CD14+/CD31+ monocytes expanded by UM171 correct hemophilia A in zebrafish upon lentiviral gene transfer of factor VIII.

Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

A Histone Deacetylase Inhibitor, Panobinostat, Enhances Chimeric Antigen Receptor T-cell Antitumor Effect Against Pancreatic Cancer.

PURPOSE: In this article, we describe a combination chimeric antigen receptor (CAR) T-cell therapy that eradicated the majority of tumors in two immunocompetent murine pancreatic cancer models and a human pancreatic cancer xenograft model. EXPERIMENTAL DESIGN: We used a dual-specific murine CAR T cell that expresses a CAR against the Her2 tumor antigen, and a T-cell receptor (TCR) specific for gp100. As gp100 is also known as pMEL, the dual-specific CAR T cells are thus denoted as CARaMEL cells. A vaccine containing live vaccinia virus coding a gp100 minigene (VV-gp100) was administered to the recipient mice to stimulate CARaMEL cells. The treatment also included the histone deacetylase inhibitor panobinostat (Pano). RESULTS: The combination treatment enabled significant suppression of Her2+ pancreatic cancers leading to the eradication of the majority of the tumors. Besides inducing cancer cell apoptosis, Pano enhanced CAR T-cell gene accessibility and promoted CAR T-cell differentiation into central memory cells. To test the translational potential of this approach, we established a method to transduce human T cells with an anti-Her2 CAR and a gp100-TCR. The exposure of the human T cells to Pano promoted a T-cell central memory phenotype and the combination treatment of human CARaMEL cells and Pano eradicated human pancreatic cancer xenografts in mice. CONCLUSIONS: We propose that patients with pancreatic cancer could be treated using a scheme that contains dual-specific CAR T cells, a vaccine that activates the dual-specific CAR T cells through their TCR, and the administration of Pano.

Enhancing chimeric antigen receptor T-cell immunotherapy against cancer using a nanoemulsion-based vaccine targeting cross-presenting dendritic cells.

OBJECTIVES: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells is a form of cancer immunotherapy that has achieved remarkable efficacy in patients with some haematological cancers. However, challenges remain in CAR T-cell treatment of solid tumours because of tumour-mediated immunosuppression. METHODS: We have demonstrated that CAR T-cell stimulation through T-cell receptors (TCRs) in vivo can generate durable responses against solid tumours in a variety of murine models. Since Clec9A-targeting tailored nanoemulsion (Clec9A-TNE) vaccine enhances antitumour immune responses through selective activation of Clec9A+ cross-presenting dendritic cells (DCs), we hypothesised that Clec9A-TNE could prime DCs for antigen presentation to CAR T cells through TCRs and thus improve CAR T-cell responses against solid tumours. To test this hypothesis, we used CAR T cells expressing transgenic TCRs specific for ovalbumin (OVA) peptides SIINFEKL (CAROTI) or OVA323-339 (CAROTII). RESULTS: We demonstrated that the Clec9A-TNEs encapsulating full-length recombinant OVA protein (OVA-Clec9A-TNE) improved CAROT T-cell proliferation and inflammatory cytokine secretion in vitro. Combined treatment using the OVA-Clec9A-TNE and CAROT cells resulted in durable responses and some rejections of tumours in immunocompetent mice. Tumour regression was accompanied by enhanced CAROT cell proliferation and infiltration into the tumours. CONCLUSION: Our study presents Clec9A-TNE as a prospective avenue to enhance CAR T-cell efficacy for solid cancers.

Enterotoxins can support CAR T cells against solid tumors.

Responses of solid tumors to chimeric antigen receptor (CAR) T cell therapy are often minimal. This is potentially due to a lack of sustained activation and proliferation of CAR T cells when encountering antigen in a profoundly immunosuppressive tumor microenvironment. In this study, we investigate if inducing an interaction between CAR T cells and antigen-presenting cells (APCs) in lymphoid tissue, away from an immunosuppressive microenvironment, could enhance solid-tumor responses. We combined CAR T cell transfer with the bacterial enterotoxin staphylococcal enterotoxin-B (SEB), which naturally links a proportion of T cell receptor (TCR) Vß subtypes to MHC-II, present on APCs. CAR T cell proliferation and function was significantly enhanced by SEB. Solid tumor-growth inhibition in mice was increased when CAR T cells were administered in combination with SEB. CAR T cell expansion in lymphoid tissue was demonstrated, and inhibition of lymphocyte egress from lymph nodes using FTY720 abrogated the benefit of SEB. We also demonstrate that a bispecific antibody, targeting a c-Myc tag on CAR T cells and cluster of differentiation 40 (CD40), could also enhance CAR T cell activity and mediate increased antitumor activity of CAR T cells. These model systems serve as proof-of-principle that facilitating the interaction of CAR T cells with APCs can enhance their ability to mediate antitumor activity.

Genetic Redirection of T Cells for the Treatment of Pancreatic Cancer.

Conventional treatments for pancreatic cancer are largely ineffective, and the prognosis for the vast majority of patients is poor. Clearly, new treatment options are desperately needed. Immunotherapy offers hope for the development of treatments for pancreatic cancer. A central requirement for the efficacy of this approach is the existence of cancer antigen-specific T cells, but these are often not present or difficult to isolate for most pancreatic tumors. Nevertheless, specific T cells can be generated using genetic modification to express chimeric antigen receptors (CAR), which can enable T cell responses against pancreatic tumor cells. CAR T cells can be produced ex vivo and expanded in vitro for infusion into patients. Remarkable responses have been documented using CAR T cells against several malignancies, including leukemias and lymphomas. Based on these successes, the extension of CAR T cell therapy for pancreatic cancer holds great promise. However, there are a number of challenges that limit the full potential of CAR T cell therapies for pancreatic cancer, including the highly immunosuppressive tumor microenvironment (TME). In this article, we will review the recent progress in using CAR T cells in pancreatic cancer preclinical and clinical settings, discuss hurdles for utilizing the full potential of CAR T cell therapy and propose research strategies and future perspectives. Research into the use of CAR T cell therapy in pancreatic cancer setting is rapidly gaining momentum and understanding strategies to overcome the current challenges in the pancreatic cancer setting will allow the development of effective CAR T cell therapies, either alone or in combination with other treatments to benefit pancreatic cancer patients.

Sex determination in forensic odontology: A review.

Forensic odontology is the application of dental principles to legal issues. Sex determination is a subdivision of forensic odontology and it is very important especially when information relating to the deceased is unavailable. Sex determination becomes the first priority in the process of identification of a person by a forensic investigator in the case of mishaps, chemical and nuclear bomb explosions, natural disasters crime investigations, and ethnic studies. This article reviews upon the various methods used in sex determination.

Oral pigmentation: A review.

Pigmentations are commonly found in the mouth. They represent in various clinical patterns that can range from just physiologic changes to oral manifestations of systemic diseases and malignancies. Color changes in the oral mucosa can be attributed to the deposition of either endogenous or exogenous pigments as a result of various mucosal diseases. The various pigmentations can be in the form of blue/purple vascular lesions, brown melanotic lesions, brown heme-associated lesions, gray/black pigmentations.

Odontogenic myxoma of maxilla: A rare presentation in an elderly female.

Odontogenic myxomas are rare benign neoplasm of mesenchymal origin, comprising 3-6% of all odontogenic tumors. They are slow growing, non-metastasizing, often asymptomatic with local aggressiveness due to its infiltrative nature and hence high recurrence rate, with a high incidence of occurrence in the mandible. Most frequently occurs in second to third decade of life, seldom occurs beyond these age groups. Hereby, we present a case of odontogenic myxoma occurring in the maxilla in a 65-year-old female managed by partial maxillectomy.