Human Nerve Growth Factor Receptor Research Articles

Impact of proNGF/NGF-NGFR-axis on the regulation of arterial remodelling

Abstract Background Peripheral blood mononuclear cells (MNCs) contribute to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is present in peripheral blood and the ischemic coronary artery. NGFR transduces neurotrophin signals towards cell survival or apoptosis in different cell types dependent on coupling co-receptors. However, the role of NGFR-positive (NGFR+) MNCs in arterial remodelling is unknown. Purpose To investigate the functional mechanisms under which NGFR+ MNCs are involved in arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -bone marrow (BM)-specific (KO) mice were subjected to unilateral carotid artery ligation. The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human NGFR+ MNCs from a healthy volunteer were sorted using fluorescence-activated cell sorting and characterised using RNA sequencing. The cell apoptosis (AnnexinV+7AAD-) and chemotaxis in response to the ligands, NGF and its precursor proNGF, were assessed using flow cytometry and a Transwell migration. Results In WT mice, BM-derived NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly greater in the BM-specific depletion of NGFR than in WT mice. NGFR+ cells in the neointima exhibited apoptosis (TUNEL+ and cleaved caspase-3+) accompanied by promoted F4/80+ macrophage and anti-inflammatory IL-10. By contrast, in the BM-specific NGFR-KO model, non-BM-derived SMCs increased in the neointima with augmented proliferation, and the accumulation of BM-derived cells and the expression of IL-10 were decreased. The expressions of proNGF/NGF and inflammatory cytokines, including IL-6, were not changed in a ligated artery, irrespective of NGFR+ depletion. Human NGFR+ MNCs expressed TrkB, TrkC, and sortilin co-receptors but not macrophage markers. Human NGFR+ MNCs, but not NGFR- MNCs, co-cultured with artery SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated proNGF/NGF expression in SMCs, and proNGF/NGF did not affect the proliferation of SMCs in vitro. Furthermore, NGFR+ MNCs, but not NGFR- MNCs, increased migration toward NGF and did not show chemotaxis toward proNGF. Conclusions These data imply that local NGF might draw NGFR+ MNCs to the injured artery, in which proNGF induces NGFR+ MNCs apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis to resolve inflammation and decrease neointimal formation. Thus, we propose the proNGF/NGF-NGFR axis as potentially contributing to the suppression of arterial remodelling.

Bone-marrow-derived nerve growth factor receptor-positive dendritic cells contribute to the suppression of arterial remodelling via apoptosis

Abstract Background Bone marrow (BM) contributes to the pathogenesis of arteriosclerosis. Nerve growth factor receptor (NGFR) is expressed in BM stroma and is present in peripheral blood and the ischemic coronary artery. NGFR transduces signals towards cell survival or apoptosis in different cell types according to the coupling co-receptors. The previous clinical study demonstrated that low gene expression of NGFR in peripheral leucocytes in patients with acute coronary syndrome predicted 5-year repetitive coronary interventions at a de novo lesion. Purpose To investigate the hypothesis that BM-derived NGFR-positive (NGFR+) cells are associated with arterial remodelling. Methods Adult C57BL/6J male NGFR-wild-type (WT) or -knockout (KO) mice were subjected to unilateral carotid artery ligation after BM transplantation (BMT) from green fluorescent protein-positive (GFP+) NGFR-WT or -KO mice (N = 4-6). The ligated and the opposite-sided, sham-operated arteries were assessed by immunohistology and gene expression analysis using a PCR on day 28. Also, human peripheral blood NGFR+ mononuclear cells (MNCs) from a healthy volunteer were sorted using FACS Aria II and co-cultured with smooth muscle cells (SMCs) to examine early apoptosis (AnnexinV+7AAD-) in response to the NGFR ligands; proNGF and NGF. Also, human NGFR+MNCs from three healthy volunteers underwent RNA sequencing analysis and were immunophenotypically characterised by flow cytometry. Results In WT mice, NGFR+ cells accumulated in the neointima after ligation. The neointimal area was significantly larger in the mice with BM-specific depletion of NGFR than that in WT mice. After BMT from GFP+NGFR-WT mice, NGFR+GFP+ cells accumulated in the neointima, exhibited apoptosis (TUNEL+ and cleaved caspase-3+), and promoted F4/80+GFP+ macrophage accumulation. By contrast, in the BM-specific NGFR-KO model, medial SMCs (αSMA+GFP-) occupied the neointima with augmented proliferation, and GFP+ cells were rare. Notably, apoptotic NGFR+ cells were accompanied by the accumulation of macrophages (F4/80+NGFR-) and expressed IL-10, which were abolished by BM-specific depletion of NGFR. Furthermore, human NGFR+, but not NGFR- cells, co-cultured with SMCs were susceptible to apoptosis in response to proNGF. Additionally, PDGF stimulated NGF expression in SMCs in vitro, and proNGF was expressed in a ligated artery, implying that local proNGF might cause NGFR+ cell apoptosis. Meanwhile, RNA sequencing and flow cytometric analysis demonstrated that human NGFR+MNCs resemble dendritic cells (DCs) and predominantly express the plasmacytoid DCs markers. Consistently, NGFR+ cells in the neointima were positive for HLA-DR and CD11c. Conclusions BM-derived NGFR+ cells expressed dendritic cell markers and contributed to the suppression of arterial remodelling mediated by apoptosis. Apoptotic NGFR+ cells promoted macrophage chemotaxis and coordinately inhibited medial SMC migration to, and proliferation in, the neointima.

Benzenesulfonamide Analogs: Synthesis, Anti-GBM Activity and Pharmacoprofiling.

The tropomyosin receptor kinase A (TrkA) family of receptor tyrosine kinases (RTKs) emerge as a potential target for glioblastoma (GBM) treatment. Benzenesulfonamide analogs were identified as kinase inhibitors possessing promising anticancer properties. In the present work, four known and two novel benzenesulfonamide derivatives were synthesized, and their inhibitory activities in TrkA overexpressing cells, U87 and MEF cells were investigated. The cytotoxic effect of benzenesulfonamide derivatives and cisplatin was determined using trypan blue exclusion assays. The mode of interaction of benzenesulfonamides with TrkA was predicted by docking and structural analysis. ADMET profiling was also performed for all compounds to calculate the drug likeness property. Appropriate QSAR models were developed for studying structure-activity relationships. Compound 4-[2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfon-amide (AL106) and 4-[2-(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide (AL107) showed acceptable binding energies with the active sites for human nerve growth factor receptor, TrkA. Here, AL106 was identified as a potential anti-GBM compound, with an IC50 value of 58.6 µM with a less toxic effect in non-cancerous cells than the known chemotherapeutic agent, cisplatin. In silico analysis indicated that AL106 formed prominent stabilizing hydrophobic interactions with Tyr359, Ser371, Ile374 and charged interactions with Gln369 of TrkA. Furthermore, in silico analysis of all benzenesulfonamide derivatives revealed that AL106 has good pharmacokinetics properties, drug likeness and toxicity profiles, suggesting the compound may be suitable for clinical trial. Thus, benzenesulfonamide analog, AL106 could potentially induce GBM cell death through its interaction with TrkA and might be an attractive strategy for developing a drug targeted therapy to treat glioblastoma.

Human NGFR+/VCAM-1+/Mcam+ Bone Marrow-Derived Stromal Cells (NVML) Provide Enhanced Support for Normal Hematopoiesis and Are Disrupted in Myelodysplastic Syndrome

The bone marrow microenvironment (BMME) is indispensable for support of normal and malignant hematopoiesis. Mesenchymal stem/stromal cells (MSCs) give rise to multiple non-hematopoietic components that play a pivotal role in myelodysplastic syndromes (MDS). However, therapeutic targeting of MSCs has been challenging, in part due to the limited definition of the heterogeneity of MSC populations from human bone marrow. Using bone marrow from normal healthy controls obtained through aspirates rather than surgical specimens (normal bone marrow; NBM), we successfully identified two novel distinctive subsets of human MSCs by combining three MSC cell surface markers: CD271/ Nerve Growth Factor Receptor (NGFR), CD106/ Vascular Cell Adhesion Molecule-1 (VCAM-1) and CD146/ Melanoma Cell Adhesion Molecule (MCAM). We first isolated three BM-derived non-endothelial lineage (CD45/CD235ab/CD34-CD31-) cell populations from NBM: CD271+/CD146- (NGFR+/Lineage- cells: NLCs), CD271+/CD146+/CD106+ (NGFR+/VCAM1+/MCAM+/Lineage- NVML cells), and CD271+/CD146+/CD106- (NGFR+/MCAM+/Lineage-; NML cells). Gene profiling by RNA-seq revealed that NLCs and NVML cells represent distinct populations, whereas NML cells are a transitional population. Transcriptional analysis suggested that NVML cells, compared to NLCs, have stem cell characteristics, including quiescence and multipotentiality, and express higher levels of key genes critical for the support of hematopoietic stem and progenitor cells (HSPCs), including CXCL12 (SDF1A), Kit ligand (KITL) and Angiopoietin-1 (ANGPT1). This signature was compatible with BM-derived Leptin receptor+ MSCs with high capacity of supporting HSPCs based on previously reported single cell RNA-seq analysis in mice (Baryawno et al 2019, Cell). Consistent with their transcriptome, functional analysis showed that NVML cells had higher colony forming units (CFU-F) and tri-lineage differentiation capacity compared to NLCs. Normal NLCs or NVML cells were also co-cultured with NBM or human MDS cell line (MDS-L) in the absence of cytokines/chemokines to evaluate the ability of these novel human MSC populations to provide a supportive niche for HSPCs. NVML cells provided increased support for normal hematopoietic progenitors compared to baseline and NLCs (fold increase over baseline 1.78±0.78(NLCs, p<0.05) vs 2.71±1.71(NVML cells, p<0.001), NLCs vs NVML cells, p<0.05). In contrast, NLCs showed increased ability of supporting MDS-L (fold increase over baseline 2.35±1.35 (NLCs, p<0.01) vs 1.5±0.5 (NVML cells, p=0.26), NLCs vs NVML cells, p=0.053). We next profiled the transcriptome of NLCs and NVML cells from 17 patients with MDS in comparison with age-matched healthy controls (n=8, >50 years old). In spite of mutational heterogeneity in this MDS patient cohort, principal component analysis (PCA) revealed a distinctive shift of MDS-derived NVML cells compared to controls. For upregulated transcripts, pathways and gene ontology analyses of differentially expressed genes in NVML cells from MDS compared to controls identified signals related to DNA/RNA processing, while most of the down-regulated transcripts identified cytokines/chemokines critical for HSPCs. When the transcriptional profiles of NVML cells from MDS patients were compared to age-matched NVML controls, critical genes for HSPC support highly expressed in normal NVML (e.g. SDF1A, ANGPT1) were significantly down-regulated. Moreover, the transcriptional signature of NVML cells from MDS patients was similar to that of normal NLCs. Together these data suggest that, in spite of mutational heterogeneity in the hematopoietic clone, MDS may differentially impact these novel human marrow MSC subsets, decreasing normal HSPC support by disrupting NVML cells, which acquired the transcriptional signature of MDS-supportive normal NLCs. These data highlight a novel paradigm to enrich key human niche cells that provide support for HSPCs. Moreover, our data suggest that these human MSC cell subsets are differentially disrupted in MDS. Mutation specific effects are also likely to impact MDS-dependent disruption of the BMME. Therefore, our novel strategy to enrich critical human BM MSC subsets that differentially support normal and malignant hematopoiesis represents a new paradigm to accelerate therapeutic targeting of the BMME in MDS and other hematologic malignancies.

Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo

Chimeric antigen receptor (CAR) T cell therapy has demonstrated unprecedented success with high remission rates for heavily pretreated patients with hematological malignancies. The hinge connecting the extracellular antigen recognition unit to the transmembrane domain provides the length and flexibility of the CAR constructs and ensures that the CAR can reach the target antigen and mediate recognition and killing of target cells. The hinge can also include specific amino acid sequences to improve CAR expression, influence T cell proliferation, and facilitate CAR T cell detection, enrichment, and even elimination. Here, we report the generation of two novel hinge domains derived from the low-affinity p75 chain of the human nerve growth factor receptor (NGFR), termed N3 and N4, which, when incorporated into the CAR backbone, allow detection as well as high-grade enrichment of CAR T cells with GMP-compatible immunomagnetic reagents. After optimizing the MACS protocol for excellent CAR T cell purity and yield, we demonstrated that N3- and N4-hinged CAR T cells are as efficacious as their CD8-hinged counterparts in vitro against hematological blasts and also in vivo in the control of acute monocytic leukemia in an immunodeficient mouse xenograft model. Thus, both hinges could potentially be an integral part of future CAR designs and universally applicable in clinical applications.

Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.

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

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.

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.

Cytological characterization of murine bone marrow and spleen hematopoietic compartments for improved assessment of toxicity in preclinical gene marking models

Gene therapy has proven its potential to cure diseases of the hematopoietic system, but potential adverse reactions related to insertional mutagenesis by integrating gene vectors and chromosomal instability in long-lived repopulating cells have emerged as a major limitation. Preclinical gene therapy in murine models is a powerful model for assessment of gene marking efficiency and adverse reactions. However, changes in the hematologic composition after transplantation with retrovirally modified hematopoietic stem cells have not been well investigated in large cohorts of animals by systematic cytological analyses. In the present study, cytological analyses of bone marrow and spleen were performed in a large cohort (n = 58) of C57BL/6J mice over an extended observation period after gene marking. Interestingly, we observed hematological malignancies in four out of 30 animals transplanted with dLNGFR (truncated form of the human p75 low-affinity nerve growth factor receptor) and tCD34 modified stem/progenitor cells. Our data demonstrate that cytological analysis provides important information for diagnosis of hematological disorders and thus should be included in preclinical studies and performed in each investigated animal. Together with histological analysis, flow cytometric analysis, and other analyses, the quality and predictive value of preclinical gene therapy studies will be improved.

Establishment of an indicator cell system for hepatitis C virus

Although a cell culture system for HCV JFH-1 strain has been developed, no robust cell culture system for serum-derived HCV is available. In this study, we have established systems capable of monitoring infection with JFH-1 virus based on specific reporter gene expression through proteolysis of chimeric transcription factors by HCV NS3/4A protease. We utilized a transcriptional factor Gal4-TBP that synergistically enhances transcription of the GAL4UAS and HIV-1 LTR tandem promoter with the Tat protein. We constructed chimeric Tat and Gal4-TBP transcription factors containing the HCV NS3/4A cleavage sequence of a mitochondria-resident IPS-1, but not those of the HCV polyprotein, and manipulated them to localize in the ER. Upon infection with JFH-1 virus, the transcription factors were efficiently cleaved by HCV protease, migrated into the nucleus and activated the reporter gene under the tandem promoter. Upon infection with JFH-1 virus, the Huh7OK1/TG-Luc cell line carrying the transcription factors and a luciferase gene under the promoter expressed luciferase in a dose-dependent manner in close correlation with HCV RNA replication. Huh7OK1/TG-LNGFR cells carrying the transcription factors and a cDNA of human low affinity nerve growth factor receptor under the promoter were selectively concentrated by immunomagnetic cell sorting upon infection with JFH-1 virus. These results indicate that the chimeric constructs bearing the ER-resident IPS-1 sequence are specifically recognized and efficiently cleaved by HCV protease and are harnessed for detection of HCV replication and for recovery of HCV-infected cells. This strategy may be applicable for the establishment of cell culture systems for the isolation of serum-derived HCV.

Mesenchymal stem cells from human umbilical cords ameliorate mouse hepatic injury in vivo

To investigate human umbilical cord-derived mesenchymal stem cells (hUCMSCs) for use in the reversal of mouse hepatic injury. Human umbilical cord-derived mesenchymal stem cells, characterized by flow cytometry, were transplanted into carbon tetrachloride (CCl(4))-injured mice, and then followed for determination of localization and differentiation. Reverse transcriptase-polymerase chain reaction for the human 17alpha gene and fluorescence in situ hybridization analysis for the human X chromosome were used to locate exogenous hUCMSCs in mouse livers. Peripheral blood and liver specimens were collected at 7, 14 and 21 days after transplantation. For evaluating the recovery of injured liver tissues, serum aminotransferase was measured, and the pathological state of the hepatocytes was assessed. The hUCMSCs were positive for the human MSC-specific markers CD13, CD29, CD44, CD105 and nerve growth factor receptor, but negative for the haematopoietic lineage markers CD31, CD34, CD38, CD45 and HLA-DR. Under conditions favouring differentiation in vivo, the expression of tryptophan 2,3-dioxygenase, human alpha-fetoprotein, cytokeratin 18, fibroblast secretory protein 1 and alpha-smooth-muscle-actin was detectable after hUCMSCs administration to mice subjected to liver injury. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labelling and proliferating cell nuclear antigen staining showed that transplanted hUCMSCs could inhibit hepatocyte apoptosis and facilitate proliferation. Serum aminotransferases were decreased after transplantation of hUCMSCs into the injured mice, and hepatocyte denaturation was reduced. Human umbilical cord-derived mesenchymal stem cells can enhance recovery of CCl(4)-injured mouse liver, providing evidence that such therapy could be useful for liver disorders or injury.

Gfi1-mediated Stabilization of GATA3 Protein Is Required for Th2 Cell Differentiation

The differentiation of naive CD4 T cells into Th2 cells requires the T cell receptor-mediated activation of the ERK MAPK cascade. Little is known, however, in regard to how the ERK MAPK cascade regulates Th2 cell differentiation. We herein identified Gfi1 (growth factor independent-1) as a downstream target of the ERK MAPK cascade for Th2 cell differentiation. In the absence of Gfi1, interleukin-5 production and the change of histone modification at the interleukin-5 gene locus were severely impaired. Furthermore, the interferon gamma gene showed a striking activation in the Gfi1(-/-) Th2 cells. An enhanced ubiquitin/proteasome-dependent degradation of GATA3 protein was observed in Gfi1(-/-) Th2 cells, and the overexpression of GATA3 eliminated the defect of Th2 cell function in Gfi1-deficient Th2 cells. These data suggest that the T cell receptor-mediated induction of Gfi1 controls Th2 cell differentiation through the regulation of GATA3 protein stability.

Zhangfei, a novel regulator of the human nerve growth factor receptor, trkA

The replication of herpes simplex virus (HSV) in epithelial cells, and during reactivation from latency in sensory neurons, depends on a ubiquitous cellular protein called host cell factor (HCF). The HSV transactivator, VP16, which initiates the viral replicative cycle, binds HCF as do some other cellular proteins. Of these, the neuronal transcription factor Zhangfei suppresses the ability of VP16 to initiate the replicative cycle. It also suppresses Luman, another cellular transcription factor that binds HCF. Interactions of nerve growth factor (NGF) and its receptor tropomyosin-related kinase (trkA) appear to be critical for maintaining HSV latency. Because the neuronal transcription factor Brn3a, which regulates trkA expression, has a motif for binding HCF, we investigated if Zhangfei had an effect on its activity. We found that Brn3a required HCF for activating the trkA promoter and Zhangfei suppressed its activity in non-neuronal cells. However, in neuron-like NGF-differentiated PC12 cells, both Brn3a and Zhangfei activated the trkA promoter and induced the expression of endogenous trkA. In addition, capsaicin, a stressor, which activates HSV in in vitro models of latency, decreased levels of Zhangfei and trkA transcripts in NGF-differentiated PC12 cells.

Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation

Regulatory T cells (T(reg)) express Foxp3, a forkhead family member that is necessary and sufficient for T(reg) lineage choice and function. Ectopic expression of Foxp3 in non-T(reg) leads to repression of the interleukin 2 (IL-2) and interferon gamma (IFNgamma) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNgamma loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNgamma genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.

Leukemia-Specific Antigens Alone Are Insufficient for GVL in MHC-Matched Allogeneic Stem Cell Transplantation: An Essential Role for Minor H Antigens.

In MHC-matched allogeneic transplantation (alloSCT), donor T cells recognize minor H antigens (miHAs), which are peptide products of polymorphic genes presented by MHC. It has been presumed that leukemias must express miHAs to be targeted by alloreactive T cells. For graft-versus-leukemia (GVL) in MHC-disparate alloSCTs, tumors must express an allogeneic MHC (Reddy, 2005); however, whether miHAs need to be expressed has not been addressed. Indeed, recent work suggests that non-miHA antigens can be targeted. In alloSCT for CML, CD8 cells reactive against a non-polymorphic epitope of proteinase 3 (PR3) are amplified, a response not possible in MHC-disparate alloSCT. This response has been suggested to be augmented by the simultaneous alloresponse. If so, leukemic cells may not need to express alloantigen at all. A related question is how well antigens exclusively expressed in leukemic cells are targeted. Such antigens are derived from translocations, mutations, or from selectively overexpressed proteins, as is the case with PR3. For these antigens to be targeted by CD8 cells, the leukemia itself must have antigen presenting cell (APC) function or the antigen must be cross-presented by other APCs. To address these questions, we developed a model in which leukemias express leukemia-specific antigens and do or do not express miHAs. We have been studying GVL against a murine model of chronic phase CML (mCP-CML) induced by retroviral introduction of the human p210 cDNA into mouse bone marrow (BM) cells. mCP-CML resembles human CP-CML in that it is myeloproliferative syndrome with splenomegaly and a high white blood count consisting of maturing myeloid cells and few blasts. The retroviral constructs also express the human nerve growth factor receptor or enhanced green fluorescence protein (GFP), allowing us to track transduced cells by FACS. These leukemias therefore express two potential neoantigens: p210 and either NGFR or GFP. mCP-CML cells also overexpress murine PR3. By infecting recipient B6 or donor C3H.SW (C3) BM we created equivalent leukemias that did or did not express miHAs. In B6→B6 transplants, B6 CD8 cells did not mediate GVL against either B6 NGFR+ or GFP+ mCP-CML. Thus NGFR, GFP, PR3 and p210 are insufficient for a GVL effect. To determine whether they can be targeted when T cells are also primed by miHAs, we performed GVL experiments using the C3H.SW(H-2b)→B6(H-2b) strain pairing and compared GVL against C3 or B6 mCP-CML. To distinguish the two, we used p210/GFP and p210/NGFR to infect C3 and B6 BM respectively in one experiment and reversed the retroviruses in a second. Irradiated hosts received C3 BM, either B6 or C3 mCP-CML or a mix of both with or without 106 C3 CD8 cells. Mice that did not receive donor CD8 cells died of mCP-CML regardless of which mCP-CML they received. Most recipients of B6 mCP-CML and donor CD8 cells cleared mCP-CML. In contrast, recipients of C3 CD8 cells and C3 or a mix of B6 and C3 mCP-CML died from C3 mCP-CML with similar kinetics as did mice that received no CD8 cells. Thus, concurrent immunity against miHAs did not provoke a meaningful CD8 response against leukemia-specific antigens. We also infected C3 and B6 BM with GFP/p210 to try to augment the anti-GFP response. Nonetheless, all recipients of a mix of C3 and B6 mCP-CML died without evidence of GVL. In summary, these experiments demonstrate that p210, NGFR, GFP and PR3 are insufficient for GVL regardless of whether there is a simultaneous allogeneic response and thus leukemias must express miHAs.

Novel Cytosine Deaminase Fusion Gene Enhances the Effect of Radiation on Breast Cancer in Bone by Reducing Tumor Burden, Osteolysis, and Skeletal Fracture

Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression. The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CD(y)). CD-expressing tumor cells (4TCD(y)) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCD(y), but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCD(y) intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).

Coexpression of the Uracil Phosphoribosyltransferase Gene with a Chimeric Human Nerve Growth Factor Receptor/Cytosine Deaminase Fusion Gene, Using a Single Retroviral Vector, Augments Cytotoxicity of Transduced Human T Cells Exposed to 5-Fluorocytosine

Donor T lymphocytes genetically engineered to express a "suicide gene" to facilitate negative selection represent a promising strategy for the management of graft-versus-host disease occurring after allogeneic hematopoietic cell transplantation (HCT). For this purpose, the herpes simplex virus thymidine kinase (HSV-tk) gene, although well studied, has limitations. Cytosine deaminase (CD), an alternative gene for negative selection, converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). Sensitivity of cells to 5-FU can be further increased by expression of uracil phosphoribosyltransferase (UPRT), which catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate. By using a chimeric gene (NG/CD) expressing the truncated human nerve growth factor receptor (NGFR) for positive selection fused to the Saccharomyces cerevisiae CD gene, we investigated strategies to achieve optimal T cell eradication by CD and UPRT expression, utilizing a single retroviral vector. Three vector strategies were compared on the basis of NGFR expression by flow cytometry, western analysis, and enzymatic activity. A construct (NG/CDiU) expressing UPRT and NG/CD, using a bicistronic message, provided the greatest UPRT activity and killing, reducing the lethal dose of 5-FC sufficient to eradicate 90% of cells from 38.7 microg/ml (300 microM) (NG/CD expression alone) to 0.13 microg/ml (1 microM). This approach provides an effective alternative to the HSV-tk system for eradication of donor T lymphocytes after allogeneic HCT.

A Bex‐cycle built for two

Neurotrophins promote neuronal survival, but it has been recognized that they also stimulate differentiation. Indeed, in early studies by Hamburger, Levi‐Montelcini and Cohen, nerve growth factor (NGF) not only promoted survival, but also caused marked neurite outgrowth when added to explants of sensory ganglia (Cohen et al , 1954). The ability of NGF to promote differentiation was particularly well illustrated when Greene & Tischler (1976) showed that a pheochromocytoma cell line (PC12) treated with NGF exited the cell cycle and differentiated into a sympathetic neuron‐like phenotype. Since then, it has been established that the survival and differentiation effects of the neurotrophins are primarily mediated by binding to members of the Trk family of tyrosine kinase receptors (Huang & Reichardt, 2003); however, the first receptor identified for NGF was a 75‐kDa protein referred to as p75 (Chao et al , 1986; Radeke et al , 1987). In the past few years, the p75 receptor has received a lot of attention because of its multifunctional signalling ability. This receptor can promote cell survival or induce apoptosis, regulate neurite outgrowth and promote peripheral myelination, depending on the specific cellular context, the expression of co‐receptors and the activating ligand (Barker, 2004). The role of p75 in promoting differentiation and cell‐cycle arrest has been largely overlooked in recent years, even though this was one of the first cellular responses to be shown to result from the binding of NGF to the receptor. Long considered to be simply a co‐receptor for the Trks, p75 came to be recognized as a bona fide signalling molecule when it was shown to activate sphingomyelinase. This resulted in the production of ceramide, which led to the differentiation of T9 glioma cells as indicated by their cell‐cycle arrest and process outgrowth (Dobrowsky et al , 1994). Although the focus of most subsequent research …

Immunotherapy of EBV Post Solid Organ Transplantation Lymphoproliferative Disease with Genetically Engineered Haploidentical T Cells.

Epstein Barr Virus (EBV) associated post-transplantation lymphoproliferative disease (PTLD) is commonly treated with reduction of immune suppression, Rituximab and chemotherapy, but few options exist for individuals with resistant disease. Donor lymphocyte infusions (DLI) from the patient's original donor have provided responses in PTLD following allogeneic marrow transplantation, but HLA matched allogeneic donors are generally not available for solid organ transplantation recipients. A renal-pancreas transplant recipient with refractory PTLD was treated with an infusion of haploidentical T cells genetically engineered to allow eradication of these cells by the administration of ganciclovir should severe GVHD or organ rejection be observed. A retrovirus capable of expressing the herpes simplex virus thymidine kinase (HSV-tk) gene to provide negative selection and the human nerve growth factor receptor (NGFR) for positive selection was used to transduce donor T cells (1.2 x 109) 48 hours post activation with anti-CD3/CD28 beads. The transduction frequency was 37.2% based on NGFR expression; following selection with a biotinylated anti-NGFR antibody and anti-biotin magnetic beads using the Miltenyi CliniMACS a 98.1% NGFR+ product was obtained. Engineered T cells (106/kg) were infused following a lympho-depletion regimen consisting of fludarabine 125 mg/m2 and cyclophosphamide 60 mg/kg. Platelet (>50,000) and neutrophil (>500) recovery occurred 10 and 12 days post cell infusion, respectively. A bone marrow evaluation on day +21 revealed 8.2% of nucleated cells in the marrow to be of donor origin by VNTR analysis. On day +30 peripheral blood VNTR testing documented 28.6% of mononucleated cells to be donor in origin. CT imaging 4 weeks after infusion of the engineered T cells documented decreased size of the primary tumor in the breast. After the initial neutrophil and platelet recovery, neutropenia and thrombocytopenia recurred. A bone marrow evaluation on day 37 revealed decreasing cellularity, possibly consistent with GVHD associated aplasia; VNTR analysis of this marrow sample revealed 20.9% of cells to be of donor origin. The decision was made to treat the patient with ganciclovir (GCV) to eliminate donor T cells contributing to aplasia. Unfortunately, the patient expired within 48 hours of beginning GCV due to an infectious complication. On autopsy, no evidence of previously biopsy proven EBV PTLD was found in the lung and the buccal surface of the mouth. In addition, much of the primary breast mass was free of PTLD. Lymphocytic infiltration of hepatic portal tracts was observed, consistent with GVHD. No evidence of rejection was noted in the transplanted kidney or pancreas. In summary, an excellent response of a refractory EBV PTLD was obtained using therapy including haploidentical T cells engineered for negative selection, and extended persistence of the cells was documented was for greater than 1 month. In the future, a secondary drop in blood counts will be used to start CGV for negative selection of these cells in vivo. This novel strategy may provide effective therapy to transplant recipients without donors who invariable die of their underlying secondary malignancy.