Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications.

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA® and MSD CETSA® - that require less than 100 µL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

A vaccine inducing solely cytotoxic T lymphocytes fully prevents Zika virus infection and fetal damage.

As vaccine-induced non-neutralizing antibodies may cause antibody-dependent enhancement of Zika virus (ZIKV) infection, we test a vaccine that induces only specific cytotoxic T lymphocytes (CTLs) without specific antibodies. We construct a DNA vaccine expressing a ubiquitinated and rearranged ZIKV non-structural protein 3 (NS3). The protein is immediately degraded and processed in the proteasome for presentation via major histocompatibility complex (MHC) class I for CTL generation. We immunize Ifnar1-/- adult mice with the ubiquitin/NS3 vaccine, impregnate them, and challenge them with ZIKV. Our data show that the vaccine greatly reduces viral titers in reproductive organs and other tissues of adult mice. All mice immunized with the vaccine survived after ZIKV challenge. The vaccine remarkably reduces placenta damage and levels of pro-inflammatory cytokines, and it fully protects fetuses from damage. CD8+ CTLs are essential in protection, as demonstrated via depletion experiments. Our study provides a strategy to develop safe and effective vaccines against viral infections.

Inflammation and oxidative stress induced by lipid peroxidation metabolite 4-hydroxynonenal in human corneal epithelial cells.

PURPOSE: Oxidative stress is widely known to be a major contributor in the pathogenesis of dry eye disease (DED). 4-Hydroxynonenal (4-HNE), a well-known byproduct frequently measured as an indicator of oxidative stress-induced lipid peroxidation, has been shown to be elevated in both human and murine corneal DED samples. This study aims to investigate if 4-HNE is responsible for the oxidative stress in human corneal epithelial cells (HCECs) and explores the underlying mechanism by which it confers its effects. METHODS: SV40-immortalized HCECs were cultured in minimum essential media (MEM) with 1% penicillin/streptomycin and 10% fetal bovine serum. HCECs were exposed to media with or without 4-HNE and cell culture supernatants were collected at 4 and 24 h. Cellular reactive oxygen species (ROS) measurement was performed using a 2',7'-dichlorofluorescein diacetate (DCFDA) assay kit according to the manufacturer's instructions. Protein levels of antioxidant enzymes copper/zinc superoxide dismutase 1 (SOD1) and NAD(P)H quinone dehydrogenase 1 (NQO1) were analyzed by Western blot. NF-κB activation and expression of IL-6 and IL-8 were measured using an NF-κB p65 Total SimpleStep ELISA Kit and Proteome Profiler Human Cytokine Array Kit. Cell viability was evaluated by LDH cytotoxicity assay. RESULTS: Treatment with 4-HNE decreased cell viability of HCECs. Band intensities corresponding to levels of ROS production showed a significant increase in ROS generation after treatment with 4-HNE. 4-HNE decreased SOD1 levels and upregulated NQO1 expression in HCECs. A significant increase in activation of NF-κB and production of pro-inflammatory cytokines IL-6 and IL-8 was observed after treatment with 4-HNE. Exposure to N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the oxidative effects of 4-HNE on HCECs. CONCLUSION: 4-HNE induces oxidative stress in corneal epithelial cells by increasing levels of ROS generation and modifying the expression of antioxidant enzyme levels, decreasing cell viability of HCECs in vitro. This study demonstrates a potential pathway by which 4-HNE functions to confer its detrimental effects and provides a new therapeutic target for the treatment of DED.

Zidovudine protects hyperosmolarity-stressed human corneal epithelial cells via antioxidant pathway.

Dry Eye Disease (DED) is a very common disorder that can result in severe disability and vision loss. Although the pathogenesis of DED is not fully understood, hyperosmolarity, inflammation, and tear film instability are recognized as hallmarks of DED. Recently, Nucleoside Reverse Transcriptase Inhibitors (NRTIs), a class of medication used to treat HIV, have been shown to inhibit inflammation in a mouse model of retinal atrophy. In this study, we investigated whether Zidovudine (AZT) can inhibit human corneal epithelial cell (HCEC) inflammatory responses under hyperosmotic conditions. HCECs were cultured in hyperosmotic media containing AZT. Cell viability, cytokine production, and reactive oxygen species (ROS) production were measured. We found that AZT decreased nuclear factor kappa B (NF-κB) and Interleukin-6 (IL-6) levels, increased Superoxide Dismutase 1 (SOD1) production, decreased ROS production, and increased cell viability. These results support the novel use of AZT in the reduction of ocular surface inflammation and the promotion of corneal health in the context of DED.

'It's going to shorten your life': framing of oncologist-patient communication about prognosis.

In this qualitative study, we used grounded theory techniques to analyze transcripts of 29 first-time encounters between oncologists and patients referred to them with previously diagnosed, incurable cancer. We found that 23 (79%) of the transcripts included 166 examples of prognostic talk. The language used ranged from general to personal, with 25% of statements mentioning no patients (e.g., 'the tumor will usually come back'), 13% mentioning patients other than the current patient (e.g., 'some patients with tumor response will be around for 2-5 years'), 11% mentioning the current patient in the context of others (e.g., 'on average, people like you live a couple of years'), and 51% directly focusing on the patient (e.g., 'this thing's going to kill you eventually'). More statements pertained to treatment-related prognosis than to disease-related prognosis (67 versus 33%). While 31% of statements focused on negative outcomes (loss framing), the remainder either focused on positive outcomes (gain framing) or discussed a combination of positive and negative outcomes (mixed framing). When discussions focused on negative outcomes, general and indirect language was commonly used, perhaps to buffer the patient from news of the poor prognosis.

Pushing up daisies: implicit and explicit language in oncologist-patient communication about death.

GOALS OF WORK: Although there are guidelines regarding how conversations with patients about prognosis in life-limiting illness should occur, there are little data about what doctors actually say. This study was designed to qualitatively analyze the language that oncologists and cancer patients use when talking about death. SUBJECTS AND METHODS: We recruited 29 adults who had incurable forms of cancer, were scheduled for a first-time visit with one of six oncologists affiliated with a teaching hospital in Australia, and consented to having their visit audiotaped and transcribed. Using content analytic techniques, we coded various features of language usage. MAIN RESULTS: Of the 29 visits, 23 (79.3%) included prognostic utterances about treatment-related and disease-related outcomes. In 12 (52.2%) of these 23 visits, explicit language about death ("terminal," variations of "death") was used. It was most commonly used by the oncologist after the physical examination, but it was sometimes used by patients or their kin, usually before the examination and involving emotional questioning about the patient's future. In all 23 (100%) visits, implicit language (euphemistic or indirect talk) was used in discussing death and focused on an anticipated life span (mentioned in 87.0% of visits), estimated time frame (69.6%), or projected survival (47.8%). CONCLUSIONS: Instead of using the word "death," most participants used some alternative phrase, including implicit language. Although oncologists are more likely than patients and their kin to use explicit language in discussing death, the oncologists tend to couple it with implicit language, possibly to mitigate the message effects.