensitivity to RAF/MEK inhibitors. Our study reveals directly how drug adaptation happens in individual tumor cells leading to emergence of heterogeneous cell sub-populations with reduced drug-sensitivity that may be selectively targeted by drug combinations. Citation Format: Mohammad Fallahi-Sichani, Verena Becker, Benjamin Izar, Gregory J. Baker, Jia-Ren Lin, Sarah A. Boswell, Levi A. Garraway, Peter K. Sorger. Single-cell analysis reveals an adaptive, slowly-dividing, de-differentiated, drug-resistant cell state selectively inhibitable by drug combinations [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR17.

Allogeneic Genetically Modified T Cells (HSV-TK) As Adjunctive Treatment in Haploidentical Hematopoietic Stem-Cell Transplantation (haplo-HSCT) of Adult Patients with High-Risk Hematological Malignancies: A Pair-Matched Analysis from the Acute Leukemia Working Party of EBMT

Introduction. Current approaches to haplo-HSCT rely either on T-cell depletion to overcome the HLA disparity or on the administration of the lymphotoxic agent cyclophosphamide several days after stem cell infusion, with the goal of selectively depleting activated alloreactive lymphocytes in vivo. In both approaches, haplo-HSCT can be associated with prolonged immunodeficiency post-transplantation. Thus, effective approaches to hastening immune reconstitution following transplantation are needed. Zalmoxis¨ is an Advanced Therapy Medicinal Product based on somatic T-cells genetically modified to express the Herpes Simplex Thymidine Kinase (HSV-TK) suicide gene and a truncated form of the human Low Affinity Nerve Growth Factor Receptor (ΔLNGFR) genes (for identification of transduced cells). The expression of the HSV-TK gene, as a suicide gene allows the selective killing of dividing cells upon administration of the pro-drug ganciclovir (GCV). If GvHD occurs, ganciclovir/valganciclovir can be administered. Here we report the results of a pair-matched analysis which compared the outcome of patients who received HSV-TK cells infusion post haplo-HSCT versus those who did not receive any cellular therapy post-transplant.Patients and Methods. The HSV-TK patients' group included 45 patients who were treated as part of 2 prospective trials with various types of high-risk hematologic malignancies. These patients were compared to patients treated with haplo-HSCT reported to the acute leukemia working party registry of the EBMT. Inclusion criteria for the pair-matched analysis encompassed haplo-HSCT transplants performed in adult patients diagnosed with AML/ALL/sAML in CR or relapse at transplantation. To equate the distribution of baseline characteristics between the HSV-TK and control group and to reduce bias in treatment effect estimation, a pair-matched analysis was performed.This analysis, in which pairs of HSV-TK and control subjects sharing similar baseline characteristics were formed, used the following parameters as pair matching factors: patient age, diagnosis (AML, ALL and sAML), disease status at HSCT (CR1, CR2, CR3 or relapse) and time from diagnosis to HSCT. The planned ratio of HSV-TK patients to control patients was one to four. Efficacy outcome measures of this pair-matched analysis were OS, LFS, NRM and relapse incidence (RI). Cumulative incidence rates of chronic GVHD were also analyzed.Results. Overall, 37 HSV-TK-treated patients matched with 140 controls (71 from PT-Cy cohort and 69 from TCD cohort transplanted between 2005 and 2013). The recommended dose and schedule of HSV-TK cells was 1x107 cells/kg given as IV infusion every 30 days for a maximum of 4 times until a circulating T-cell count higher than 100 per μL. The 1st administration should occur between day 21 to day 49 after HSCT. Baseline characteristics of the HSV-TK treated and the control patient population are summarized in the below table.OS at 1-year was significantly improved in the HSV-TK-group compared with the control group (p=0.01). The survival rates were 49% and 37% for HSV-TK- and control group, respectively. The NRM at 1-year was also improved upon treatment with HSV-TK, with 43% for the control group and 22% for the HSV-TK-group (p=0.014). A difference in favor of the HSV-TK-group could also be observed for the 1-year incidence of chronic GvHD with 25% for the control group vs 9% for the HSV-TK-group (p=0.04). The LFS and the RI were not different between the groups. Interestingly, these differences remained similar whether considering the TCD or the PT-CY subgroups). Together the data suggest that the benefit seen in OS is driven by a reduction in the NRM. A further analysis of NRM data revealed that in the control group 34 of 140 (24%) patients died due to infection and 8 of 140 (6%) succumbed due to GvHD. In the HSV-TK population 4 (11%) patients died because of infection and no patient died due to GvHD. This suggests that the reduction in NRM mortality in the HSV-TK population is caused both by a reduction in death due to infection and due to GvHD. Concerning safety, no death was attributed to HSV-TK cells. Acute GvHD resolved in all cases, and activation of the suicide gene by treatment with GCV has contributed to the control of GvHD.Conclusion. The above pair-matched analyzis confirmed the positive impact and benefit of HSV-TK cells as adjunctive treatment in haplo-HSCT with an acceptable safety pattern. [Display omitted] DisclosuresBonini:Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

Establishing Modified Human CYP4B1 As a Novel Suicide Gene System for Adoptive T-Cell Therapies

Retargeting autologous T-cells to recognize tumor-associated antigens by genetic modifications with viral vectors that express either enhanced T-cell receptors (TCRs) or chimeric antigen receptors (CARs) has shown successful activity against B-cells malignancies. Equipping the transduced T-cell with a suicide gene can prevent potential safety problems as on-target off-target toxicity, off-target reactivity and cytokine-release syndrome (Casucci et al., 2015). The concept of a suicide gene has already been utilized to prevent a Graft-versus-host-disease (GvHD) following donor lymphocyte infusion (DLI). The first suicide gene system used in clinical phase I/II studies was the human Herpes simplex virus type 1 thymidine kinase with its prodrug ganciclovir that was used to control Graft-versus-host-disease following donor lymphocyte infusion (DLI) for leukemia relapse after allogeneic stem cell transplantation. More recent examples for suicide gene systems are icasp9, an inducible apoptosis switch with a synthetic dimerizer protein as prodrug, and CD20 with rituximab as a prodrug. We recently published a novel potential human suicide gene system by rendering the inactive P450 cytochrome CYP4B1 with 13 amino acid changes into the highly active CYP4B1P+12 in combination with 4-ipomeanol (4-IPO) (Wiek et al., 2015). 4-IPO is furan produced by sweet potatoes infected with the common mold Fusarium javanicum and has been identified as the major lung toxin causing lung edema in livestock (Wilson et al., 1970). A second natural occurring substance causing lung edema in horses is the major component of the Perilla frutenscens fruit oil called perilla ketone (PK) (Wilson et al., 1977). Importantly, the only difference between PK and 4-IPO is a methyl group at C5 in PK, where 4-IPO has a hydroxyl group. Therefore in this work, we tested the hypothesis whether PK could function as a potential new prodrug for the CYP4B1P+12 suicide gene.Initially we compared the cytotoxicity caused by PK and 4-IPO in CYP4B1P+12 transduced primary human T-cells from healthy donors. For these experiments, we used third generation lentiviral vectors with T2A sites to express the suicide gene in combination with a truncated version of the low affinity nerve growth factor receptor (DNGFR) as a selection marker for Miltenyi MACS microbeads. Through the selection process, we were able to enrich the suicide gene expressing T-cell population from 61±6.0% to ≥95±1.8%.After selection, we challenged the CYP4B1P+12 expressing T-cells with increasing concentrations (2.9-90 µM) of both substrates and analyzed the survival of the cells. After 24h, 65.8±5.26% of CYP4B1P+12 positive T-cells incubated with 2.9 µM 4-IPO were still alive, while incubation with 90 µM lead to only 17.5±1.45% living T-cells. In contrast, incubation with 2.9 µM PK resulted in only 16.02±1.08% living cells, while 90 µM PK induced cell death in 94.2±0.79% of cells. Next, we demonstrated that a lower concentration, 0.9 µM, already led to only 13.2±0.46% alive cells after 24h. Monitoring apoptosis in the transduced T-cells by flow cytometry demonstrated that incubation with 90 µM 4-IPO for 4h decreased the rate of transduced T-cells to only 87.4±2.89%, while 10h and 24h incubation resulted in 20.4±4.9% and 5.8±0.93% living annexinV-negative cells. In contrast, 2h incubation with only 9 µM PK led to 88.7±2.07%, 4h to 60.2±5.47% and 10h to only 5.5±1.85% living annexinV-negative T-cells. Incubation of the T-cells with 90 µM did not change these numbers. Hence, we were able show that PK is not only more potent as 4-IPO, it also proves to have a faster kinetic in inducing apoptosis.After these promising in vitro results of using PK as a novel more potent prodrug for the CYP4B1P+12 human suicide gene, we performed dose escalation studies of PK in mice with targeted disruption of the Cyp4b1 gene. Using up to 25 mg PK/kg mouse daily up for 3d did not induce any clinical symptoms in the Cyp4b1-/- animals, while Cyp4b1+/+ animals needed to be sacrificed 4h and 7h after a single i.p. injection of 10 mg/kg or 5 mg/kg PK, respectively, due to severe respiratory distress. Results of in vivo studies analyzing the elimination of CYP4B1P+12 positive syngenic T-cells in Cyp4b1-/- mice after injection with either 4-IPO or PK will be finalized and presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

Smooth muscle-derived neurotrophins regulate development and function of vagal gastrointestinal (GI) afferents

Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) regulate development of overlapping populations of vagal sensory neurons. During development, they are expressed in GI smooth muscle, a tissue innervated by vagal mechanoreceptors. To investigate the contributions of this BDNF and NT-3 to vagal development, they were ablated from smooth muscle (smBDNF-KO, smNT-3-KO). As GI vagal afferents signal meal termination (satiation), adult meal patterns were utilized as a functional assay of altered GI vagal development. smNT-3-KO mice exhibited a decrease in meal-induced c-Fos activation in the solitary tract nucleus and area postrema compared to controls, suggesting vagal afferent innervation of the gut was decreased. Consistent with this, smNT-3-KO mice showed increased meal duration, and increased size of their first daily meal, suggesting vagal satiation signaling was reduced. In contrast, smBDNF-KO mice had increased intestinal vagal mechanoreceptor innervation. Increased numbers of vagal sensory neurons and intestinal fiber bundles in the knockout mice suggested this was due to increased survival of intestinal vagal mechanoreceptors. Thus, BDNF may normally suppress survival of these mechanoreceptors, counter to expectations for a growth factor. smBDNF-KO mice ate meals of shorter duration and smaller size than controls, suggesting the increased intestinal innervation provided increased satiation signaling. The opposite effects of smBDNF-KO and smNT-3-KO on GI vagal sensory development/function may result from interactions of smooth muscle-derived BDNF, but not NT-3, with the low affinity nerve growth factor receptor, p75. This receptor is expressed by vagal afferent neurons during development and its activation by BDNF typically results in cell death.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

Chromosomal rearrangements involving the NTRK1 gene in colorectal carcinoma

Chromosomal rearrangements of the NTRK1 gene, which encodes the high affinity nerve growth factor receptor (tropomyosin related kinase, TRKA), have been observed in several epithelial cancers, such as colon cancer, papillary thyroid carcinoma or non small cell lung cancer. The various NTRK1 fusions described so far lead to constitutive activation of TRKA kinase activity and are oncogenic. We further investigated here the existence and the frequency of NTRK1 gene rearrangements in colorectal cancer. Using immunohistochemistry and quantitative reverse transcriptase PCR, we analyzed a series of human colorectal cancers. We identified two TRKA positive cases over 408, with NTRK1 chromosomal rearrangements. One of these rearrangements is a TPM3–NTRK1 fusion already observed in colon cancer, while the second one is a TPR–NTRK1 fusion never described in this type of cancer. These findings further confirm that translocations in the NTRK1 gene are recurring events in colorectal cancer, although occurring at a low frequency (around 0.5%).

462. Automated Manufacturing Process for the Production of Genetically Modified Lymphocytes

The MM-TK is a medicinal product constituted of T lymphocytes ex vivo genetically modified with the γ retroviral vector SFCMM-3 Mut2 #48 to express a mutated form of the Herpes Simplex Virus Thymidine kinase (HSV-TK Mut2) and a truncated form of the human Low affinity Nerve Growth Factor Receptor (ΔLNGFR) genes. It has development as adjunctive therapy in leukaemia patients or undergone bone marrow transplantation. The current manufacturing process is performed following semi-automated procedure using instrument and tubing set not marketing compliant. In order to optimize this and to standardize cellular therapeutic production for clinical trial and for the market, MolMed have strengthened an alliance with Miltenyi Biotec for tailoring of CliniMACS Prodigy platform for MM-TK DS manufacturing.A closed and automated cell process was developed using the new device with a specific software designed in order to manufacture a robust, safe and consistent product.The manufacturing process essentially consists in the stimulation of a large number of PBMCs (about 3×109 cells) isolated from an haploidentical donor, with anti-CD3 monoclonal antibody and IL-2, transduction of the stimulated cells with the SFCMM-3 Mut2 #48 retroviral vector in RetroNectin® coated bags and immunoselection of positively transduced cells using the CD271-LS Microbeads reagent (Miltenyi Biotec). Selected TK cells are then further expanded for 7 days before final formulation in freezing medium. The whole manufacturing process lasts 13 days.The following steps have been automated: cell concentration and cell washing at different manipulation steps and immunomagnetic selection of the transduced cells.Four development full-scale runs demonstrate that automated cell processing for the genetic manipulation of T lymphocytes can be performed in the closed- and automated-system designed by MolMed, reaching good results for all in-process parameters evaluated: cell recovery of 80-90%; cell viability more than 80% at each step; total process yield of 72%. The DS/DP product has been evaluated for the quality attributes as product and process related impurities (CD14% + CD16% + CD19% and residual retronectin, residual anti-CD3, residual CD271 microbeads) and potency of the product (ΔLNGFR expression; sensitivity to Ganciclovir; Vector Copy Number; immunophenotype as CD3%, CD4%, CD8%, CD45%,CD25, CD69, HLA-DR, CD95; CD45RA and CD62L). The results will support the discussion with Regulatory Autorities and permit the introduction of the new optimized closed manufacturing process in the late phase of the clinical trial and for the market.

Nogo-A and its functions beyond axonal inhibition: the controversial role of Nogo-A in Parkinson's disease.

Nogo-A belongs to the reticulon family (RTN4) and is generally assumed to be one of the most potent myelin associated neurite outgrowth inhibitors in the central nervous system (CNS). Together with other inhibitors such as the myelin associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), several semaphorins and ephrins as well as chondriotin sulphate proteoglycans, Nogo-A contributes to a nonpermissive environment in the brain and spinal cord. Based on their seminal observation that neurons grown in close proximity to CNS derived oligodendrocytes showed a robust decrease in neurite outgrowth and number of processes, Schwab and Caroni (1988) postulated for nonpermissive substrate properties present in the CNS myelin. In further studies, they identified a high molecular weight component from the CNS myelin called NI-250 which was later renamed Nogo-A. In the course it could be demonstrated that Nogo-A is expressed on the surface of oligodendrocytes and is responsible for the inhibition of axonal sprouting. Since the nonpermissive environment plays a very important role in brain injuries and neurodegenerative diseases, Nogo-A and its receptors were extensively studied.

Mo2036 Remodeling of Colonic Sensory Afferents in a Rat Model of Post-Infectious Gut Dysfunction: Implication of Neurotrophic Factors

Background: Inflammation-induced visceral hypersensitivity is associated, at least partially, to peripheral sensitization of sensory afferents by locally released neurotrophic factors. Aims: Here, we explore the potential role of neurotrophic factors in long-term changes in visceral afferents innervating non-primary areas of inflammation (colon) in a rat model of Trichinella spiralis (TS)-induced gut dysfunction. Methods: Inflammatory-like changes, mucosal mast cells (MMCs) dynamics, and expression of nerve growth factor (NGF) and glial cell linederived neurotrophic factors (GDNF, artemin and neurturin) were determined in the colon of control and TS-infected rats, up to day 30 post-infection (PI). Markers of sensory afferents (high affinity nerve growth factor receptor, TrkA; GDNF family receptor alpha 3, GFRα3; calcitonin gene-related peptide, CGRP; and transient receptor potential vanilloid channel1, TRPV1) were assessed in thoracolumbar (T12-L2, TL) and lumbosacral (L6-S2, LS) DRGs. Functionality of colonic afferents at 14 and 30 days PI was determined by assessing changes in TRPV1 levels in TL and LS DRGs following intracolonic capsaicin. Results: TS infection induced a primary jejunitis with concomitant inflammatory-like changes in the colon: an increase in histological scores, IL-13 up-regulation and MMC infiltration (Table 1). Inflammation was partly resolved at day 30 PI, except for a persistent MMC infiltrate (Table 1). Colonic expression of neurotrophic factors showed a transient reduction by day 14 PI (25%50%), with the exception of artemin that showed a biphasic response with an up-regulation (by 30%; P<0.05) at day 30 PI (Table 2). Overall, TS infection did not affect DRG expression of none of the genes assessed, except for a transient down-regulation of TPRV1 in TL DRGs at day 14 PI. Afferent stimulation with intracolonic capsaicin induced a down-regulation of TRPV1 expression in TL and LS DRGs, an effect significantly enhanced in LS DRGs of TSinfected rats, at either 14 or 30 days PI (Table 2). Intracolonic capsaicin also up-regulated peripheral artemin in non-infected rats, an effect absent in TS-infected animals at day 30 PI (Table 2). Capsaicin-induced changes in colonic expression of artemin correlated negatively with the down-regulation of TRPV1 in LS DRGs. Conclusions: During intestinal inflammation, changes in expression of neurotrophic factors and remodeling of visceral afferents are observed outside the primary region affected by the inflammatory insult. These observations suggest that extended remodelation of sensory afferents, probably related to local changes in neurotropins, might occur during inflammatory conditions of the gut. Similar mechanisms might be operating in states of widespread alterations of visceral sensitivity. Table 1 Colonic inflammation-related changes during T. spiralis infection

Graft Versus Leukemia With Little Graft Versus Host Disease? Long Term Follow-Up Of a Study Using Transduced Donor T-Cells For Donor Leukocyte Transfusion

▪Fourteen patients with myeloid leukemia (12 with acute and 2 with chronic myelogenous leukemia) were transplanted from their HLA-identical (n = 9) or haploidentical (n = 5) family donors with CD34-enriched stem cells (HSCT) without further immunosuppression. In order to induce a graft versus leukemia (GvL) effect and to investigate the possibility of controlling graft-versus-host disease (GvHD), the standard transplantation protocol was adapted to include transfusion of gene-modified donor T-cells after HSCT in 11 patients, 3 did not receive any T-cells.Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3 which expresses the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene and the truncated low affinity nerve growth factor receptor (ΔLNGFR) for selection purposes. After transfusion, SFCMM-3 transduced T-cells were detectable in all 11 patients by PCR and FACS analyses immediately after transfusion and during the follow up period (range: 1.1-11 years). Two of 9 patients developed acute GvHD: one of the skin, grade 1, 56 days after transfusion of the transduced cells, the other grade II which was successfully treated with ganciclovir. Loss of bcr-abl gene expression was achieved in one patient after expansion of transduced cells. Donor chimerism was stabilized after transfusion of transduced cells in all patients treated. In one patient a cytomegalovirus-reactivation was treated by the transfusion of gene-modified donor T-cells also indicating effectiveness of treatment. To date, 5 patients have relapsed, and died, one after a second HSCT. Two of the patients without transduced T-cells died due to relapse and 7 patients are alive and well and in complete clinical remission.Thus we have shown that transfusion of transduced T-cells is effective and safe in a long follow-up of 11 years post transfusion. Disclosures:No relevant conflicts of interest to declare.

Human periodontal ligament stem cells repair mental nerve injury.

Human periodontal ligament stem cells are easily accessible and can differentiate into Schwann cells. We hypothesized that human periodontal ligament stem cells can be used as an alternative source for the autologous Schwann cells in promoting the regeneration of injured peripheral nerve. To validate this hypothesis, human periodontal ligament stem cells (1 × 10(6)) were injected into the crush-injured left mental nerve in rats. Simultaneously, autologous Schwann cells (1 × 10(6)) and PBS were also injected as controls. Real-time reverse transcriptase polymerase chain reaction showed that at 5 days after injection, mRNA expression of low affinity nerve growth factor receptor was significantaly increased in the left trigeminal ganglion of rats with mental nerve injury. Sensory tests, histomorphometric evaluation and retrograde labeling demonstrated that at 2 and 4 weeks after injection, sensory function was significantly improved, the numbers of retrograde labeled sensory neurons and myelinated axons were significantly increased, and human periodontal ligament stem cells and autologous Schwann cells exhibited similar therapeutic effects. These findings suggest that transplantation of human periodontal ligament stem cells show a potential value in repair of mental nerve injury.