Prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation comparing tuberculin skin test and interferon-gamma release assay.

The aim of this study was to evaluate the prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation candidates, using tuberculin skin test and QuantiFERON-TB Gold-Plus, in a high-burden tuberculosis country. Adult candidates for hematopoietic stem cell transplantation performed both tests before and those submitted to transplantation were followed up for 12 months. The prevalence of latent Mycobacterium tuberculosis infection was 17.1% and a moderate agreement between QuantiFERON-TB Gold-Plus and tuberculin skin test was observed in this population. Previous tuberculosis exposure was a risk factor for latent Mycobacterium tuberculosis infection. No cases of tuberculosis were diagnosed during follow-up period.

STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication.

Stimulator of interferon genes (STING) signaling is a promising target in cancer immunotherapy, with many ongoing clinical studies in combination with immune checkpoint blockade (ICB). Existing STING-based therapies largely focus on activating CD8+ T cell or NK cell-mediated cytotoxicity, while the role of CD4+ T cells in STING signaling has yet to be extensively studied in vivo. Here, a distinct CD4-mediated, protein-based combination therapy of STING and ICB as an in situ vaccine, is reported. The treatment eliminates subcutaneous MC38 and YUMM1.7 tumors in 70-100% of mice and protected all cured mice against rechallenge. Mechanistic studies reveal a robust TH 1 polarization and suppression of Treg of CD4+ T cells, followed by an effective collaboration of CD4+ T, CD8+ T, and NK cells to eliminate tumors. Finally, the potential to overcome host STING deficiency by significantly decreasing MC38 tumor burden in STING KO mice is demonstrated, addressing the translational challenge for the 19% of human population with loss-of-function STING variants.

Charge shielding effects of PEG bound to NH2-terminated PAMAM dendrimers - an experimental approach.

Cationic poly(amido amine) (PAMAM) dendrimers exhibit great potential for use in drug delivery, but their high charge density leads to an inherent cytotoxicity. To increase biocompatibility, many studies have attached poly(ethylene glycol) (PEG) chains to the dendrimer surface. It is unclear how these tethered PEG chains influence the physicochemical properties of the dendrimer. Here, we develop a fluorescence-based assay utilizing anionic biological tissue to quantify the electrostatic binding affinity of a library of PEG-PAMAM conjugates with various PEG chain lengths and grafting densities. We find that covalently bound PEG chains reduce the electrostatic binding affinity more significantly than what can be achieved through covalent bonds only. Contrary to previous thought, this reduction is not explained by the steric hindrance effects of PEG chains, suggesting that other, non-covalent interactions between PEG and PAMAM are present. Using acetylated PAMAM conjugates, we convert electrostatic binding affinity to the number of charged amines accessible to the physiological environment. These data, coupled with 1H-NMR, allows us to study more closely the non-covalent interactions between PEG and PAMAM. We find that increasing PEG chain length increases the number of non-covalent interactions. Additionally, at low grafting densities, increasing the number of PEG chains on the PAMAM surface also increases the non-covalent interactions. At higher grafting densities, however, PEG chains sterically repel one another, forcing chains to elongate away from the surface and reducing the number of interactions between PAMAM and individual PEG chains. The data presented here provides a framework for a more precise mechanistic understanding of how the length and density of tethered PEG chains on PAMAM dendrimers influence drug delivery properties.

Engineering a Two-Component Hemostat for the Treatment of Internal Bleeding through Wound-Targeted Crosslinking.

Primary hemostasis (platelet plug formation) and secondary hemostasis (fibrin clot formation) are intertwined processes that occur upon vascular injury. Researchers have sought to target wounds by leveraging cues specific to these processes, such as using peptides that bind activated platelets or fibrin. While these materials have shown success in various injury models, they are commonly designed for the purpose of treating solely primary or secondary hemostasis. In this work, a two-component system consisting of a targeting component (azide/GRGDS PEG-PLGA nanoparticles) and a crosslinking component (multifunctional DBCO) is developed to treat internal bleeding. The system leverages increased injury accumulation to achieve crosslinking above a critical concentration, addressing both primary and secondary hemostasis by amplifying platelet recruitment and mitigating plasminolysis for greater clot stability. Nanoparticle aggregation is measured to validate concentration-dependent crosslinking, while a 1:3 azide/GRGDS ratio is found to increase platelet recruitment, decrease clot degradation in hemodiluted environments, and decrease complement activation. Finally, this approach significantly increases survival relative to the particle-only control in a liver resection model. In light of prior successes with the particle-only system, these results emphasize the potential of this technology in aiding hemostasis and the importance of a holistic approach in engineering new treatments for hemorrhage.

Synergistic combination therapy delivered via layer-by-layer nanoparticles induces solid tumor regression of ovarian cancer.

The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC-BCL2, BCL-XL, and MCL1. Co-inhibition of BCL2 and BCL-XL (ABT-263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer-by-layer nanoparticles (LbL NPs) that co-encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near-complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co-encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.

Layer-by-layer interleukin-12 nanoparticles drive a safe and effective response in ovarian tumors.

Ovarian cancer is especially deadly, challenging to treat, and has proven refractory to known immunotherapies. Cytokine therapy is an attractive strategy to drive a proinflammatory immune response in immunologically cold tumors such as many high grade ovarian cancers; however, this strategy has been limited in the past due to severe toxicity. We previously demonstrated the use of a layer-by-layer (LbL) nanoparticle (NP) delivery vehicle in subcutaneous flank tumors to reduce the toxicity of interleukin-12 (IL-12) therapy upon intratumoral injection. However, ovarian cancer cannot be treated by local injection as it presents as dispersed metastases. Herein, we demonstrate the use of systemically delivered LbL NPs using a cancer cell membrane-binding outer layer to effectively target and engage the adaptive immune system as a treatment in multiple orthotopic ovarian tumor models, including immunologically cold tumors. IL-12 therapy from systemically delivered LbL NPs shows reduced severe toxicity and maintained anti-tumor efficacy compared to carrier-free IL-12 or layer-free liposomal NPs leading to a 30% complete survival rate.

Low calf circumference adjusted for body mass index is associated with prolonged hospital stay.

BACKGROUND: Calf circumference (CC) is of emerging importance because of its practicality, high correlation with skeletal muscle, and potential predictive value for adverse outcomes. However, the accuracy of CC is influenced by adiposity. CC adjusted for BMI (BMI-adjusted CC) has been proposed to counteract this problem. However, its accuracy to predict outcomes is unknown. OBJECTIVES: To evaluate the predictive validity of BMI-adjusted CC in hospital settings. METHODS: A secondary analysis of a prospective cohort study in hospitalized adult patients was conducted. The CC was adjusted for BMI by reducing 3, 7, or 12 cm for BMI (in kg/m2) of 25-29.9, 30-39.9, and ≥40, respectively. Low CC was defined as ≤34 cm for males and ≤33 cm for females. Primary outcomes included length of hospital stay (LOS) and in-hospital death, and secondary outcomes were hospital readmissions and mortality within 6 mo after discharge. RESULTS: We included 554 patients (55.2 ± 14.9 y, 52.9% men). Among them, 25.3% presented with low CC, whereas 60.6% had BMI-adjusted low CC. In-hospital death occurred in 13 patients (2.3%), and median LOS was 10.0 (5.0-18.0) d. Within 6 mo from discharge, 43 patients (8.2%) died, and 178 (34.0%) were readmitted to the hospital. BMI-adjusted low CC was an independent predictor of LOS ≥ 10 d (odds ratio = 1.70; 95% confidence interval: 1.18, 2.43], but it was not associated with the other outcomes. CONCLUSIONS: BMI-adjusted low CC was identified in more than 60% of hospitalized patients and was an independent predictor of longer LOS.

Stuck in the past? Rumination-related memory integration.

Memories connected to ruminative concerns repetitively capture attention, even in situations designed to alter them. However, recent research on memory updating suggests that memory for benign substitutes (e.g., reinterpretations) might be facilitated by integration with the ruminative memories. As a first approach, two experiments (Ns = 72) mimicked rumination-related memories with rumination-themed stimuli and an imagery task. College undergraduates screened for ruminative status first studied and imaged ruminative cue-target word pairs, and then in a second phase they studied the same cues re-paired with benign targets (along with new and repeated pairs). On the test of cued recall of benign targets, they judged whether each recalled word had been repeated or changed across the two phases (or was new in the second phase). When target changes were not remembered, recall of benign targets revealed proactive interference that was insensitive to ruminative status. However, when participants remembered change and the ruminative targets, their recall of benign targets was facilitated, particularly if they identified as ruminators (Experiment 1). When the test simply asked for recall of either or both targets (Experiment 2), ruminators recalled both targets more frequently than did others. These outcomes suggest that ruminative memories might provide bridges to remembering associated benign memories, such as reinterpretations, under conditions consistent with everyday ruminative retrieval.

Experience-Dependent Plasticity in Nucleus Laminaris of the Barn Owl.

Barn owls experience increasing interaural time differences (ITDs) during development, because their head width more than doubles in the month after hatching. We therefore hypothesized that their ITD detection circuit might be modified by experience. To test this, we raised owls with unilateral ear inserts that delayed and attenuated the acoustic signal, then measured the ITD representation in the brainstem nucleus laminaris (NL) when they were adult. The ITD circuit is composed of delay line inputs to coincidence detectors, and we predicted that plastic changes would lead to shorter delays in the axons from the manipulated ear, and complementary shifts in ITD representation on the two sides. In owls that received ear inserts starting around P14, the maps of ITD shifted in the predicted direction, but only on the ipsilateral side, and only in those tonotopic regions that had not experienced auditory stimulation prior to insertion. The contralateral map did not change. Experience-dependent plasticity of the ITD circuit occurs in NL, and our data suggest that ipsilateral and contralateral delays are independently regulated. Thus, altered auditory input during development leads to long-lasting changes in the representation of ITD.

Inhibitor of Apoptosis Proteins Antagonist Induces T-cell Proliferation after Cross-Presentation by Dendritic Cells.

Cross-presentation of tumor antigens by dendritic cells (DC) is crucial to prime, stimulate and restimulate CD8+ T cells. This process is important in initiating and maintaining an antitumor response. Here, we show that the presence of conventional type 1 DCs (cDC1), a DC subtype that excels in cross-presentation, in the tumor correlated with response to neoadjuvant immune checkpoint blockade (ICB) in melanoma. This led us to hypothesize that patients failing to respond to ICB could benefit from enhanced cross-presentation of tumor antigens. We therefore established a cross-presentation assay to screen over 5,500 compounds for enhancers of DC cross-presentation using induced T-cell proliferation as the readout. We identified 145 enhancers, including AZD5582, an antagonist of inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2, and XIAP. AZD5582 treatment led to DC activation of the noncanonical NF-kB pathway, enhanced antigen import from endolysosomes into the cytosol, and increased expression of genes involved in cross-presentation. Furthermore, it upregulated expression of CD80, CD86, MHC class II, CD70 and secretion of TNF by DCs. This enhanced DC activation and maturation program was observed also in tumor-bearing mice upon AZD5582 treatment, culminating in an increased frequency of systemic tumor antigen-specific CD8+ T cells. Our results merit further exploration of AZD5582 to increase antigen cross-presentation for improving the clinical benefit of ICB in patients who are unlikely to respond to ICB.

Acidity-mediated induction of FoxP3+ regulatory T cells.

Glucose limitation and increased lactic acid levels are consequences of the elevated glycolytic activity of tumor cells, and constitute a metabolic barrier for the function of tumor infiltrating effector immune cells. The immune-suppressive functions of regulatory T cells (Tregs) are unobstructed in lactic-acid rich environments. However, the impact of lactic acid on the induction of Tregs remains unknown. We observed increased TGFß-mediated induction of Forkhead box P3+ (FoxP3+ ) cells in the presence of extracellular lactic acid, in a glycolysis-independent, acidity-dependent manner. These CD4+ FoxP3+ cells expressed Treg-associated markers, including increased expression of CD39, and were capable of exerting suppressive functions. Corroborating these results in vivo, we observed that neutralizing the tumor pH by systemic administration of sodium bicarbonate (NaBi) decreased Treg abundance. We conclude that acidity augments Treg induction and propose that therapeutic targeting of acidity in the tumor microenvironment (TME) might reduce Treg-mediated immune suppression within tumors.

How Personalization Affects Motivation in Gamified Review Assessments.

Personalized gamification aims to address shortcomings of the one-size-fits-all (OSFA) approach in improving students' motivations throughout the learning process. However, studies still focus on personalizing to a single user dimension, ignoring multiple individual and contextual factors that affect user motivation. Unlike prior research, we address this issue by exploring multidimensional personalization compared to OSFA based on a multi-institution sample. Thus, we conducted a controlled experiment in three institutions, comparing gamification designs (OSFA and Personalized to the learning task and users' gaming habits/preferences and demographics) in terms of 58 students' motivations to complete assessments for learning. Our results suggest no significant differences among OSFA and Personalized designs, despite suggesting user motivation depended on fewer user characteristics when using personalization. Additionally, exploratory analyses suggest personalization was positive for females and those holding a technical degree, but negative for those who prefer adventure games and those who prefer single-playing. Our contribution benefits designers, suggesting how personalization works; practitioners, demonstrating to whom the personalization strategy was more or less suitable; and researchers, providing future research directions. Supplementary Information: The online version contains supplementary material available at 10.1007/s40593-022-00326-x.

A new label-free optical imaging method for the lymphatic system enhanced by deep learning.

Our understanding of the lymphatic vascular system lags far behind that of the blood vascular system, limited by available imaging technologies. We present a label-free optical imaging method that visualizes the lymphatic system with high contrast. We developed an orthogonal polarization imaging (OPI) in the shortwave infrared range (SWIR) and imaged both lymph nodes and lymphatic vessels of mice and rats in vivo through intact skin, as well as human mesenteric lymph nodes in colectomy specimens. By integrating SWIR-OPI with U-Net, a deep learning image segmentation algorithm, we automated the lymph node size measurement process. Changes in lymph nodes in response to cancer progression were monitored in two separate mouse cancer models, through which we obtained insights into pre-metastatic niches and correlation between lymph node masses and many important biomarkers. In a human pilot study, we demonstrated the effectiveness of SWIR-OPI to detect human lymph nodes in real time with clinical colectomy specimens. One Sentence Summary: We develop a real-time high contrast optical technique for imaging the lymphatic system, and apply it to anatomical pathology gross examination in a clinical setting, as well as real-time monitoring of tumor microenvironment in animal studies.

Designer co-beta-peptide copolymer selectively targets resistant and biofilm Gram-negative bacteria.

New antimicrobials are urgently needed to combat Gram-negative bacteria, particularly multi-drug resistant (MDR) and phenotypically resistant biofilm species. At present, only sequence-defined alpha-peptides (e.g. polymyxin B) can selectively target Gram-negative bacterial lipopolysaccharides. We show that a copolymer, without a defined sequence, shows good potency against MDR Gram-negative bacteria including its biofilm form. The tapered blocky co-beta-peptide with controlled N-terminal hydrophobicity (#4) has strong interaction with the Gram-negative bacterial lipopolysaccharides via its backbone through electrostatic and hydrogen bonding interactions but not the Gram-positive bacterial and mammalian cell membranes so that this copolymer is non-toxic to these two latter cell types. The new #4 co-beta-peptide selectively kills Gram-negative bacteria with low cytotoxicity both in vitro and in a mouse biofilm wound infection model. This strategy provides a new concept for the design of Gram-negative selective antimicrobial peptidomimetics against MDR and biofilm species.

Delusions and Hallucinations Are Associated With Greater Severity of Delirium.

BACKGROUND: The 3 core domains of delirium (cognitive, higher level thinking, circadian) do not include the less common noncore psychotic symptoms. However, psychosis might inform about perturbations of neural circuitry, outcomes, or suggest tailored clinical management. OBJECTIVE: We assessed relationships between psychosis and other characteristics of delirium in patients without dementia or antipsychotics treatment. METHODS: Cross-sectional analysis of 366 adults with delirium per the Delirium Rating Scale Revised-98, whose items distinguish hallucinations and delusions from other types of misperceptions and abnormal thought content, assessed during the preceding 24 hours to capture symptom severity fluctuation. The relationship of psychosis with other delirium characteristics was assessed using bivariate comparisons and analysis of variance as appropriate for groups with no psychosis and any psychosis (hallucinations and/or delusions), and subgroups with only hallucinations, only delusions, or both. A discriminant logistic model assessed variables associated with presence of any psychotic features versus none. RESULTS: Delirium with any psychotic features occurred in 44.5% (163 of 366). Of the 366, 119 (32.5%) had only hallucinations (Hall), 14 (3.8%) had only delusions (Del), and 30 (8.2%) had both (Both). In the psychotic group (n = 163), 73.0% were Hall, 8.6% Del, and 18.4% Both. All psychotic patient groupings had significantly greater delirium severity on the Delirium Rating Scale Revised-98. Delusions and hallucinations were discordant for occurring together. The discriminant model found increased odds of having psychosis as 3 symptom severities increased (visuospatial ability, thought process, and sleep-wake cycle) where these each represented a delirium core domain. The noncore symptom of lability of affect had high odds ratio for psychosis, while motor retardation reduced odds of psychosis in this model. CONCLUSIONS: Consistent with prior reports, psychosis occurred in less than half of delirious patients with delusions being infrequent, and an association with affective lability was found. Given that previous functional magnetic resonance imaging research found a correlation between neural network dysconnectivity with greater severity of delirium, psychotic symptoms might be a clinical marker for greater underlying cerebral cortical neural circuitry dysfunction.

Predicting transport of intra-articularly injected growth factor fusion proteins into human knee joint cartilage.

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application. STATEMENT OF SIGNIFICANCE: The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application.

Surface Presentation of Hyaluronic Acid Modulates Nanoparticle-Cell Association.

Nanoparticle (NP) drug carriers have revolutionized medicine and increased patient quality of life. Clinically approved formulations typically succeed because of reduced off-target toxicity of the cargo. However, increasing carrier accumulation at disease sites through precise targeting remains one of the biggest challenges in the field. Novel multivalent ligand presentations and self-assembled constructs can enhance cell association, but an inability to draw direct comparisons across formulations has hindered progress. Furthermore, how nanoparticle structure influences function often is unclear. In this report, we leverage the well-characterized hyaluronic acid (HA)-CD44 binding pair to investigate how the surface architecture of modified NPs impacts their association with ovarian cancer cells that overexpress CD44. We functionalized anionic liposomes with 5 kDa HA by either covalent conjugation via surface coupling or electrostatic self-assembly using the layer-by-layer (LbL) adsorption method. Comparing these two methods, we observed a consistent enhancement of NP-cell association with the self-assembly LbL technique, particularly with higher molecular weight (≥10 kDa) HA. To further optimize association, we increased the surface-available HA. We synthesized a bottlebrush glycopolymer composed of a polynorbornene backbone and pendant 5 kDa HA and layered this macromolecule onto NPs. Flow cytometry revealed that the LbL HA bottlebrush NP outperformed the LbL linear display of HA. Cellular visualization by deconvolution optical microscopy corroborated results from all three constructs. Using exogenous HA to block NP-CD44 interactions, we found the LbL HA bottlebrush NP had a 4-fold higher binding avidity than the best-performing LbL linear HA NP. We further observed that decreasing the density of HA bottlebrush side chains to 75% had minimal impact on LbL NP stability or cell association, though we did see a reduction in binding avidity with this side-chain-modified NP. Our studies indicate that LbL surfaces are highly effective for multivalent displays, and the mode in which they present a targeting ligand can be optimized for NP cell targeting.