Low-affinity Nerve Growth Factor Research Articles

Seasonal variation of NGF in seminal plasma and expression of NGF and its cognate receptors NTRK1 and p75NTR in the sex organs of rams

Nerve growth factor (NGF) has long been known as the main ovulation-inducing factor in induced ovulation species, however, recent studies suggested the NGF role also in those with spontaneous ovulation. The first aim of this study was to evaluate the presence and gene expression of NGF and its cognate receptors, high-affinity neurotrophic tyrosine kinase 1 receptor (NTRK1) and low-affinity p75 nerve growth factor receptor (p75NTR), in the ram genital tract. Moreover, the annual trend of NGF seminal plasma values was investigated to evaluate the possible relationship between the NGF production variations and the ram reproductive seasonality. The presence and expression of the NGF/receptors system was evaluated in the testis, epididymis, vas deferens ampullae, seminal vesicles, prostate, and bulbourethral glands through immunohistochemistry and real-time PCR (qPCR), respectively. Genital tract samples were collected from 5 adult rams, regularly slaughtered at a local abattoir. Semen was collected during the whole year weekly, from 5 different adult rams, reared in a breeding facility, with an artificial vagina. NGF seminal plasma values were assessed through the ELISA method. NGF, NTRK1 and p75NTR immunoreactivity was detected in all male organs examined. NGF-positive immunostaining was observed in the spermatozoa of the germinal epithelium, in the epididymis and the cells of the secretory epithelium of annexed glands, NTRK1 receptor showed a localization pattern like that of NGF, whereas p75NTR immunopositivity was localized in the nerve fibers and ganglia. NGF gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis than in the other tissues. NTRK1 gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.05) in all the other tissues examined. Gene expression of p75NTR was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis and bulbourethral glands. NGF seminal plasma concentration was greater from January to May (p < 0.01) than in the other months. This study highlighted that the NGF system was expressed in the tissues of all the different genital tracts examined, confirming the role of NGF in ram reproduction. Sheep are short-day breeders, with an anestrus that corresponds to the highest seminal plasma NGF levels, thus suggesting the intriguing idea that this factor could participate in an inhibitory mechanism of male reproductive activity, activated during the female anestrus.

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.

Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting.

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.

Abstract 2405: Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland

Abstract Lacrimal gland adenoid cystic carcinoma (LGACC) is a rare but highly lethal indolent cancer originating in the cells of secretory glands, primarily of the head and neck. With the 10-year survival rate of 20%, the only life extending technique is to remove the eye and surrounding socket contents followed by extensive chemoradiation. Despite radical treatment, LGACC has a poorer prognosis than head and neck adenoid cystic carcinoma in part due to its propensity for perineural invasion (PNI) and intracranial spread. Analysis of LGACC tumors has revealed signatures enriched for pathways known to be involved in normal LG development, suggesting LGACC tumor cells re-engage or hijack developmental programs during its progression. This project sought to evaluate if developmental pathways involved in embryologic LG innervation were similarly recapitulated by LGACC tumors, and whether any of those markers could be correlated to perineural invasion phenotypes. We performed microarray analysis of embryonic and adult murine lacrimal glands and generated a differential expression signature associated with normal LG developmental pathways. Among other pathways, Notch and FGF signaling are significantly upregulated during LG organogenesis and our group and others have shown that activating mutations in these pathways are correlated with poor prognosis in LGACC. Interestingly, among the highest differentially expressed genes in the embryonic LG was the low-affinity nerve growth factor receptor (p75NTR). We thus subsequently evaluated the expression of p75NTR in LGACC tumors and its correlation to PNI. Our immunofluorescent imaging of histologically confirmed tumors with PNI demonstrated that p75NTR is highly correlated to cancer cells that have invaded the nerve. This correlation of p75NTR expression in nerve infiltrating LGACC cells can inform our investigation into the mechanisms for LGACC PNI and elucidate possible therapeutic targets to limit this aggressive manifestation in LGACC. Citation Format: Acadia H. Moeyersoms, Ryan A. Gallo, Madison Weiss, Michelle M. Maeng, David T. Tse, Daniel Pelaez. Expression of the low affinity neurotrophin receptor (p75NTR) correlates with perineural invasion in adenoid cystic carcinoma of the lacrimal gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2405.

Distribution of α-synuclein in normal human jejunum and its relations with the chemosensory and neuroendocrine system.

Alpha-synuclein (α-syn) is a presynaptic neuronal protein and its structural alterations play an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). It has been originally described in the brain and aggregated α-syn has also been found in the peripheral nerves including the enteric nervous system (ENS) of PD patients. ENS is a network of neurons and glia found in the gut wall which controls gastrointestinal function independently from the central nervous system. Moreover, two types of epithelial cells are crucial in the creation of an interface between the lumen and the ENS: they are the tuft cells and the enteroendocrine cells (EECs). In addition, the abundant enteric glial cells (EGCs) in the intestinal mucosa play a key role in controlling the intestinal epithelial barrier. Our aim was to localize and characterize the presence of α-syn in the normal human jejunal wall. Surgical specimens of proximal jejunum were collected from patients submitted to pancreaticoduodenectomy and intestinal sections underwent immunohistochemical procedure. Alpha-syn has been found both at the level of the ENS and the epithelial cells. To characterize α-syn immunoreactive epithelial cells, we used markers such as choline acetyltransferase (ChAT), useful for the identification of tuft cells. Then we evaluated the co-presence of α-syn with serotonin (5-HT), expressed in EECs. Finally, we used the low-affinity nerve growth factor receptor (p75NTR), to detect peripheral EGCs. The presence of α-syn has been demonstrated in EECs, but not in the tuft cells. Additionally, p75NTR has been highlighted in EECs of the mucosal layer and co-localized with α-syn in EECs but not with ChAT-positive cells. These findings suggest that α-syn could play a possible role in synaptic transmission of the ENS and may contribute to maintain the integrity of the epithelial barrier of the small intestine through EECs.

Genetically Engineered Regulatory T Cells for Treatment of Graft-Versus-Host-Disease

Graft-versus-host-disease (GVHD) undermines survival and complicates survivorship of recipients of allogeneic hematopoietic stem-cell transplantation (HSCT). Adoptive transfer of natural T regulatory cells (nTregs) expressing the lineage-specific transcription factor FOXP3 has been successfully used to prevent or treat GVHD in humans and mice. However, human applications of propagated nTregs have been hampered by the need for extensive ex vivo expansion to obtain sufficient numbers. Furthermore, such nTregs could lose expression of FOXP3 compromising their suppressive function after adoptive transfer. Therefore, we have developed a method to generate large numbers of engineered Tregs (eTregs) that stably express FOXP3 and show potent immune suppressive functions in vitro and in a mouse model of GVHD.Primary human CD4+ T cells were transduced with a lentiviral construct to co-express FOXP3 and truncated low-affinity nerve growth factor receptor (dLNGFR). The genetically modified T cells were purified using dLNGFR-specific magnetic beads and propagated by cross-linking CD3/CD28 in the presence of IL-2 for two weeks, resulting in a substantial increase in cell number while maintaining stable expression of FOXP3. Flow cytometry demonstrated that ≥90% purified eTregs were CD4+CD25+CD127neg FOXP3+, reminiscent of bona fide Tregs. In addition, the propagated eTregs maintained TCR Vβ diversity and expressed markers associated with Treg function, including CTLA-4, GITR and CD39.The eTregs vigorously suppressed the proliferation of TCR-stimulated allogeneic CD4+ and CD8+ effector T cells in vitro in a dose-dependent fashion. The suppression was superior to FOXP3+ nTregs isolated from peripheral blood mononuclear cells (PBMC) and numerically expanded by cross-linking CD3/CD28 in the presence of IL-2. We stimulated eTregs with phorbol 12-myristate 13-acetate and a Ca2+ ionophore to determine intracellular IL-2 and IFN-γ by flow cytometry. The production of these cytokines was significantly reduced as compared to stimulated control effector T cells. These results indicated that the eTregs were hyporesponsive or anergic, which is consistent with the findings from nTregs by us and others.To determine the therapeutic potential of eTregs, we used a xenogeneic model of GVHD (xGVHD), induced in sublethally irradiated immunocompromised (NSG) mice. We co-infused allogeneic human PBMC with graded numbers of eTregs and nTregs and demonstrated that the eTregs could significantly protect mice from xGVHD-induced weight loss as well as mortality, as compared to untreated control mice (Figure). Of note, fewer numbers of eTregs were needed compared to nTregs to achieve these biological endpoints. Analysis of peripheral blood from the mice demonstrated the engraftment of eTregs with maintained TCR Vβ diversity and histological analyses of tissues showed substantially reduced infiltrations of human CD45+ cells in the colon, liver and bone marrow in eTreg-treated mice as compared to untreated controls. In addition, cytokines such as human TNF-α and IFN-γ, associated with the pathogenesis of human GVHD, were markedly decreased in the plasma of eTreg-treated mice.In summary, we demonstrated that lentivirus-mediated gene transfer of FOXP3/dLNGFR into primary human CD4+ T cells enabled the generation of potent eTregs that were capable of controlling xGVHD in mice. Our results warrant further study in patients after allogeneic HSCT with GVHD. [Display omitted] DisclosuresTu:Intrexon Corporation: Employment, Equity Ownership. Fox:Intrexon Corporation: Employment, Equity Ownership. Ly:Intrexon Corporation: Employment, Equity Ownership. Steinhart:Intrexon Corporation: Employment, Equity Ownership. Laporte:Intrexon Corporation: Employment. Brennan:Intrexon Corporation: Employment, Equity Ownership. Bolinger:Intrexon Corporation: Employment, Equity Ownership. Cooper:Argos Therapeutics: Equity Ownership; AmpliPhi Biosciences: Equity Ownership; Intrexon Corporation: Equity Ownership, Patents & Royalties; Procter & Gamble: Equity Ownership; Targazyme, Inc.: Equity Ownership; Sangamo Biosciences: Patents & Royalties; Immatics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Miltenyi Biotec: Honoraria; Organovo Holdings: Equity Ownership; Ziopharm Oncology: Employment, Equity Ownership, Research Funding; Ferring: Consultancy. Waldner:Intrexon Corporation: Employment, Equity Ownership.

The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.

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.

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome

AbstractImmunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

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.

Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors.

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

Differentiation potentials of CD271 positive cells from caprine skin tissue-derived mesenchymal stem cells

Background & Aim Mesenchymal stem cells (MSCs) are undifferentiated cells, found throughout the body after development, which multiply by cell division to replenish dying cells and regenerate damaged tissues. They can be found in adult animals and humans, unlike embryonic stem cells. MSCs characterized by a fibroblast-like morphology possess self-renewal and can be directed to differentiate into cells of mesodermal lineages under specific conditions. However, MSCs are a heterogeneous population of cells with varying magnitudes of differentiation potential among single clone of MSCs. It was documented that in vitro single cell cloning of human MSCs demonstrated that approximately 30% of the clonal cells were multipotent and thus true MSCs. CD271 (low-affinity nerve growth factor receptor) has been identified as a marker of the most homogeneous MSCs subset. Methods, Results & Conclusion Using immunomagnetic selection method, we separated CD271 positive (CD271-P) or negative cells (CD271-P) from caprine ear skin tissue-derived MSCs (capESK-MSCs). Two types of cells were examined for cellular morphology, proliferation properties, expression of cell surface markers and mesodermal differentiation capabilities at early passage. CD271-P and CD271-N cells from capESK-MSCs exhibited abilities to attach to culture plate and expand in vitro. CD271-P cells had spindle-shaped morphology, but CD271-N cells had spindle and round shape morphology. CD271-P and CD271-N cells exhibited not significant difference in cumulative population doubling level during 10 consecutive passages. In addition, CD271-P and CD271-N cells displayed similar immunophenotypic markers (CD34, CD45, CD73, CD90 and CD105). However, CD271-P and CD271-N cells exhibited difference in differentiation potentials. Two types of cells exhibited adipogenic, osteogenic and chondrogenic differentiation potentials. Specifically, CD271-P cells have a greater capacity for chondrogenic differentiation potential compared to CD271-N cells at passage four. CD271 has been identified as a marker of the most homogeneous MSCs. It is capable of promoting differentiation along adipogenic, osteogenic and chondrogenic lineages and producing significantly higher levels of cytokines as compared to the total population of MSCs. In this study, we observed homogenous cell population, proliferating ability and mesodermal differentiation potentials from CD271-P cells from capESK-MSCs.

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.

Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.

Highly Efficient and Selective CAR-Gene Transfer Using CD4- and CD8-Targeted Lentiviral Vectors

Chimeric antigen receptor (CAR)-modified T cells have revealed promising results in the treatment of cancer, but they still need to overcome various hurdles, including a complicated manufacturing process. Receptor-targeted lentiviral vectors (LVs) delivering genes selectively to T cell subtypes may facilitate and improve CAR T cell generation, but so far they have resulted in lower gene delivery rates than conventional LVs (vesicular stomatitis virus [VSV]-LV). To overcome this limitation, we studied the effect of the transduction enhancer Vectofusin-1 on gene delivery to human T cells with CD4- and CD8-targeted LVs, respectively, encoding a second-generation CD19-CAR in conjunction with a truncated version of the low-affinity nerve growth factor receptor (ΔLNGFR) as reporter. Vectofusin-1 significantly enhanced the gene delivery of CD4- and CD8-LVs without a loss in target cell selectivity and killing capability of the generated CAR T cells. Notably, delivery rates mediated by VSV-LV were substantially reduced by Vectofusin-1. Interestingly, a transient off-target signal in samples treated with Vectofusin-1 was observed early after transduction. However, this effect was not caused by uptake and expression of the transgene in off-target cells, but rather it resulted from cell-bound LV particles having ΔLNGFR incorporated into their surface. The data demonstrate that gene transfer rates in the range of those mediated by VSV-LVs can be achieved with receptor-targeted LVs.

Application of a Bioactive/Bioresorbable Three-Dimensional Porous Uncalcined and Unsintered Hydroxyapatite/Poly-D/L-lactide Composite with Human Mesenchymal Stem Cells for Bone Regeneration in Maxillofacial Surgery: A Pilot Animal Study.

A novel three-dimensional (3D) porous uncalcined and unsintered hydroxyapatite/poly-d/l-lactide (3D-HA/PDLLA) composite demonstrated superior biocompatibility, osteoconductivity, biodegradability, and plasticity, thereby enabling complex maxillofacial defect reconstruction. Mesenchymal stem cells (MSCs)—a type of adult stem cell—have a multipotent ability to differentiate into chondrocytes, adipocytes, and osteocytes. In a previous study, we found that CD90 (Thy-1, cluster of differentiation 90) and CD271 (low-affinity nerve growth factor receptor) double-positive cell populations from human bone marrow had high proliferative ability and differentiation capacity in vitro. In the present study, we investigated the utility of bone regeneration therapy using implantation of 3D-HA/PDLLA loaded with human MSCs (hMSCs) in mandibular critical defect rats. Microcomputed tomography (Micro-CT) indicated that implantation of a 3D-HA/PDLLA-hMSC composite scaffold improved the ability to achieve bone regeneration compared with 3D-HA/PDLLA alone. Compared to the sufficient blood supply in the mandibular defection superior side, a lack of blood supply in the inferior side caused delayed healing. The use of Villanueva Goldner staining (VG staining) revealed the gradual progression of the nucleated cells and new bone from the scaffold border into the central pores, indicating that 3D-HA/PDLLA loaded with hMSCs had good osteoconductivity and an adequate blood supply. These results further demonstrated that the 3D-HA/PDLLA-hMSC composite scaffold was an effective bone regenerative method for maxillofacial boney defect reconstruction.