Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System.

Chimeric antigen receptor (CAR) T cell therapy has proven clinically beneficial against B cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma. However, suboptimal clinical outcomes have been associated with decreased expansion and persistence of adoptively transferred CAR T cells, antigen-negative relapses, and impairment by an immunosuppressive tumor microenvironment. Improvements in CAR T cell design are required to enhance clinical efficacy, as well as broaden the applicability of this technology. Here, we demonstrate that interleukin-18 (IL-18)-secreting CAR T cells exhibit enhanced in vivo expansion and persistence and significantly increase long-term survival in syngeneic mouse models of both hematological and solid malignancies. In addition, we demonstrate that IL-18-secreting CAR T cells are capable of modulating the tumor microenvironment, as well as enhancing an effective endogenous anti-tumor immune response. IL-18-secreting CAR T cells represent a promising strategy to enhance the clinical outcomes of adoptive T cell therapy

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 \ub1 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys

Actin inhibition increases megakaryocyte proplatelet formation through an apoptosis-dependent mechanism.

BACKGROUND:Megakaryocytes assemble and release platelets through the extension of proplatelet processes, which are cytoplasmic extensions that extrude from the megakaryocyte and form platelets at their tips. Proplatelet formation and platelet release are complex processes that require a combination of structural rearrangements. While the signals that trigger the initiation of proplatelet formation process are not completely understood, it has been shown that inhibition of cytoskeletal signaling in mature megakaryocytes induces proplatelet formation. Megakaryocyte apoptosis may also be involved in initiation of proplatelet extension, although this is controversial. This study inquires whether the proplatelet production induced by cytoskeletal signaling inhibition is dependent on activation of apoptosis. METHODS:Megakaryocytes derived from human umbilical cord blood CD34+ cells were treated with the actin polymerization inhibitor latrunculin and their ploidy and proplatelet formation were quantitated. Apoptosis activation was analyzed by flow cytometry and luminescence assays. Caspase activity was inhibited by two compounds, ZVAD and QVD. Expression levels of pro-survival and pro-apoptosis genes were measured by quantitative RT-PCR. Protein levels of Bcl-XL, Bax and Bak were measured by western blot. Cell ultrastructure was analyzed by electron microscopy. RESULTS:Actin inhibition resulted in increased ploidy and increased proplatelet formation in cultured umbilical cord blood-derived megakaryocytes. Actin inhibition activated apoptosis in the cultured cells. The effects of actin inhibition on proplatelet formation were blocked by caspase inhibition. Increased expression of both pro-apoptotic and pro-survival genes was observed. Pro-survival protein (Bcl-xL) levels were increased compared to levels of pro-apoptotic proteins Bak and Bax. Despite apoptosis being activated, the megakaryocytes underwent minimal ultrastructural changes during actin inhibition. CONCLUSIONS:We report a correlation between increased proplatelet formation and activation of apoptosis, and that the increase in proplatelet formation in response to actin inhibition is caspase dependent. These findings support a role for apoptosis in proplatelet formation in this model

Actin polymerization inhibitor (Latrunculin) increases megakaryocyte maturation and proplatelet formation.

<p>A,B) FACS profiles of propidium iodide staining indicating ploidy of control (A) and latrunculin treated (B) cells. C) Actin inhibition increases the levels of polyploid megakaryocytes in culture. Y axis represents the % of cells with ploidy ≥8N. D,E) Wright-Giemsa stained megakaryocytes showing increased polyploidization with actin-inhibitor treatement: D) Untreated control E) Actin polymerization inhibitor treated. F) Actin inhibition increases the levels of megakaryocytes proplatelet formation in culture. Y-axis represents the sum of all proplatelet (PP) segments counted inside of grids in 10 random field images (40x magnification). G,H) Light microscopy images showing increased proplatelet formation after actin polymerization inhibitor treatment: G) Untreated control H) Actin polymerization inhibitor treated. * p<0.05</p

Actin inhibitor (Latrunculin) induces apoptosis activation in cultured megakaryocytes.

<p>A) FACS analysis of apoptosis activation by measuring Mitochondrial Outer Membrane Potential (MOMP) and phosphatidyl serine (PS) externalization B) Luminescence activity measuring Caspase 3 and 7 activity were measured using Caspase-Glo 3/7 Assay. Actin inhibition induces overexpression of both pro-apoptotic and pro-survival genes and increases BCL-XL protein synthesis C) RT-PCR analysis of mRNA expression of pro-apoptotic genes: BAX, BAK, BNIP3 and BNIP3L and pro-survival genes: BCL-2, BCL-XL, IGF1R and CFLAR relative to untreated control. BCL-XL protein showed increased synthesis with actin polymerization inhibitor treatment D) Western Blot (density ratio = 1.34 compared to untreated control) and E) Intracellular protein quantification with FACS. * p<0.05; ** p<0.005</p

Megakaryocyte ultrastructure showing early signs of apoptosis in actin polymerization inhibitor treated megakaryocytes, such as enlarged peripheral margin with concentration of granules towards the center of the cell and more frequently displayed cytoplasmic blebbing.

<p>A) Untreated control B) Actin polymerization inhibitor treated.</p

Actin polymerization inhibitor (Latrunculin) treatment does not affect proplatelet formation and platelet structure.

<p>A) Preplatelet-like cell protrusions stained with β- tubulin and showing characteristic circumferential loops of microtubules. B) Caspase inhibition with ZVAD-FMK or Q-VD-OPH inhibits proplatelet formation from actin polymerization inhibitor treated megakaryocytes. Y-axis represents the sum of all proplatelet (PP) segments counted inside of grids in 10 random field images (40x magnification). C,D) Light microscopy images showing decreased proplatelet formation after caspase inhibitor treatment: C) Actin polymerization inhibitor treated D) ZVAD-FMK E) Q-VD-OPH. * p<0.05</p