The development of a 14-day non-viral engineered CAR T-cell process

Immunotherapy utilizing chimeric antigen receptor (CAR) T cells is a promising strategy for the treatment of several types of cancer. Many preclinical and clinical studies engineer CAR T cells through a viral vector, presenting the potential for genotoxicity or insertional mutagenesis. We propose a 14-day non-viral process where we introduce the gene of interest via electroporation; integration can be achieved with the Sleeping Beauty transposon system. Minicircle (MC) DNA constructs containing the CAR, a surface marker (EGFRt), and a double mutant of dihydrofolate reductase (DHFRdm) are electroporated into previously frozen, unstimulated CD4/CD8 T cells with an RNA construct coding for the Sleeping Beauty transposase. After electroporation, cells are bead-stimulated with CD3/CD28 without the use of feeder cells throughout the process. CAR+ cells expressing DHFRdm are rendered insensitive to an FDA-approved small molecule drug, methotrexate (MTX), which allows for chemical selection of the cells of interest while avoiding a magnetic bead sort. The entire process is completed in 2 weeks with a media formulation that contains a serum-free replacement. Please click Additional Files below to see the full abstract

Regulation of melanoma cell survival and function by matricellular signaling and microenvironmental factors

Metastatic melanoma is an aggressive, drug resistant form of skin cancer. Despite the recent development of several new therapeutics, patients typically relapse within 6 months of starting treatment. Consequently, researchers are working to understand the mechanisms of melanoma drug resistance and the key factors that impact its survival. Because cell-ECM (extracellular matrix) interactions alter outside-in signaling and relay information about the surrounding tissue to a cell, the cellular microenvironment can regulate cell function and this, in turn, may influence melanoma responsiveness to pharmacological inhibition. Preclinical studies have shown differential drug efficacy between traditional tissue culture-treated polystyrene (TCPS) and multicellular spheroids embedded within a soft collagen matrix. While researchers attribute more drug resistant behavior to three-dimensional (3D) cell culture, it is unclear whether interactions with soft (or stiff) matrices can promote this response, if dimensionality (2D versus 3D) of the culture environment matters, or whether the existence of cell-cell contacts is paramount to drug responsiveness. This thesis research aimed to use hydrogel systems as synthetic ECM mimics to systematically study the influence of matrix elasticity and dimensionality on the behavior of melanoma cells at different stages of disease progression in response to the clinically relevant drug, Zelboraf (PLX4032, vemurafenib). Peptide-functionalized poly(ethylene glycol) (PEG) hydrogels provided control over bulk properties while probing specific questions regarding the local cell-ECM microenvironment and interactions. Human cell lines derived from radial growth phase and metastatic melanoma were cultured on hydrogel surfaces or encapsulated within synthetic ECM mimics with tunable elasticity and biological functionality (e.g., integrin binding epitopes, protease degradability). First, the role of matrix rigidity on PLX4032 sensitivity was assessed on 2D hydrogels of varying moduli. The influence of dimensionality and cell-cell contacts on melanoma drug responsiveness to PLX4032 in 2D and 3D culture was measured. Finally, cells encapsulated within hydrogels were evaluated for both proteolytic activity in response to PLX4032 and the functional impact of this enzymatic activity. This thesis research demonstrated the importance of the tumor microenvironment in regulating PLX4032 responses and its implications for preclinical studies and clinical treatment

SH3YL1 regulates dorsal ruffle formation by a novel phosphoinositide-binding domain

The newly identified SYLF lipid-binding domain of SH3YL1 mediates phosphoinositide binding during dorsal membrane morphogenesis

Successful management of pelvic recurrence of MSI-High endometrial cancer by total pelvic exenteration followed by administration of pembrolizumab:A case report

Surgery can be curative treatment for pelvic locoregional recurrence of endometrial cancer; however, a cure is contingent on complete resection. Here, we report the case of a patient in whom recurrent endometrial tumor remained in the pelvis after resection; long-term control was achieved with postoperative administration of pembrolizumab.The patient had recurrent endometrial cancer of stage IA and was treated with chemotherapy and radiation, but tumor persisted in the pelvic cavity. We therefore attempted total pelvic exenteration, but the tumor was adherent to the pelvic wall and complete resection could not be achieved. However, postoperative administration of pembrolizumab controlled the residual tumor for more than two years without regrowth. We believe that since the resected tumor was MSI-High, the residual tumor responded well to pembrolizumab. It is not known whether cytoreductive surgery contributes to a long-term response to pembrolizumab, but at least in our patient, pembrolizumab appeared to be a very effective drug therapy for MSI-High endometrial cancer that was refractory to chemotherapy and radiotherapy

Dental exchange class in the 2018 introduction to dentistry practice impressions analysis of junior high school students and dental university students

Summary[Purpose]In the introduction to dentistry practice in 2018, the first–year students of Matsumoto Dental University (MDU) had the opportunity to participate in the dental exchange class with students from junior high schools and elementary schools in Shiojiri City. Among them, the MDU students received a report on the class from the first and sec ond graders of Koryo Junior High School. The MDU students returned the impressions of the class to the junior high school students. The class was a valuable experience for the students and the teachers of MDU. We thought we should analyze the students’ impressions and use them for future reference.[ Methods ] The number of the students’ impressions was 131 for the first–year, and 149 for the second–year students of Koryo Junior High School, while the number of MDU students’ impressions was 56 for the first–year students and 50 for the second–year students.Based on these impressions, (1) we selected the main words of the impressions as keywords,counted them, and organized them into items based on our subjective opinion. (2)The impressions were text–mined and multivariate analysis was performed. (3) Visualizationwas attempted by cooccurrence network analysis.[Results] The result of (1), the key words were 354 words for the first–year students of junior high school and 353 words for the second–year students. The MDU students had 144 words for the first–year students and 154 words for the second–year students. The importanceof the key words and the frequency of their occurrence were found in (2). The importance of the key word “tooth” was the most important and thefrequency of its appearance in the first year of junior high school, while “understanding” was the most important and “tooth” was the most frequent in the second year of junior high school. The MDU students gave the highest importance and frequency of occurrence of “presentation” to both the first and second–year students. In task (3), we connected the key words in the network and visualized the relationship between the words.[Discussion] By looking at the number of key words, it can be inferred that the first and second–year students were able to understand the task. The investigation implies that theMDU students might have gained valuable experience from this exchange class.[Conclusion] Throughout the exchange class,1. The first and second–year students of Koryo Junior High School gained professional knowledge of dentistry.2. Students at Koryo Junior High School gained a better understanding of the MDU.3. First year students of the MDU gained valuable experience as presenters by understanding, preparing and devising class assignments

Involvement of folate and vitamin B12 deficiency in patients with normocytic anemia

Objectives: Deficiencies in folate (FA) and vitamin B12 (VB12) are causes of macrocytic anemia. However, in clinical practice, FA and/or VB12 deficiency can occur in patients with normocytic anemia. This study aimed to determine the prevalence of FA/VB12 deficiency in patients with normocytic anemia and the importance of vitamin replacement therapy in these patients. Methods: We retrospectively reviewed electronic medical record information of patients whose hemoglobin and serum FA/VB12 concentrations were measured at the Department of Hematology (N=1,388) and other departments (N=1,421) of Fujita Health University Hospital. Results: In the Hematology Department, 530 (38%) patients showed normocytic anemia. Of these, 49 (9.2%) had FA/VB12 deficiency. Twenty of 49 (41%) patients had some hematological malignancies and 27 (55%) had benign hematological disorders. Of the nine patients who received vitamin replacement therapy, one showed a partial improvement in the hemoglobin concentration of ≥1 g/dL. Conclusions: In the clinical setting, the measurement of FA/VB12 concentrations in patients with normocytic anemia may be useful. Replacement therapy may be a treatment option to consider in patients with low FA/VB12 concentrations. However, physicians need to pay attention to the presence of background diseases, and the mechanisms of this situation require further investigation