Optimizing Ex Vivo CAR-T Cell-Mediated Cytotoxicity Assay through Multimodality Imaging.

CAR-T cell-based therapies have demonstrated remarkable efficacy in treating malignant cancers, especially liquid tumors, and are increasingly being evaluated in clinical trials for solid tumors. With the FDA's initiative to advance alternative methods for drug discovery and development, full human ex vivo assays are increasingly essential for precision CAR-T development. However, prevailing ex vivo CAR-T cell-mediated cytotoxicity assays are limited by their use of radioactive materials, lack of real-time measurement, low throughput, and inability to automate, among others.

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors.

Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging.

CRISPR/Cas9 Edited RAS & MEK Mutant Cells Acquire BRAF and MEK Inhibitor Resistance with MEK1 Q56P Restoring Sensitivity to MEK/BRAF Inhibitor Combo and KRAS G13D Gaining Sensitivity to Immunotherapy.

mutation drives uncontrolled cell growth in most melanomas. While tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the , and mutations to the BRAF inhibitor resistance.

Stoichiometric Analyses of Soluble CD4 to Native-like HIV-1 Envelope by Single-Molecule Fluorescence Spectroscopy.

Analyses of HIV-1 envelope (Env) binding to CD4, and the conformational changes the interactions induce, inform the molecular mechanisms and factors governing HIV-1 infection. To address these questions, we used a single-molecule detection (SMD) approach to study the nature of reactions between soluble CD4 (sCD4) and soluble HIV-1 trimers. SMD of these reactions distinguished a mixture of one, two, or three CD4-bound trimer species.

Patterns of conserved gp120 epitope presentation on attached HIV-1 virions.

A complete picture of HIV antigenicity during early replication is needed to elucidate the full range of options for controlling infection. Such information is frequently gained through analyses of isolated viral envelope antigens, host CD4 receptors, and cognate antibodies. However, direct examination of viral particles and virus-cell interactions is now possible via advanced microscopy techniques and reagents.

Monotherapy with either dolutegravir or raltegravir fails to durably suppress HIV viraemia in humanized mice.

Objectives: To compare the effectiveness of HIV integrase inhibitor monotherapy between raltegravir and dolutegravir as an approach to simplify therapy.

Methods: We evaluated and compared the efficacy of 20 week monotherapy with dolutegravir or raltegravir in humanized mice (HSC-NSG) infected with HIVBaL. Plasma HIV RNA was measured by quantitative RT-PCR (limit of detection of 150 copies/45 μL of plasma) and drug levels by LC-MS/MS.

Structural definition of an antibody-dependent cellular cytotoxicity response implicated in reduced risk for HIV-1 infection.

Unlabelled: The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection.

Targeting of the purine biosynthesis host cell pathway enhances the activity of tenofovir against sensitive and drug-resistant HIV-1.

Background: Targeting host-cell pathways to increase the potency of nucleoside/nucleotide analog reverse transcriptase inhibitors (NRTIs) is an important strategy for clinical investigation. Resveratrol is a natural product that inhibits cellular ribonucleotide reductase, prolonging the S phase of the cell cycle and preferentially lowering dATP levels.

Methods: We performed in vitro evaluation of resveratrol on the antiviral activity of adenosine analog tenofovir (TFV) against sensitive and drug-resistant human immunodeficiency virus type 1 (HIV-1), from subtypes B and C, in primary cells.

Synergistic inhibition of R5 HIV-1 by maraviroc and CCR5 antibody HGS004 in primary cells: implications for treatment and prevention.

CCR5 blockers inhibit CCR5-tropic (R5) HIV-1, including strains resistant to other antiretrovirals. We demonstrate that the CCR5 antibody HGS004 and the CCR5 antagonist maraviroc have potent antiviral synergy against R5 HIV-1, translating into dose reductions of more than 10-fold for maraviroc and more than 150-fold for HGS004. These data, together with the high barrier of resistance to HGS004, suggest that combinations of maraviroc and HGS004 could provide effective preventive and therapeutic strategies against R5 HIV-1.

CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells.

R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection.

Evaluation of radiological versus chemical toxicity limits for varying enrichments of uranium for Department of Energy facilities.

On 8 June 2007, the Department of Energy amended its occupational radiation protection rule Title 10 Code of Federal Regulations Part 835, Occupational Radiation Protection. The Department of Energy revised the radiation weighting factors, tissue weighting factors, and most of the dosimetric terms used in Title 10 Code of Federal Regulations Part 835 to reflect the recommendations for assessing dose and associated terminology from ICRP Publication 60, 1990 Recommendations of the ICRP on Radiological Protection. In support of the amendment, the Department of Energy is revising its guidance documents on evaluation of radiological vs.

A standardized method for measuring the force required to join wire harnesses and sparkplugs.

Understanding the forces required to insert a sparkplug wire (wire) onto a sparkplug (plug), independent of worker variation, is important for ergonomists, engineers, and designers. This paper describes a methodology for measuring the forces required to seat a wire onto a plug. A three-axis programmable mill was used to insert wires onto plugs mounted on a force transducer.

Developing in-vitro bioassay goals for plutonium and uranium for Department of Energy facilities.

On 8 June 2007, the Department of Energy amended its occupational radiation protection rule Title 10 Code of Federal Regulations Part 835, Occupational Radiation Protection. Department of Energy revised the radiation weighting factors, tissue weighting factors and most of the dosimetric terms used in Title 10 Code of Federal Regulations Part 835 to reflect the recommendations for assessing dose and associated terminology from ICRP Publication 60, 1990 Recommendations of the ICRP on Radiological Protection. In support of the amendment, Department of Energy is revising its guidance documents on establishing bioassay result goals, which are used in assessing bioassay capabilities and establishing bioassay frequencies.

An alteration of human immunodeficiency virus gp41 leads to reduced CCR5 dependence and CD4 independence.

Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface (gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of the viral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, generally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralization sensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell lines expressing low levels of cell surface CCR5.

Folding myoglobin within a sol-gel glass: protein folding constrained to a small volume.

The unfolding and refolding reaction of myoglobin was examined in solution and within a porous silica sol-gel glass. The sol-gel pores constrain the protein to a volume that is the same size and shape as the folded native state accompanied by a few layers of water solvation. Denaturants such as low pH buffers can be diffused through the gel pores to the protein to initiate unfolding and refolding.

Investigation of flexible hose insertion forces and selected factors.

Flexible hoses are widely used in automobiles. Understanding the force required to insert a hose, independent of worker variation, is important for engineers, designers and health professionals. Various parameters affect the insertion forces including the interference between the hose and flange.

Hypophosphorylation of residue Y1045 leads to defective downregulation of EGFRvIII.

Downregulation of the EGF receptor is the net result of receptor degradation and recycling. Cbl functions by specifically targeting activated ErbB receptors for ubiquitination, facilitating ligand-induced desensitization of EGFR. The interaction between EGFR and c-Cbl has been shown to depend upon receptor phosphorylation at tyrosine residue 1045, the major docking site for c-Cbl.

Suppression of MIP-1beta transcription in human T cells is regulated by inducible cAMP early repressor (ICER).

Local production of macrophage inflammatory protein-1beta (MIP-1beta), a beta-chemokine that blocks human immunodeficiency virus type 1 (HIV-1) entry into CD4+ CC chemokine receptor 5+ target cells, may be a significant factor in resistance to HIV-1 infection and control of local viral spread. The mechanisms governing MIP-1beta expression in T cells, however, are not well understood. Our results suggest that MIP-1beta RNA expression in T cells is dynamically regulated by transcriptional factors of the cyclic adenosine monophosphate (cAMP) responsive element (CRE)-binding (CREB)/modulator family.

Characterization of a simian human immunodeficiency virus encoding the envelope gene from the CCR5-tropic HIV-1 Ba-L.

The tat, rev, vpu, and env genes from the monocytotropic CCR5-dependent HIV-1 Ba-L isolate were substituted for homologous simian immunodeficiency virus (SIV) sequences in the SIV genome. The resultant SHIV (SHIV Ba-L) replicated in CCR5-positive PM-1 cells but not in CCR5-negative CEMX174 cells. Infection of HOS cells expressing different co-receptors showed SHIV Ba-L to be strictly CCR5-dependent.

Human herpesvirus 8-encoded vGPCR activates nuclear factor of activated T cells and collaborates with human immunodeficiency virus type 1 Tat.

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-kappa B, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors.

Activation of NF-kappaB by the human herpesvirus 8 chemokine receptor ORF74: evidence for a paracrine model of Kaposi's sarcoma pathogenesis.

Infection with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38.

An HIV-1 transgenic rat that develops HIV-related pathology and immunologic dysfunction.

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of gag and pol, is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional.

Motion times, hand forces, and trunk kinematics when using material handling manipulators in short-distance transfers of moderate mass objects.

The risk of musculoskeletal injury associated with manual materials handling tasks has led in part to the use of material handling manipulators, yet there is limited empirical data to facilitate selection, design, and evaluation of these devices. A laboratory study of two types of mechanical manipulators (articulated arm and overhead hoist) was conducted of short-distance transfers of moderate loads, and the influence of various task parameters and transfer method on motion times, peak hand forces, and torso kinematics was obtained. Use of manipulators increased elemental motion times for symmetric sagittal plane transfers by 36-63%, and asymmetric transfers (in the frontal plane) by 62-115%, compared to similar transfers performed manually.

Muscle responses to simulated torque reactions of hand-held power tools.

The aim of this work was to investigate physiological responses to torque reaction forces produced by hand-held power tools used to tighten threaded fasteners. Such tools are used repetitively by workers in many industries and are often associated with upper limb musculoskeletal complaints. The tools considered for stimulation in this study had straight handles and required from 100 to 400 ms to tighten fasteners to a peak torque of 1.

The effect of keyboard keyswitch make force on applied force and finger flexor muscle activity.

The design of the force-displacement characteristics or 'feel' of keyboard keyswitches has been guided by preference and performance data; there has been very little information on how switch 'feel' alters muscle activity or applied force. This is a laboratory-based repeated measures design experiment to evaluate the effect of computer keyboard keyswitch design on applied finger force and muscle activity during a typing task. Ten experienced typists typed on three keyboards which differed in keyswitch make force (0.