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

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

Simultaneous measurement of phase retardation and optic axis of a phase compensation film using an axially-symmetric sheared polymer network liquid crystal

A new method for simultaneously measuring the phase retardation and optic axis of a uniaxial compensation film is demonstrated using an axially-symmetric sheared polymer network liquid crystal (SPNLC). By overlaying a tested compensation film with a calibrated SPNLC cell between crossed polarizers, two dark spots are clearly observed in a CCD image. From the orientation direction and distance of these two spots, the optic axis and phase retardation value of the compensation film can be determined. This method is particularly useful for those optical systems whose optic axis and phase retardation are dynamically changing

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

GPER-induced signaling is essential for the survival of breast cancer stem cells.

G protein-coupled estrogen receptor-1 (GPER), a member of the G protein-coupled receptor (GPCR) superfamily, mediates estrogen-induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non-BCSCs of three patient-derived xenografts of ER- /PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER-mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD-Ser118 to sustain BCSC characteristics. Transfection with a dominant-negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs

Axially-symmetric sheared polymer network liquid crystals

An axially-symmetric sheared polymer network liquid crystal (SPNLC) device is demonstrated and its performances characterized. Through analyzing the structure of this axially-symmetric SPNLC, we constructed a 3-D model to explain the observed phenomena. The simulation results agree well with the experiment. Two potential applications of such an axially-symmetric SPNLC, namely tunable-focus negative lens and spatial polarization converter, are discussed

The antagonism between MCT-1 and p53 affects the tumorigenic outcomes

<p>Abstract</p> <p>Background</p> <p>MCT-1 oncoprotein accelerates p53 protein degradation via a proteosome pathway. Synergistic promotion of the xenograft tumorigenicity has been demonstrated in circumstance of p53 loss alongside MCT-1 overexpression. However, the molecular regulation between MCT-1 and p53 in tumor development remains ambiguous. We speculate that MCT-1 may counteract p53 through the diverse mechanisms that determine the tumorigenic outcomes.</p> <p>Results</p> <p>MCT-1 has now identified as a novel target gene of p53 transcriptional regulation. MCT-1 promoter region contains the response elements reactive with wild-type p53 but not mutant p53. Functional p53 suppresses MCT-1 promoter activity and MCT-1 mRNA stability. In a negative feedback regulation, constitutively expressed MCT-1 decreases p53 promoter function and p53 mRNA stability. The apoptotic events are also significantly prevented by oncogenic MCT-1 in a p53-dependent or a p53-independent fashion, according to the genotoxic mechanism. Moreover, oncogenic MCT-1 promotes the tumorigenicity in mice xenografts of p53-null and p53-positive lung cancer cells. In support of the tumor growth are irrepressible by p53 reactivation <it>in vivo</it>, the inhibitors of p53 (MDM2, Pirh2, and Cop1) are constantly stimulated by MCT-1 oncoprotein.</p> <p>Conclusions</p> <p>The oppositions between MCT-1 and p53 are firstly confirmed at multistage processes that include transcription control, mRNA metabolism, and protein expression. MCT-1 oncogenicity can overcome p53 function that persistently advances the tumor development.</p

A novel randomly textured phosphor structure for highly efficient white light-emitting diodes

We have successfully demonstrated the enhanced luminous flux and lumen efficiency in white light-emitting diodes by the randomly textured phosphor structure. The textured phosphor structure was fabricated by a simple imprinting technique, which does not need an expensive dry-etching machine or a complex patterned definition. The textured phosphor structure increases luminous flux by 5.4% and 2.5% at a driving current of 120 mA, compared with the flat phosphor and half-spherical lens structures, respectively. The increment was due to the scattering of textured surface and also the phosphor particles, leading to the enhancement of utilization efficiency of blue light. Furthermore, the textured phosphor structure has a larger view angle at the full width at half maximum (87°) than the reference LEDs

Intraprostatic injection of botulinum toxin type- A relieves bladder outlet obstruction in human and induces prostate apoptosis in dogs

BACKGROUND: With the increasing interest with botulinum toxin – A (BTX-A) application in the lower urinary tract, we investigated the BTX-A effects on the canine prostate and also in men with bladder outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH). METHODS: Transperineal injection into the prostate using transrectal ultrasound (TRUS) was performed throughout the study. Saline with or without 100 U of BTX-A was injected into mongrel dogs prostate. One or 3 months later, the prostate was harvested for morphologic and apoptotic study. In addition, eight BPH patients refractory to α-blockers were treated with ultrasound guided intraprostatic injection of 200 U of BTX-A. RESULTS: In the BTX-A treated dogs, atrophy and diffuse apoptosis was observed with H&E stain and TUNEL stain at 1 and 3 months. Clinically, the mean prostate volume, symptom score, and quality of life index were significantly reduced by 18.8%, 73.1%, and 61.5% respectively. Maximal flow rate significantly increased by 72.0%. CONCLUSION: Intraprostatic BTX-A injection induces prostate apotosis in dogs and relieves BOO in humans. It is therefore a promising alternative treatment for refractory BOO due to BPH