A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

Effectiveness of a Cognitive Behavioral Randomized Controlled Trial for People Living with HIV Who are Heavy Drinkers: The Holistic Health Recovery Program (HHRP) Trial in Miami.

This study evaluates the impact of an intervention targeting high-risk behaviors among diverse, alcohol-using adults living with HIV (N = 267) from 2009 to 2013 in Miami, FL. The intervention took place in a group setting for eight sessions over 4 weeks and was compared to a didactic health promotion group. Assessments were conducted pre-intervention, post-intervention, and at 3, 6, and 12 months follow-up. Intervention participants (48% of sample) evidenced greater knowledge about HIV, more condom self-efficacy, and greater intentions to use condoms after participation. This was particularly noteworthy because associations among knowledge about HIV, more condom self-efficacy, and greater intentions to use condoms were negatively associated with intervention status at baseline. Participants also reported fewer heavy drinking days after participating in the intervention than those in the control group. Greater HIV knowledge, more condom self-efficacy and intentions to use condoms predicted more condom assertiveness; greater intentions to use condoms predicted fewer unprotected sexual behaviors. These findings underscore the importance of taking a comprehensive, multi-systemic approach to address risky behaviors in high-risk, diverse populations.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Evidence of movement variability patterns during a repetitive pointing task until exhaustion.

Human movement is characterized by its variability: the same task is never performed twice in exactly the same way. This variability is believed to play a functional role in movement performance and adaptability, as well as in preventing musculoskeletal damage. This article focuses on the time-evolution of movement variability throughout a repetitive pointing task until exhaustion. The kinematics of 13 subjects performing the pointing task is analyzed. Principal Component Analysis of joint angles identifies joint coordinations for each pointing cycle, and cycle-by-cycle comparison highlights movement variability. Non-supervised clustering reveals that subjects adopt successive coordination patterns at an intra-individual level. Inter-individual variability is characterized by the number and type of such patterns: from 3 to 5 patterns, mobilizing the trunk, the shoulder and the upper limbs differently. Movement variability exists even in a seemingly basic and constrained task. It appears in the very early stages of fatigue onset, and may correspond to adaptative coordination responses throughout task performance. This observation should encourage workstation designers to better account for movement variability in order to preserve operators' health and safety.

Temporal leeway: can it help to reduce biomechanical load for older workers performing repetitive light assembly tasks?

Current industrial production systems allow assembly of customised products which include additional elements distinguishing them from a reference model. This customisation can result in significant additional time constraints which compel workers to complete their tasks faster, which may pose problems for older workers. The objective of this laboratory study was to investigate the impact of restrictive or flexible pacing during assembly of customised products among groups of younger and older participants. The data gathered were used to analyse cycle-time, assembly performance, muscular load, and kinematic adaptations. The flexible pacing condition was found to improve production performance, increasing customised assembly cycle-time and reducing biomechanical load, for both young and older participants. However, as the task required fine manual dexterity, older participants were subjected to a higher biomechanical load, even in the flexible pacing scenario. These results should encourage assembly-line designers to allow flexible time constraints as much as possible and to be particularly attentive to the needs of older workers.

[Myocarditis, a treacherous condition]. / Myocarditis, een verraderlijk ziektebeeld.

Myocarditis, a treacherous condition It is important to recognize myocarditis at an early stage. To illustrate this, we present two male patients aged 39 and 51, respectively, who were admitted with febrile disease and signs of circulatory instability. Initially, myocarditis was not suspected in these patients. After the sudden death of the younger patient it was discovered that he had had fulminant myocarditis. Its nonspecific, heterogeneous clinical presentation, and potentially disastrous outcome make myocarditis a treacherous condition, which mainly affects younger adults. Although its aetiology is broad, the primary cause in the western world is a viral infection leading to lymphocytic myocarditis. Fulminant forms are rare, but this diagnosis needs to be considered in patients with an atypical illness and impaired haemodynamics or electrocardiogram (ECG) abnormalities. Early and liberal consultation of a cardiologist is important. Primary diagnostics include blood testing (e.g. troponin, creatinine kinase), ECG, echocardiography and exclusion of coronary ischaemia. The diagnosis can be confirmed by cardiovascular MRI or endomyocardial biopsy.

Bioluminescence of Vibrio fischeri: bacteria respond quickly and sensitively to pulsed microwave electric (but not magnetic) fields.

Biological systems with intrinsic luminescent properties serve as powerful and noninvasive bioreporters for real-time and label-free monitoring of cell physiology. This study employs the bioluminescent marine bacterium Vibrio fischeri to investigate the effects of separated microwave electric (E) and magnetic (H) fields. Using a cylindrical TM010 mode aluminum resonant cavity, designed to spatially separate E and H fields of a pulsed microwave (2.45 GHz) input, we sampled at 100-ms intervals the 490-nm emission of bioluminescence from suspensions of the V. fischeri. E-field exposure (at 4.24 and 13.4 kV/m) results in rapid and sensitive responses to 100-ms pulses. H-field excitation elicits no measurable responses, even at 100-fold higher power input levels (equivalent to 183 A/m). The observed effects on bacterial light output partially correlate with measured E-field-induced temperature increases. In conclusion, the endogenous bioluminescence of V. fischeri provides a sensitive and noninvasive method to assess the biological effects of microwave fields.

Prenatal maternal stress is associated with lower cortisol and cortisone levels in the first morning urine of 45-month-old children.

Prenatal stress (PS) has been related to altered hypothalamic-pituitary-adrenal (HPA) axis activity later in life. So far, studies in children assessing HPA axis functioning have focused on salivary cortisol, reflecting daytime activity. The present work is part of a prospective study and aims to extend knowledge about the association between PS and HPA axis regulation in children. To do so, we investigated cortisol, cortisone, and the ratio cortisone/(cortisone + cortisol) in the first morning urine of 45-month-old children in relation to several measures of maternal stress during pregnancy. Urinary cortisol and cortisone were measured by online turbulent flow chromatography coupled with high performance liquid chromatography-tandem mass spectrometry. PS was defined as: perceived stress for aim 1 (Perceived Stress Scale; n = 280); presence of self-reported (n = 371) and expert-rated psychopathology for aim 2 (Mini International Neuropsychiatric Interview; n = 281); continuous measures of anxiety and depression for exploratory aim 3 (State-Trait Anxiety Inventory and Edinburgh Postnatal Depression Scale; n = 280). Aim 1: Perceived maternal PS showed negative associations with cortisol and cortisone levels. Aim 2: The presence of expert-rated maternal psychopathology was associated with reduced morning cortisone. Aim 3: Continuous measures of anxiety and depression showed negative associations with cortisol and cortisone levels. After correcting for multiple testing, perceived maternal PS (aim 1) and prenatal level of anxiety (aim 3) were significant predictors of children's urinary cortisol and cortisone in the morning (and, in the case of cortisone, also prenatal level of depression). The ratio cortisone/(cortisone + cortisol) as a global marker for the balance between the enzymes metabolizing cortisol to cortisone and vice versa (11ß-hydroxysteroid dehydrogenases type 1 and 2; 11ß-HSD1 and 2) was not associated with any measure of maternal PS (aims 1-3). The present study provides insight into possible programming effects of PS on nocturnal HPA axis activity and a proxy of 11ß-HSD in a large sample. The results suggest that the nocturnal rate of cortisol production is lower in children exposed to PS, but do not support the hypothesis of divergent 11ß-HSD activity.

Poor maternal anthropometric characteristics and newborns' birth weight and length: a cross-sectional study in Benin.

Background: Maternal undernutrition is known to negatively impact newborns' birth weight and length, but this finding is poorly documented in the Beninese population. This study aimed to assess the effect of maternal anthropometry on mean birth weight and length in a Beninese cohort of newborns. Methods: A cross-sectional study was carried out in Tori Bossito, Republic of Benin. Pregnant women attending maternity wards between June 2007 and July 2008 were recruited. At delivery the women's characteristics, including weight and height, were gathered and newborns' birth weights and lengths were measured. Statistical analysis was performed using multiple linear regression. Results: A total of 526 mother-infant pairs were enrolled; 29.8% of women had low weight status and 26.2% had short stature (<155 cm). The mean birth weight was 2985 g (standard deviation [SD] 384) the mean birth length was 48.7 cm (SD 2.2). Maternal low weight status (coefficient=-151.81, p<0.001) and short stature (coefficient=-135.49, p<0.001) reduced the mean birth weight. Similar results were found for mean birth length, which was decreased by maternal low weight status (coefficient=-0.42, p=0.04) and short stature (coefficient=-0.51, p=0.01). Conclusion: Maternal undernutrition expressed by low anthropometry remains problematic in the Beninese population and induces transmission of malnutrition. Nutritional interventions are required to break this vicious cycle.

Stress reactivity in preschool-aged children: Evaluation of a social stress paradigm and investigation of the impact of prenatal maternal stress.

Prenatal maternal stress is an established risk factor for somatic and psychological health of the offspring. A dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis in offspring has been suggested as an important mechanism. However, the impact of prenatal stress on stress reactivity in preschool-aged children is not yet well understood. This is partly due to the fact that for this age group there is no stress test as well established as for older children and adults. In the present work a previously published stress test (Kryski et al., 2011) was evaluated in a large sample of 45-month-old children (n = 339). Furthermore, the relation between measures of prenatal maternal stress and cortisol reactivity was investigated. Prenatal stress was defined as psychopathology (self-report available for n = 339; expert-rating available for a subsample of n = 246) and perceived stress (n = 244) during pregnancy. The stress paradigm elicited significant increases in salivary cortisol 30 and 40 min after the test, and 60.8% of the children were classified as responders. Lower cortisol levels after the stress test were observed in the group of children with prenatal stress defined as maternal psychopathology (both self-reported and expert-rated). Maternal perceived stress as a continuous measure was not significantly associated with cortisol levels. However, when comparing children in the highest quartile of maternal perceived stress to all other children, significantly lower cortisol values were observed in the prenatally stressed group. The present study confirms the paradigm by Kryski et al. as an effective stress test for preschool-aged children. Moreover, it provides further evidence that prenatal stress impacts HPA axis reactivity. Future studies should target the timing, nature, and intensity of prenatal stressors and their effect on the stress response in offspring at different developmental stages.

3D X-Ray Imaging of Continuous Objects beyond the Depth of Focus Limit.

X-ray ptychography is becoming the standard method for sub-30 nm imaging of thick extended samples. Available algorithms and computing power have traditionally restricted sample reconstruction to 2D slices. We build on recent progress in optimization algorithms and high performance computing to solve the ptychographic phase retrieval problem directly in 3D. Our approach addresses samples that do not fit entirely within the depth of focus of the imaging system. Such samples pose additional challenges because of internal diffraction effects within the sample. We demonstrate our approach on a computational sample modeled with 17 million complex variables.

A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG-asparaginase, and doxorubicin.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains a major cause of death in children. AMP-activated protein kinase (AMPK) affects the unfolded protein response (UPR), leading to increased vulnerability to endoplasmic reticulum (ER) stress in ALL cells. In vitro, metformin causes ALL cell death via AMPK-mediated inhibition of the UPR. It was evaluated whether ER stress could be induced in relapsed ALL through a phase I study investigating the safety and feasibility of metformin in combination with relapse induction chemotherapy. PROCEDURE: Metformin was administered twice daily for 28 days in addition to vincristine, dexamethasone, PEG-asparaginase and doxorubicin (VXLD). Dose escalation of metformin was evaluated using a 3+3 design. Pharmacokinetics (PK), pharmacodynamic (PD) evaluation of the AMPK and ER stress/UPR pathways, and treatment response were assessed. RESULTS: Fourteen patients were enrolled; all were evaluable for toxicity. The recommended phase 2 dose (RP2D) was Dose level 2, 1,000 mg/m2 /day. A single dose-limiting toxicity (DLT), hypoglycemia with acidosis, was observed at the RP2D and two DLTs, diarrhea and acidosis, were observed at Dose Level 3. Nine patients were evaluable for response as defined by the protocol, receiving at least 85% of planned metformin doses. Five complete remissions, one partial response, and one stable disease were observed. PD evaluation showed induction of ER stress, activation of AMPK, and inhibition of the UPR. CONCLUSIONS: The VXLD with metformin was tolerable with a RP2D for metformin of 1,000 mg/m2 /day and yielded responses in a heavily pretreated population. ER stress was induced and toxicities attributable to metformin occurred in all dose levels.

Inhibition of the NEDD8 conjugation pathway induces calcium-dependent compensatory activation of the pro-survival MEK/ERK pathway in acute lymphoblastic leukemia.

De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C ß2 (PKC-ß2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.

Polarization domain walls in optical fibres as topological bits for data transmission.

Domain walls are topological defects which occur at symmetry-breaking phase transitions. While domain walls have been intensively studied in ferromagnetic materials, where they nucleate at the boundary of neighbouring regions of oppositely aligned magnetic dipoles, their equivalent in optics have not been fully explored so far. Here, we experimentally demonstrate the existence of a universal class of polarization domain walls in the form of localized polarization knots in conventional optical fibres. We exploit their binding properties for optical data transmission beyond the Kerr limits of normally dispersive fibres. In particular, we demonstrate how trapping energy in well-defined train of polarization domain walls allows undistorted propagation of polarization knots at a rate of 28 GHz along a 10 km length of normally dispersive optical fibre. These results constitute the first experimental observation of kink-antikink solitary wave propagation in nonlinear fibre optics.

All-optical sampling and magnification based on XPM-induced focusing.

We theoretically and experimentally investigate the design of an all-optical magnification and sampling function free from any active gain medium or additional amplified spontaneous noise emission. The proposed technique is based on the co-propagation of an arbitrary shaped signal together with an orthogonally polarized intense fast sinusoidal beating within a normally dispersive optical fiber. This process allows us to experimentally demonstrate a 40-GHz sampling operation as well as an 8-dB magnification of an arbitrary shaped nanosecond signal around 1550 nm in a 5-km long optical fiber. The experimental observations are in good agreement with numerical and theoretical analysis.

Ankyrin-3 as a molecular marker of early-life stress and vulnerability to psychiatric disorders.

Exposure to early-life stress (ELS) may heighten the risk for psychopathology at adulthood. Here, in order to identify common genes that may keep the memory of ELS through changes in their methylation status, we intersected methylome analyses performed in different tissues and time points in rats, non-human primates and humans, all characterized by ELS. We identified Ankyrin-3 (Ank3), a scaffolding protein with a strong genetic association for psychiatric disorders, as a gene persistently affected by stress exposure. In rats, Ank3 methylation and mRNA changes displayed a specific temporal profile during the postnatal development. Moreover, exposure to prenatal stress altered the interaction of ankyrin-G, the protein encoded by Ank3 enriched in the post-synaptic compartment, with PSD95. Notably, to model in humans a gene by early stress interplay on brain phenotypes during cognitive performance, we demonstrated an interaction between functional variation in Ank3 gene and obstetric complications on working memory in healthy adult subjects. Our data suggest that alterations of Ank3 expression and function may contribute to the effects of ELS on the development of psychiatric disorders.

The NEDD8-activating enzyme inhibitor pevonedistat activates the eIF2α and mTOR pathways inducing UPR-mediated cell death in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for adults remain dismal. Further, effective treatment strategies for relapsed/refractory ALL remain elusive. We previously uncovered that ALL cells are prone to apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms. We investigated the antineoplastic activity of pevonedistat®, a novel NEDD8-activating enzyme inhibitor that targets E3 cullin-RING ligases (CRLs) dependent proteasomal protein degradation, in ALL. Herein, we report that pevonedistat induces apoptosis in ALL cells by dysregulating the translational machinery leading to induction of proteotoxic/ER stress and UPR-mediated cell death. Mechanistically, pevonedistat led to P-eIF2a dephosphorylation causing atypical proteotoxic/ER stress from failure to halt protein translation via the UPR and upregulation of mTOR/p70S6K. Additional studies revealed that pevonedistat re-balanced the homeostasis of pro- and anti-apoptotic proteins to favor cell death through altered expression and/or activity of Mcl-1, NOXA, and BIM, suggesting that pevonedistat has a "priming" effect on ALL by altering the apoptotic threshold through modulation of Mcl-1 activity. Further, we demonstrated that pevonedistat synergizes with selected anti-leukemic agents in vitro, and prolongs survival of NSG mice engrafted with ALL cells, lending support for the use of pevonedistat as part of a multi-agent approach.

Patient positioning in radiotherapy based on surface imaging using time of flight cameras.

PURPOSE: To evaluate the patient positioning accuracy in radiotherapy using a stereo-time of flight (ToF)-camera system. METHODS: A system using two ToF cameras was used to scan the surface of the patients in order to position them daily on the treatment couch. The obtained point clouds were registered to (a) detect translations applied to the table (intrafraction motion) and (b) predict the displacement to be applied in order to place the patient in its reference position (interfraction motion). The measures provided by this system were compared to the effectively applied translations. The authors analyzed 150 fractions including lung, pelvis/prostate, and head and neck cancer patients. RESULTS: The authors obtained small absolute errors for displacement detection: 0.8 ± 0.7, 0.8 ± 0.7, and 0.7 ± 0.6 mm along the vertical, longitudinal, and lateral axes, respectively, and 0.8 ± 0.7 mm for the total norm displacement. Lung cancer patients presented the largest errors with a respective mean of 1.1 ± 0.9, 0.9 ± 0.9, and 0.8 ± 0.7 mm. CONCLUSIONS: The proposed stereo-ToF system allows for sufficient accuracy and faster patient repositioning in radiotherapy. Its capability to track the complete patient surface in real time could allow, in the future, not only for an accurate positioning but also a real time tracking of any patient intrafraction motion (translation, involuntary, and breathing).