Electrical impedance spectroscopy enabled in-depth lubrication condition monitoring

Electrical contact resistance or capacitance as measured between two interfaces of a lubricated contact has been used in tribometers, partially reflecting the lubrication condition. In contrast, the electrical impedance spectroscopy (EIS) provides rich information of magnitude/phase spectrum, which is thoroughly investigated using a combination of electrical circuit models (equivalent to the lubricated contact) and in-situ measurements with a ball-on-disc contact. Results indicate a promising potential of EIS in lubrication condition monitoring, including the variation of lubricant film thickness as estimated using high-frequency magnitude response; the transition between full-film, mixed, and boundary lubrication regimes, as differentiated using extracted electrical resistance together with phase spectrum; the forming of anti-wear boundary film, where extra resistor/capacitor are added; and the degradation of lubricant, such as fuel dilution, oil oxidization, and water emulsifying

EBAG9-silencing exerts an immune checkpoint function without aggravating adverse effects

Chimeric antigen receptor (CAR) T cells have revolutionized treatment of B-cell malignancies. However, enhancing the efficacy of engineered T cells without compromising their safety is warranted. The estrogen receptor-binding fragment-associated antigen 9 (EBAG9) inhibits release of cytolytic enzymes from cytotoxic T lymphocytes. Here, we examined the potency of EBAG9-silencing for the improvement of adoptive T cell therapy. Micro-RNA-mediated EBAG9 downregulation in transplanted CTLs from immunized mice improved their cytolytic competence in a tumor model. In tolerant female recipient mice that received organ transplants, a minor histocompatibility antigen was turned into a rejection antigen by Ebag9 deletion, indicating an immune checkpoint function for EBAG9. Considerably less EBAG9-silenced human CAR T cells were needed for tumor growth control in a xenotransplantation model. Transcriptome profiling did not reveal additional risks regarding genotoxicity or aberrant differentiation. A single-step retrovirus transduction process links CAR or TCR expression with miRNA-mediated EBAG9 downregulation. Despite higher cytolytic efficacy, release of cytokines associated with cytokine release syndrome remains unaffected. Collectively, EBAG9-silencing enhances effector capacity of TCR- and CAR-engineered T cells, results in improved tumor eradication, facilitates efficient manufacturing, and decreases the therapeutic dose

Comparison of FACS and PCR for detection of BCMA-CAR-T cells

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02-0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred

CXCR5 CAR-T cells simultaneously target B cell non-Hodgkin's lymphoma and tumor-supportive follicular T helper cells

CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME

Experimental investigation into the effects of diesel dilution on engine lubrication

The dilution of lubricant due to contamination with diesel fuel is an increasingly prevalent, potentially important and poorly understood issue. Thisstudy addressestwo fundamental questions: 1) How doesthe change in lubricant rheology due to diesel dilution affect engine lubrication? 2) How is the chemical performance of lubricant components (base oil and performance additives) impacted by diesel dilution under different lubrication regimes (boundary/full film, hydrodynamic/elastohydrodynamic). This is achieved by testing three lubricant samples: 1) neat fully formulated 0W-30 engine oil, 2) fully formulated 0W-30 oil diluted with diesel at a concentration of 15%, denoted “0W-30D”, and 3) neat, fully-formulated 0W-16, with the same base oil components and performance additives as the 0W-30, but blended to give a viscosity equal to that of the diluted an equivalent “0W-30D”. Tribometer tests, including 1) low pressure, low shear viscosity, 2) Ultra-high Shear Viscosity (USV), 3) elastohydrodynamic film thickness, 4) Stribeck friction and 5) boundary friction and wear, are then conducted. To further emulate engine lubrication conditions, Stribeck curve measurements are performed on the three lubricants using a journal bearing test rig, fitted with a connecting-rod and commercial diesel engine shells. Results suggest that diesel dilution only slightly affects chemical additive performance (with friction modifiers being more inhibited than anti-wear additives) but does reduce both viscosity and film thickness. However, care must be taken in using viscometrics to predict dilution behaviour because 1) the pressure viscosity coefficient is also affected by diesel dilution which has implications for elastohydrodynamically lubrication contacts, 2) shear thinning means that viscosity modifier additives effects lose their functions at high shear rates; whereas diesel contamination affects viscosity behaviour throughout the whole shear rate range

Accelerating clinical scale production of BCMA CAR T cells with defined maturation stages

The advent of CAR T cells targeting CD19 or BCMA on B cell neoplasm demonstrated remarkable efficacy, but rapid relapses or primary refractoriness remains challenging. A leading cause for CAR T cell failure is their lack of expansion and limited persistence. Long-lived, self-renewing multipotent T memory stem cells (T(SCM)) and T central memory cells (T(CM)) likely sustain superior tumor regression, but their low frequencies in blood from cancer patients imposes a major hurdle for clinical CAR T production. We designed a clinically compliant protocol for generating BCMA CAR T cells starting with increased T(SCM)/T(CM) cell input. A CliniMACS Prodigy process was combined with flow cytometry-based enrichment of CD62L(+)CD95(+) T cells. Although starting with only 15% of standard T cell input, the selected T(SCM)/T(CM) material was efficiently activated and transduced with a BCMA CAR-encoding retrovirus. Cultivation in the presence of IL-7/IL-15 enabled the harvest of CAR T cells containing an increased CD4(+) T(SCM) fraction and 70% CD8(+) T(SCM) cells. Strong cell proliferation yielded cell numbers sufficient for clinical application, while effector functions were maintained. Together, adaptation of a standard CliniMACS Prodigy protocol to low input numbers resulted in efficient retroviral transduction with a high CAR T cell yield