MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects.

Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.

Author Correction: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1+CD38hi cells and anti-PD-1 resistance.

Understanding resistance to antibody to programmed cell death protein 1 (PD-1; anti-PD-1) is crucial for the development of reversal strategies. In anti-PD-1-resistant models, simultaneous anti-PD-1 and vaccine therapy reversed resistance, while PD-1 blockade before antigen priming abolished therapeutic outcomes. This was due to induction of dysfunctional PD-1+CD38hi CD8+ cells by PD-1 blockade in suboptimally primed CD8 cell conditions induced by tumors. This results in erroneous T cell receptor signaling and unresponsiveness to antigenic restimulation. On the other hand, PD-1 blockade of optimally primed CD8 cells prevented the induction of dysfunctional CD8 cells, reversing resistance. Depleting PD-1+CD38hi CD8+ cells enhanced therapeutic outcomes. Furthermore, non-responding patients showed more PD-1+CD38+CD8+ cells in tumor and blood than responders. In conclusion, the status of CD8+ T cell priming is a major contributor to anti-PD-1 therapeutic resistance. PD-1 blockade in unprimed or suboptimally primed CD8 cells induces resistance through the induction of PD-1+CD38hi CD8+ cells that is reversed by optimal priming. PD-1+CD38hi CD8+ cells serve as a predictive and therapeutic biomarker for anti-PD-1 treatment. Sequencing of anti-PD-1 and vaccine is crucial for successful therapy.

Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor.

An absolute or relative deficiency of pancreatic ß-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect ß-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces ß-cell replication and attenuate ß-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic ß-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional ß-cell mass.

PI3K Isoforms in CD8+ T Cell Development and Function.

CD8+ T cells are an essential part of the immune system and play a vital role in defending against tumors and infections. The phosphoinositide-3-kinase (PI3K), especially class I, is involved in numerous interrelated signaling pathways which control CD8+ T cell development, maturation, migration, activation, and differentiation. While CD8+ T lymphocytes express all class I PI3K isoforms (PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ), isoform-specific functions, especially for PI3Kα and PI3Kß have not been fully elucidated. A few studies suggest the important role of p110δ and p110γ in CD8+ T cell activation, signaling, chemotaxis and function and several clinical trials are currently testing the effect of isoform-specific inhibitors in various types of cancers, including Indolent Non-Hodgkin Lymphoma, Peripheral T cell Lymphoma, Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma, non-small cell lung carcinoma (NSCLC), head & neck cancer, and breast cancer. This chapter summarizes current knowledge of the roles of various PI3K isoforms and downstream signaling pathways in regulating CD8+ T cell fate, including cell proliferation, migration, and memory generation. We also discuss certain clinical trials employing PI3K inhibitors for cancer therapy, their limitations, and future perspectives.

The impact of the COVID-19 pandemic on hospital-treated self-harm in Sydney (Australia).

OBJECTIVES: This study investigated trends in hospital-treated self-harm and hospital presenting suicidal ideation in the period before and after COVID-19 public health responses by key socio-demographic groups among those presenting to hospitals in the Western Sydney (Australia) population catchment. METHODS: Emergency department presentations for the period January 2016 to June 2021 were used to specify a series of interrupted time-series models to compare the observed and expected event rates of (1) hospital-treated self-harm and (2) hospital presenting suicidal ideation in the period following the onset of COVID-19 public health measures in March 2020. Rate differences between observed and expected rates in the post-implementation period were also estimated in models stratified by sex, age group, country of birth and socio-economic status. RESULTS: There was no significant increase in hospital-treated self-harm in the period post-implementation of public health orders (March 2020) compared to the previous period, although there were lower than expected rates of emergency department presentations among non-Australian-born males, males aged 0-14 years and 25-44 years, and females aged 45-64 years. In contrast, there was a significant increase in hospital presenting suicidal ideation, particularly among women (rate difference per 100,000 = 3.91, 95% confidence interval = [1.35, 6.48]) and those aged 15-24 years (both males and females, rate differences ranging from 8.91 to 19.04), and among those residing in lower socio-economic status areas (both males and females, rate differences ranging from 0.90 to 2.33). CONCLUSION: There was no increase in hospital-treated self-harm rates in the 15 months post-implementation of COVID-19 public health orders in Western Sydney; however, there was a significant increase in hospital presenting suicidal ideation. The limited change in suicidal behaviour may reflect the success of social and economic supports during this period, the benefits of which may have been different for young people, and those of lower socio-economic status.

Development- and age-related alterations in the expression of AMPA receptor subunit GluR2 and its trafficking proteins in the hippocampus of male mouse brain.

AMPA type glutamate receptor (AMPAR) on the post synaptic membrane plays important role in the process of synaptic plasticity involving various scaffolding and trafficking proteins. However, their alterations during development- and aging are not well understood. Here, we report that the expression of AMPAR-GluR2 subunit is gradually up regulated in the hippocampus from 0 day to adult (20 week) and down regulated thereafter in 70 week old male mice. This pattern of GluR2 during development (0-, 7- and 15 day), maturation (45 day) and adult age resembles with similar expression pattern of the scaffolding protein PSD95. Expression pattern of Stargazin (TARPγ-2) largely follows almost similar pattern up to adult age but is up regulated in old age. Pattern of PICK1 expression, however, is opposite to our GluR2 data till adult age but its expression is significantly down regulated in old age. Our data on alterations in the expression of GluR2 in the hippocampus during development and aging indicates a high- and low positive correlations with PSD95 and Stargazin, respectively whereas negative correlation with PICK1 except in old age where expression of Stargazin is higher and that of PICK1 is lower. Our findings suggest that increasing expression pattern of GluR2 during developmental periods and at adult age may be associated with achieving cognitive abilities whereas its low expression in old age may be linked with cognitive decline and proteins like PSD95, Stargazin and PICK1 might be differentially associated with development- and age-dependent alterations in AMPAR-dependent synaptic plasticity and hence learning and memory.

Alterations in the Sp1 binding and Fmr-1 gene expression in the cortex of the brain during maturation and aging of mouse.

Fragile X mental retardation protein (FMRP) has been implicated in learning, memory and cognition, therefore, information on alterations in FMRP expression during maturation and aging may provide a clue towards understanding mechanisms of age-dependent cognitive changes in the brain. In the present paper, we have studied Fmr-1 gene expression and its correlation with interaction of a tans-acting factor Sp1with Fmr-1 promoter in the cerebral cortex of female mice at post natal period during maturation and aging. Our data reveal that level of Fmr-1 transcript in the cerebral cortex is significantly up regulated at day 7 after birth compared to day 0 (the day of birth) and is gradually down regulated from day 15 onward to old age. The pattern of Fmr-1 transcript levels corresponds with the level of FMRP, however, its level is significantly up regulated in old age compared to adult mice. Our EMSA data revealed the formation of a single complex as a result of binding of Sp1with Fmr-1 promoter sequence. Its intensity gradually decreased from the day 0 (day of birth) till day 15, remained unaltered in young, significantly decreased in adult and significantly increased in old age. Our data suggests that age-dependent alteration in the Fmr-1 gene expression is associated with Sp1 interaction with Fmr-1 promoter which in turn might be related with cognitive development during brain maturation and aging.

Brain Injury and Neurodegeneration: Molecular, Functional, and Translational Approach.

Recently, we have achieved substantial progress in our understanding of brain injury and neurodegeneration [...].

Trends in Primary Mental Health Care Service Use and Subsequent Self-Harm in Western Sydney Australia: Policy and Workforce Implications.

Background: This study investigated the trends in primary mental health care (PMHC) service use and hospital-treated self-harm in Western Sydney (Australia). Methods: A data linkage study and descriptive ecological study of PMHC referrals investigated the trends in referrals, treatment attendance, hospital-treated self-harm, and health care practitioners (HCPs) for the period of 2013−2018 (n = 19,437). Results: There was a substantial increase in referrals from 2016. The majority of referrals were females (60.9%), those aged <45 years (71.3%), and those presenting with anxiety or affective disorders (78.9%). Referrals of those at risk of suicide increased from 9.7% in 2013 to 17.8% in 2018. There were 264 (2.2%) cases of subsequent hospital-treated self-harm, with higher rates among those at risk of suicide and those who attended <6 sessions. The number of HCPs per referral also increased from 2013, as did waiting times for treatment initiation. Conclusion: Individuals presenting to PMHC services at risk of suicide, and who subsequently presented to a hospital setting following self-harm, were more likely to either not attend services following a referral or to attend fewer services. This trend occurred in the context of an increase in the number of clients per HCP, suggesting workforce capacity has not kept pace with demand.

Organosulfur/Selenium-Based Highly Fluorogenic Molecular Probes for Live-Cell Nucleolus Imaging.

We present fluorogenic cationic organo chalcogens that are highly selective to RNA. We have demonstrated that the conformational dynamics and subsequently the optical properties of these dyes can be controlled to facilitate efficient bioimaging. We report the application of organoselenium and organosulfur-based cell-permeable red-emissive probes bearing a favorable cyclic sidearm for selective and high contrast imaging of cell nucleoli. The probes exhibit high quantum yield upon interacting with RNA in an aqueous solution. An in-depth multiscale simulation study reveals that the prominent rotational freezing of the electron-donating sidearm of the probes in the microenvironment of RNA helps in attaining more planar conformation when compared to DNA. It exerts a greater extent of intramolecular charge transfer and hence leads to enhanced fluorescence emission. A systematic structure-interaction relationship study highlighted the impact of heavy-chalcogens toward the improved emissive properties of the probes.

IDO Vaccine Ablates Immune-Suppressive Myeloid Populations and Enhances Antitumor Effects Independent of Tumor Cell IDO Status.

The immunosuppressive tumor microenvironment (TME) does not allow generation and expansion of antitumor effector cells. One of the potent immunosuppressive factors present in the TME is the indoleamine-pyrrole 2,3-dioxygenase (IDO) enzyme, produced mainly by cancer cells and suppressive immune cells of myeloid origin. In fact, IDO+ myeloid-derived suppressor cells (MDSC) and dendritic cells (DC) tend to be more suppressive than their IDO- counterparts. Hence, therapeutic approaches that would target the IDO+ cells in the TME, while sparing the antigen-presenting functions of IDO- myeloid populations, are needed. Using an IDO-specific peptide vaccine (IDO vaccine), we explored the possibility of generating effector cells against IDO and non-IDO tumor-derived antigens. For this, IDO-secreting (B16F10 melanoma) and non-IDO-secreting (TC-1) mouse tumor models were employed. We showed that the IDO vaccine significantly reduced tumor growth and enhanced survival of mice in both the tumor models, which associated with a robust induction of IDO-specific effector cells in the TME. The IDO vaccine significantly enhanced the antitumor efficacy of non-IDO tumor antigen-specific vaccines, leading to an increase in the number of total and antigen-specific activated CD8+ T cells (IFNγ+ and granzyme B+). Treatment with the IDO vaccine significantly reduced the numbers of IDO+ MDSCs and DCs, and immunosuppressive regulatory T cells in both tumor models, resulting in enhanced therapeutic ratios. Together, we showed that vaccination against IDO is a promising therapeutic option for both IDO-producing and non-IDO-producing tumors. The IDO vaccine selectively ablates the IDO+ compartment in the TME, leading to a significant enhancement of the immune responses against other tumor antigen-specific vaccines.

Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells.

Plaques of amyloid fibrils composed of neuronal protein α-synuclein are one of the hallmarks of Parkinson's disease, and their selective imaging is crucial to study the mechanism of its pathogenesis. However, the existing fluorescent probes for amyloids are efficient only in solution and tissue systems, and they are not selective enough for the visualization of amyloid fibrils in living cells. In this study, we present two molecular rotor-based probes RB1 and RB2. These thiazolium probes show affinity to α-synuclein fibrils and turn-on fluorescence response upon interactions. Because of its extended π-conjugation and high rotational degree of freedom, RB1 exhibits a 76 nm red-shift of absorption maxima and 112-fold fluorescence enhancement upon binding to amyloid fibrils. Owing to its strong binding affinity to α-synuclein fibrils, RB1 can selectively stain them in the cytoplasm of living HeLa and SH-SY5Y cells with high optical contrast. RB1 is a cell-permeable and noncytotoxic probe. Taken together, we have demonstrated that RB1 is an amyloid probe with an outstanding absorption red-shift that can be used for intracellular imaging of α-synuclein fibrils.

Pharmacological and Therapeutic Approaches in the Treatment of Epilepsy.

Epilepsy affects around 50 million people across the globe and is the third most common chronic brain disorder. It is a non-communicable disease of the brain that affects people of all ages. It is accompanied by depression, anxiety, and substantially increased morbidity and mortality. A large number of third-generation anti-epileptic drugs are available, but they have multiple side-effects causing a decline in the quality of life. The inheritance and etiology of epilepsy are complex with multiple underlying genetic and epigenetic mechanisms. Different neurotransmitters play intricate functions to maintain the normal physiology of various neurons. If there is any dysregulation of neurotransmission due to aberrant transmitter levels or their receptor biology, it can result in seizures. In this review, we have discussed the roles played by various neurotransmitters and their receptors in the pathophysiology of epilepsy. Drug-resistant epilepsy (DRE) has remained one of the forefront areas of epilepsy research for a long time. Understanding the mechanisms underlying DRE is of utmost importance because of its high incidence rate among epilepsy patients and increased risks of psychosocial problems and premature death. Here we have enumerated various hypotheses of DRE. Further, we have discussed different non-conventional therapeutic strategies, including combination therapy and non-drug treatment. The recent studies supporting the modern approaches for the treatment of epilepsy have been deliberated with particular reference to the mTOR pathway, breakdown of the blood-brain barrier, and inflammatory pathways.

Reversible spatial and temporal control of lipid signaling.

Herein, we introduce versatile molecular tools that enable specific delivery and visualization of photoswitchable lipids at cellular membranes, namely at the plasma membrane and internal membranes. These molecules were prepared by tethering ortho-nitrobenzyl-based fluorescent cages with a signaling lipid bearing an azobenzene photoswitch. They permit two sequential photocontrolled reactions, which are uncaging of a lipid analogue and then its repeated activation and deactivation. We used these molecules to activate GPR40 receptor transiently expressed in HeLa cells and demonstrated downstream modulation of intracellular Ca2+ levels.

Maternal obesity increases offspring's mammary cancer recurrence and impairs tumor immune response.

Over 50% of women at a childbearing age in the United States are overweight or obese, and this can adversely affect their offspring. We studied if maternal obesity-inducing high fat diet (HFD) not only increases offspring's mammary cancer risk but also impairs response to antiestrogen tamoxifen. Female rat offspring of HFD and control diet-fed dams, in which estrogen receptor-positive (ER+) mammary tumors were induced with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), exhibited similar initial responses to antiestrogen tamoxifen. However, after tamoxifen therapy was completed, almost all (91%) tumors recurred in HFD offspring, compared with only 29% in control offspring. The increase in local mammary tumor recurrence in HFD offspring was linked to an increase in the markers of immunosuppression (Il17f, Tgfß1, VEGFR2) in the tumor microenvironment (TME). Protein and mRNA levels of the major histocompatibility complex II (MHC-II), but not MHC-I, were reduced in the recurring DMBA tumors of HFD offspring. Further, infiltration of CD8+ effector T cells and granzyme B+ (GZMB+) cells were lower in their recurring tumors. To determine if maternal HFD can pre-program similar changes in the TME of allografted E0771 mammary tumors in offspring of syngeneic mice, flow cytometry analysis was performed. E0771 mammary tumor growth was significantly accelerated in the HFD offspring, and a reduction in the numbers of GZMB and non-significant reduction of interferon γ (IFNγ) secreting CD8+ T cells in the TME was seen. Thus, consumption of a HFD during pregnancy increases susceptibility of the female rat and mouse offspring to tumor immune suppression and mammary tumor growth and recurrence.

Targeting the endocannabinoid system: a predictive, preventive, and personalized medicine-directed approach to the management of brain pathologies.

Cannabis-inspired medical products are garnering increasing attention from the scientific community, general public, and health policy makers. A plethora of scientific literature demonstrates intricate engagement of the endocannabinoid system with human immunology, psychology, developmental processes, neuronal plasticity, signal transduction, and metabolic regulation. Despite the therapeutic potential, the adverse psychoactive effects and historical stigma, cannabinoids have limited widespread clinical application. Therefore, it is plausible to weigh carefully the beneficial effects of cannabinoids against the potential adverse impacts for every individual. This is where the concept of "personalized medicine" as a promising approach for disease prediction and prevention may take into the account. The goal of this review is to provide an outline of the endocannabinoid system, including endocannabinoid metabolizing pathways, and will progress to a more in-depth discussion of the therapeutic interventions by endocannabinoids in various neurological disorders.

Nitrobenzyl-based fluorescent photocages for spatial and temporal control of signalling lipids in cells.

Here we present a set of fluorescent cages prepared by tethering fluorescent dyes to a photolabile group. The developed molecules enable caging of signalling lipids, their delivery to specific cellular membranes, with further imaging, quantification, and controlled photorelease of active lipids in living cells.

Failure of Angiographic Management in Cases of Postrenal Intervention Bleed: Risk Factors and Management Approach.

OBJECTIVE: To evaluate the factors predicting the risk for failed angiographic management (AM), we retrospectively studied cases of digital subtraction angiography (DSA) and superselective angiography (SSA) to control severe/delayed bleeding following renal interventions, which may otherwise be life threatening and often require nephrectomy. METHODS: We have retrospectively evaluated the data of 154 patients who underwent DSA and or SSA during January 2006 to June 2016. Twenty-one patients (Group A) with failed AM were compared to patients with success AM (n = 133, Group B). RESULTS: Out of 21 patients in whom AM failed, 20 should be managed with subsequent sessions of DSA/SSA and only 1 had to undergo nephrectomy. On univariate analysis, low hemoglobin (P = .025), multiple tracts (n > 1) during percutaneous nephrolithotomy (P = .01), multiple bleeding site (>1 = 0.01 and >2 = 0.001) and patients, who needed inotropes (P = .008) were found to predict risk for failure. On multivariate analysis, multiple bleeding site >2 (P = .003, odds ratio 5.23, 95% confidence interval = 1.3-22.5) and patients on inotropes (P = .02, odds ratio 2.56, 95% confidence interval = 2.15-4.75) were found to independently predict the failure. CONCLUSION: Patients with multiple bleeding lesions and who are on inotropic (leading to intrarenal vasoconstriction) are at high risk for failure of AM. Most of them can be successfully managed by subsequent session AM.

Detecting the hidden burden of pre-diabetes and diabetes in Western Sydney.

AIMS: Examining pre-diabetes and diabetes rates using glycated haemoglobin (HbA1c) in emergency department (ED) and in general practice (GP) in western Sydney. METHODS: Epidemiological study of HbA1c measurements in individuals ≥18 years receiving a blood test (1) in the hospital setting of the ED at Blacktown/Mt Druitt hospital (1/06/2016 to 31/05/2018) and (2) in primary care involving Bridgeview Medical Practice (BVMP) (1/03/2017 to 01/02/2018) as well as other general practices (June 2018 only). RESULTS: Totals of 55,568 individuals from ED and 5911 individuals from GP. The prevalence of diabetes in tested individuals was 17.3% (n = 9704) in ED and 17.4% (n = 1027) in GP. The prevalence of pre-diabetes in ED was 30.2% (n = 16,854) and 26.6% (n = 1576) in GP. Regression controlling for age, season, and gender revealed a weekly increase of 1.1% in odds for diabetes and 1.5% for pre-diabetes (p < 0.001), in line with the yearly absolute increase of 1% in rate for both tested and coded hospital patients. In BVMP the rate of diabetes rose by 22% during the testing period from 8.9% to 11%. CONCLUSIONS: There exists a high burden of diabetes both in hospitals and general practice. Testing in ED and general practice revealed similarly high burdens of diabetes across different areas of the healthcare system. In the appropriate hospital and primary care setting, HbA1c can be used to identify individuals with diabetes that may benefit from targeted intervention.