Maintaining CD4/CD8 ratio and Th1-CTL subsets of chimeric antigen receptor (CAR)-T cells in serum-free culture conditions

Chimeric antigen receptor (CAR) T cells therapy is a promising strategy that significantly controlled the progress of cancer diseases. CAR-T cells could kill cancer cells through cellular immune response; therefore, CD8+ cytotoxic T cells are critical for CAR-T cell therapy. However, recent papers reported that CD4+ T helper cells were important for the response and maintenance of CAR-T cells in vivo. Here, we developed a serum-free CAR-T cell preparation process that maintained the T cell population and controlled the T cell subsets. The CD4+ and CD8+ T cell population in CAR-T cells were maintained at averagely 59.4 % and 34.6%, and the major T cell subsets were Th1 cells and cytotoxic T lymphocytes (CTLs), implying the potentially high cellular immune response. To verifying whether the prepared CAR-T cells were exhausted, the expression of several immune checkpoint markers was determined. Of interest, only less than 20% of CAR-T cells at endpoint were PD-1+ or CTLA4+, but more than 40% of CAR-T cells at the endpoint were TIM-3+, implying most CAR-T cells were not exhausted. These CAR-T cells produced more than 1 ng/mL of IFN-γ in the response to the antigen. Altogether, CAR-T cells could be prepared in our serum-free process in the controlling of T cell subsets, leading to potential high therapeutic potency. Please click Additional Files below to see the full abstract

Carbon monoxide may enhance bile secretion by increasing glutathione excretion and Mrp2 expression in rats

AbstractBackgroundNitric oxide (NO) donors have been reported to induce choleresis via an increased excretion of glutathione. The effects of another gas molecule, carbon monoxide (CO), on bile formation are, however, inconsistent among previous reports. We investigated the sequential changes of bile output and the biliary contents in rats with or without CO supplementation to elucidate the mechanism of CO on bile excretion.MethodsDichloromethane (DCM) was gastrically fed to male Sprague–Dawley rats to yield CO by liver biotransformation. The rats were divided into DCM-treated (n = 7), DCM plus L-NAME-treated (n = 6), and corn oil-treated-(n = 8) groups. Bile samples were collected hourly to examine the flow rate and bile content. Serum levels of nitrite and nitrate 4 hours after DCM supplementation with or without NO synthase (NOS) inhibition were measured by capillary electrophoresis. The expression of hepatic inducible NOS was evaluated by Western blotting 6 hours after DCM administration.ResultsLevels of carboxyhemoglobin rose to around 10% at 4 hours after DCM supplementation and were maintained until the end of the experiments. Bile flow increased after DCM supplementation and was associated with a concomitant increase of biliary glutathione and higher hepatic multidrug resistance-associated protein 2 (Mrp2) expression. Hepatic inducible NOS expression and serum nitrate/nitrite levels were also increased. Treatment with an NOS inhibitor (L-NAME) abolished the CO-induced glutathione excretion and choleresis, but not Mrp2 expression.ConclusionThe present study demonstrated that CO enhanced biliary output in conjunction with NO by increasing the biliary excretion of glutathione. The increment in biliary glutathione was associated with an increased expression of hepatic Mrp2

In vitro high expansion of chimeric antigen receptor (CAR)-T cells in serum-free process conditions

Manufacturing process is an important and complex factor for preparing chimeric antigen receptor (CAR) T cells for therapy. Although serum was widely applied in the culture or expansion of T cells, the quality of serum could be varied from batch to batch, leading to the variation of T cell expansion and quality. In addition, the safety of pathogens from serum and Chemistry, Manufacturing, and Control (CMC) were required to be considered. To overcome the disadvantages of serum application in T cell culture, serum-free and xeno-free culture conditions were required. We intended to develop a rapid serum-free culture condition for the expansion of immune T cells ex vivo. In our optimized serum-free condition, CAR-T cells could be expanded to about 100-200 times to the initial cell number after 6-day culture and the cell viability of all specimens was above 98%. Of interest, the percentage of CAR+ population in all specimens was increases, and the T cell pollutions could be maintained at averagely about 35-40% of CD8+ T cells and averagely about 50-55% of CD4+ T cells after culture. Taken together, our conditions could be applied in the expansion of CAR-T cells for cell therapy to support the minimum requirement of blood or cell samples from patients and to maintain the T cell population. Please click Additional Files below to see the full abstract

Fucosyltransferase 1 and 2 play pivotal roles in breast cancer cells.

FUT1 and FUT2 encode alpha 1, 2-fucosyltransferases which catalyze the addition of alpha 1, 2-linked fucose to glycans. Glycan products of FUT1 and FUT2, such as Globo H and Lewis Y, are highly expressed on malignant tissues, including breast cancer. Herein, we investigated the roles of FUT1 and FUT2 in breast cancer. Silencing of FUT1 or FUT2 by shRNAs inhibited cell proliferation in vitro and tumorigenicity in mice. This was associated with diminished properties of cancer stem cell (CSC), including mammosphere formation and CSC marker both in vitro and in xenografts. Silencing of FUT2, but not FUT1, significantly changed the cuboidal morphology to dense clusters of small and round cells with reduced adhesion to polystyrene and extracellular matrix, including laminin, fibronectin and collagen. Silencing of FUT1 or FUT2 suppressed cell migration in wound healing assay, whereas FUT1 and FUT2 overexpression increased cell migration and invasion in vitro and metastasis of breast cancer in vivo. A decrease in mesenchymal like markers such as fibronectin, vimentin, and twist, along with increased epithelial like marker, E-cadherin, was observed upon FUT1/2 knockdown, while the opposite was noted by overexpression of FUT1 or FUT2. As expected, FUT1 or FUT2 knockdown reduced Globo H, whereas FUT1 or FUT2 overexpression showed contrary effects. Exogenous addition of Globo H-ceramide reversed the suppression of cell migration by FUT1 knockdown but not the inhibition of cell adhesion by FUT2 silencing, suggesting that at least part of the effects of FUT1/2 knockdown were mediated by Globo H. Our results imply that FUT1 and FUT2 play important roles in regulating growth, adhesion, migration and CSC properties of breast cancer, and may serve as therapeutic targets for breast cancer

Extensive Reading with Guidance

A language learning mode called “word-focused extensive reading” has been proposed to facilitate word-usage learning. The user inputs a word in the computer program designed and implemented for reading to find its usage in the context of a sentence. The computer program then searches for the knowledge base for the word and provides guidance to show a summary of the salient features of word collocation. Sentences with the word in question are displayed one at a time. For each sentence the relevant collocating words are highlighted. In this way the reader sees the word collocation so as to recognize the salient features of the word and thus incrementally acquires the knowledge of word usage. The program works with large Chinese and English collections of texts. For Chinese the knowledge of word collocation was built on the basis of the Balanced Corpus of Academia Sinica. The English collocation features were collected from the British National Corpus. The program can also be useful for learning to differentiate near-synonyms

Bis{1-[(E)-o-tolyl­diazen­yl]-2-naphtho­l­ato}copper(II)

In the title complex, [Cu(C17H13N2O)2], the CuII atom is tetra­coordinated by two N atoms and two O atoms from two bidentate 1-[(E)-o-tolyl­diazen­yl]-2-naphtho­late ligands, forming a slightly distorted square-planar environment. The two N atoms and two O atoms around the CuII atom are trans to each other, with an O—Cu—O bond angle of 177.00 (9)° and an N—Cu—N bond angle of 165.63 (10)°. The average distances between the CuII atom and the coordinated O and N atoms are 1.905 (2) and 1.995 (2)Å, respectively

The experience and attitude of TMU faculty and researchers toward predatory journals and research productivity

A questionnaire related to journal submission was sent to researchers for a 2-week period to investigate the submission status as well as the problems faced by Taipei Medical University faculties and researchers. This study has two major findings including the Experience of predatory journal and Calculations of academic performance point and discuss about users’ cognition and their needs from the library and the university, as well as the library policy and services related to predatory journals. It is authors’ hope that the research results can serve as reference for other medical libraries planning to provide relevant services

Effects of ranibizumab on human corneal endothelial cells

AbstractPurposeThis study aims to evaluate corneal endothelial changes occurring over a 3-month period after intravitreal injections of ranibizumab in patients with wet age-related macular degeneration.MethodsThis is a prospective case series. A total of 29 patients (29 eyes) received a 0.5-mg intravitreal injection of ranibizumab. Specular microscopy, including measurement of central corneal thickness and endothelial cell count, was performed on each patient prior to and after completing three intravitreal injections.ResultsAll patients received three intravitreal injections and were followed up for a mean of 3 months. There was no significant change in corneal thickness (p = 0.32) or endothelial cell density (p = 0.38) after ranibizumab injections.ConclusionIntravitreal ranibizumab injections (0.5 mg) have no harmful effects on corneal endothelial cells

Pre-Emptive Treatment of Lidocaine Attenuates Neuropathic Pain and Reduces Pain-Related Biochemical Markers in the Rat Cuneate Nucleus in Median Nerve Chronic Constriction Injury Model

This study investigates the effects of lidocaine pre-emptive treatment on neuropathic pain behavior, injury discharges of nerves, neuropeptide Y (NPY) and c-Fos expression in the cuneate nucleus (CN) after median nerve chronic constriction injury (CCI). Behavior tests demonstrated that the pre-emptive lidocaine treatment dose dependently delayed and attenuated the development of mechanical allodynia within a 28-day period. Electrophysiological recording was used to examine the changes in injury discharges of the nerves. An increase in frequency of injury discharges was observed and peaked at postelectrical stimulation stage in the presaline group, which was suppressed by lidocaine pre-emptive treatment in a dose-dependent manner. Lidocaine pretreatment also reduced the number of injury-induced NPY-like immunoreactive (NPY-LI) fibers and c-Fos-LI neurons within the CN in a dose-dependent manner. Furthermore, the mean number of c-Fos-LI neurons in the CN was significantly correlated to the NPY reduction level and the sign of mechanical allodynia following CCI