A program for real-time surveillance of SARS-CoV-2 genetics.

The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.S.) from March 13th 2020 to July 3rd 2023. Our sequencing protocol ('Virseq') utilizes wet and dry lab procedures to generate mutation-resistant sequencing of the entire SARS-CoV-2 genome, capturing all major lineages. We also characterize 379 clinically relevant SARS-CoV-2 multi-strain co-infections and ensure robust detection of emerging lineages via simulation. The modular infrastructure, sequencing, and analysis capabilities we describe support the U.S. Centers for Disease Control and Prevention national surveillance program and serve as a model for rapid response to emerging pandemics at a national scale.

SARS-CoV-2 variant introduction following spring break travel and transmission mitigation strategies.

BACKGROUND: University spring break carries a two-pronged SARS-CoV-2 variant transmission risk. Circulating variants from universities can spread to spring break destinations, and variants from spring break destinations can spread to universities and surrounding communities. Therefore, it is critical to implement SARS-CoV-2 variant surveillance and testing strategies to limit community spread before and after spring break to mitigate virus transmission and facilitate universities safely returning to in-person teaching. METHODS: We examined the SARS-CoV-2 positivity rate and changes in variant lineages before and after the university spring break for two consecutive years. 155 samples were sequenced across four time periods: pre- and post-spring break 2021 and pre- and post-spring break 2022; following whole genome sequencing, samples were assigned clades. The clades were then paired with positivity and testing data from over 50,000 samples. RESULTS: In 2021, the number of variants in the observed population increased from four to nine over spring break, with variants of concern being responsible for most of the cases; Alpha percent composition increased from 22.2% to 56.4%. In 2022, the number of clades in the population increased only from two to three, all of which were Omicron or a sub-lineage of Omicron. However, phylogenetic analysis showed the emergence of distantly related sub-lineages. 2022 saw a greater increase in positivity than 2021, which coincided with a milder mitigation strategy. Analysis of social media data provided insight into student travel destinations and how those travel events may have impacted spread. CONCLUSIONS: We show the role that repetitive testing can play in transmission mitigation, reducing community spread, and maintaining in-person education. We identified that distantly related lineages were brought to the area after spring break travel regardless of the presence of a dominant variant of concern.

Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.

Targeted inhibition of protein synthesis renders cancer cells vulnerable to apoptosis by unfolded protein response.

Cellular stress responses including the unfolded protein response (UPR) decide over the fate of an individual cell to ensure survival of the entire organism. During physiologic UPR counter-regulation, protective proteins are upregulated to prevent cell death. A similar strategy induces resistance to UPR in cancer. Therefore, we hypothesized that blocking protein synthesis following induction of UPR substantially enhances drug-induced apoptosis of malignant cells. In line, upregulation of the chaperone BiP was prevented by simultaneous arrest of protein synthesis in B cell malignancies. Cytotoxicity by immunotoxins-approved inhibitors of protein synthesis-was synergistically enhanced in combination with UPR-inducers in seven distinct hematologic and three solid tumor entities in vitro. Synergistic cell death depended on mitochondrial outer membrane permeabilization via BAK/BAX, which correlated with synergistic, IRE1α-dependent reduction of BID, accompanied by an additive fall of MCL-1. The strong synergy was reproduced in vivo against xenograft mouse models of mantle cell lymphoma, Burkitt's lymphoma, and patient-derived acute lymphoblastic leukemia. In contrast, synergy was absent in blood cells of healthy donors suggesting a tumor-specific vulnerability. Together, these data support clinical evaluation of blocking stress response counter-regulation using inhibitors of protein synthesis as a novel therapeutic strategy.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

Investigating water movements around a shallow shipwreck in Big Tub Harbour of Lake Huron: Implications for managing and preserving underwater shipwrecks.

The Sweepstakes, in Fathom Five National Marine Park, is Ontario's most iconic shipwreck with over 100,000 visitors each summer. Continued exposure to water currents has directly and indirectly affected the integrity of the wreck and resulted in management interventions including efforts to stabilize the wreck and control vessel activity (both duration and speed). Despite these efforts, a scour ring is present in the sediment around the Sweepstakes, raising concerns regarding the prolonged stability of the wreck. An extensive series of field measurements were made during the summer of 2015 with the aim of differentiating between natural hydrological processes present at this site and human-derived water movements during the summer visitor season. There is a high-degree of natural current variability from processes as diverse as wind-induced surface gravity waves, internal gravity waves, and diurnal flows due to differential heating. Our results show that summer circulation driven by internal gravity waves derived from upwelling, surface waves, and differential heating was insignificant with respect to sediment resuspension and thus unlikely to produce the observed scour around the shipwreck. Scour is most likely caused by energetic winter storms, which should be a focus of future studies. While vessel induced currents were detectable at the shipwreck, they were no larger than the normal summer hydrodynamic variability, thus suggesting that management efforts continue to protect the site generally.

Clinical and Cost-Effectiveness of Lumbar Interbody Fusion Using Tritanium Posterolateral Cage (vs. Propensity-Matched Cohort of PEEK Cage).

Introduction: Surgical management of degenerative lumbar spine disorders is effective at improving patient pain, disability, and quality of life; however, obtaining a durable posterolateral fusion after decompression remains a challenge. Interbody fusion technologies are viable means of improving fusion rates in the lumbar spine, specifically various graft materials including autograft, structural allograft, titanium, and polyether ether ketone. This study assesses the effectiveness of Tritanium posterolateral cage in the treatment of degenerative disk disease. Methods: Nearest-neighbor 1:1 matched control transforaminal lumbar interbody fusion with PEEK vs. Tritanium posterior lumbar (PL) cage interbody fusion patients were identified using propensity scoring from patients that underwent elective surgery for degenerative disk diseases. Line graphs were generated to compare the trajectories of improvement in patient-reported outcomes (PROs) from baseline to 3 and 12 months postoperatively. The nominal data were compared via the χ2 test, while the continuous data were compared via Student's t-test. Results: The two groups had no difference regarding either the 3- or 12-month Euro-Qol-5D (EQ-5D), numeric rating scale (NRS) leg pain, and NRS back pain; however, the Tritanium interbody cage group had better Oswestry Disability Index (ODI) scores compared to the control group of the PEEK interbody cage at both 3 and 12 months (p=0.013 and 0.048). Conclusions: Our results indicate the Tritanium cage is an effective alternative to the previously used PEEK cage in terms of PROs, surgical safety, and radiological parameters of surgical success. The Tritanium cohort showed better ODI scores, higher fusion rates, lower subsidence, and lower indirect costs associated with surgical management, when compared to the propensity-matched PEEK cohort.

The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris.

Projected changes in bird assemblages due to climate change in a Canadian system of protected areas.

National parks often serve as a cornerstone for a country's species and ecosystem conservation efforts. However, despite the protection these sites afford, climate change is expected to drive a substantial change in their bird assemblages. We used species distribution models to predict the change in environmental suitability (i.e., how well environmental conditions explain the presence of a species) of 49 Canadian national parks during summer and winter for 434 bird species under a 2°C warming scenario, anticipated to occur in Canada around the mid-21st century. We compared these to existing species distributions in the 2010s, and classified suitability projections for each species at each park as potential extirpation, worsening, stable, improving, or potential colonisation. Across all parks, and both seasons, 70% of the projections indicate change, including a 25% turnover in summer assemblages and 30% turnover in winter assemblages. The majority of parks are projected to have increases in species richness and functional traits in winter, compared to a mix of increases and decreases in both in summer. However, some changes are expected to vary by region, such as Arctic region parks being likely to experience the most potential colonisation, while some of the Mixedwood Plains and Atlantic Maritime region parks may experience the greatest turnover and potential extirpation in summer if management actions are not taken to mitigate some of these losses. Although uncertainty exists around the precise rate and impacts of climate change, our results indicate that conservation practices that assume stationarity of environmental conditions will become untenable. We propose general guidance to help managers adapt their conservation actions to consider the potentially substantive changes in bird assemblages that are projected, including managing for persistence and change.

A human recombinant analogue to plasma-derived vaccinia immunoglobulin prophylactically and therapeutically protects against lethal orthopoxvirus challenge.

Orthopoxviruses such as variola and monkeypox viruses continue to threaten the human population. Monkeypox virus is endemic in central and western Africa and outbreaks have reached as far as the U.S. Although variola virus, the etiologic agent of smallpox, has been eradicated by a successful vaccination program, official and likely clandestine stocks of the virus exist. Moreover, studies with ectromelia virus (the etiological agent of mousepox) have revealed that IL-4 recombinant viruses are significantly more virulent than wild-type viruses even in mice treated with vaccines and/or antivirals. For these reasons, it is critical that antiviral modalities are developed to treat these viruses should outbreaks, or deliberate dissemination, occur. Currently, 2 antivirals (brincidofovir and tecovirimat) are in the U.S. stockpile allowing for emergency use of the drugs to treat smallpox. Both antivirals have advantages and disadvantages in a clinical and emergency setting. Here we report on the efficacy of a recombinant immunoglobulin (rVIG) that demonstrated efficacy against several orthopoxviruses in vitro and in vivo in both a prophylactic and therapeutic fashion. A single intraperitoneal injection of rVIG significantly protected mice when given up to 14 days before or as late as 6 days post challenge. Moreover, rVIG reduced morbidity, as measured by weight-change, as well as several previously established biomarkers of disease. In rVIG treated mice, we found that vDNA levels in blood were significantly reduced, as was ALT (a marker of liver damage) and infectious virus levels in the liver. No apparent adverse events were observed in rVIG treated mice, suggesting the immunoglobulin is well tolerated. These findings suggest that recombinant immunoglobulins could be candidates for further evaluation and possible licensure under the FDA Animal Rule.

Initial Experience with Using a Structured Light 3D Scanner and Image Registration to Plan Bedside Subdural Evacuating Port System Placement.

BACKGROUND: Chronic subdural hematoma evacuation can be achieved in select patients through bedside placement of the Subdural Evacuation Port System (SEPS; Medtronic, Inc., Dublin, Ireland). This procedure involves drilling a burr hole at the thickest part of the hematoma. Identifying this location is often difficult, given the variable tilt of available imaging and distant anatomic landmarks. This paper evaluates the feasibility and accuracy of a bedside navigation system that relies on visible light-based 3-dimensional (3D) scanning and image registration to a pre-procedure computed tomography scan. The information provided by this system may increase accuracy of the burr hole location. METHODS: In Part 1, the accuracy of this system was evaluated using a rigid 3D printed phantom head with implanted fiducials. In Part 2, the navigation system was tested on 3 patients who underwent SEPS placement. RESULTS: The error in registration of this system was less than 2.5 mm when tested on a rigid 3D printed phantom head. Fiducials located in the posterior aspect of the head were difficult to reliably capture. For the 3 patients who underwent 5 SEPS placements, the distance between anticipated SEPS burr hole location based on registration and actual burr hole location was less than 1cm. CONCLUSIONS: A bedside cranial navigation system based on 3D scanning and image registration has been introduced. Such a system may increase the success rate of bedside procedures, such as SEPS placement. However, technical challenges such as the ability to scan hair and practical challenges such as minimization of patient movement during scans must be overcome.

The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model.

Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1ß and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.

A 3D-Printed Simulator and Teaching Module for Placing S2-Alar-Iliac Screws.

BACKGROUND: The concept of the S2-alar-iliac (S2AI) screw was developed approximately one decade ago and has rapidly become an important component of spinal arthrodesis. Two challenges to placing S2AI screws are gaining an intuition for free-hand screw placement trajectory and acquisition of the appropriate radiograph to both guide screw placement and diagnose misplacement. OBJECTIVE: To present the design and manufacture of an S2AI screw placement simulator and teaching module that addresses both challenges. METHODS: This simulator involves using a 3D printer to create a life-sized pelvis. Participants first used this print to practice placing free-hand S2AI screws. Then participants used another print to practice taking radiographs showing the posterior superior iliac spine-anterior superior iliac spine corridor (teardrop) view. RESULTS: The accuracy of screw placement increased from 17 to 80% on the left side and 7 to 100% on the right side. The number of radiographs taken by each participant to obtain the teardrop view decreased after practice with the simulator compared to baseline. CONCLUSION: Practice with the S2AI simulator led to an improved intuition of an appropriate free-hand S2AI screw trajectory and a decrease in the number of radiographs needed for obtaining the correct diagnostic view.

When is the Wilcoxon-Mann-Whitney procedure a test of location? Implications for effect-size measures.

The Wilcoxon-Mann-Whitney procedure is invariant under monotone transformations but its use as a test of location or shift is said not to be so. It tests location only under the shift model, the assumption of parallel cumulative distribution functions (cdfs). We show that infinitely many monotone transformations of the measured variable produce parallel cdfs, so long as the original cdfs intersect nowhere or everywhere. Thus there are infinitely many effect sizes measured as shifts of medians, invalidating the notion that there is one true shift parameter and thereby rendering any single estimate dubious. Measuring effect size using the probability of superiority alleviates this difficulty.

Broadening of the drumhead-mode spectrum due to in-plane thermal fluctuations of two-dimensional trapped ion crystals in a Penning trap.

Two-dimensional crystals of ions stored in Penning traps are a leading platform for quantum simulation and sensing experiments. For small amplitudes, the out-of-plane motion of such crystals can be described by a discrete set of normal modes called the drumhead modes, which can be used to implement a range of quantum information protocols. However, experimental observations of crystals with Doppler-cooled and even near-ground-state-cooled drumhead modes reveal an unresolved drumhead-mode spectrum. In this work, we establish in-plane thermal fluctuations in ion positions as a major contributor to the broadening of the drumhead-mode spectrum. In the process, we demonstrate how the confining magnetic field leads to unconventional in-plane normal modes, whose average potential and kinetic energies are not equal. This property, in turn, has implications for the sampling procedure required to choose the in-plane initial conditions for molecular-dynamics simulations. For current operating conditions of the NIST Penning trap, our study suggests that the two-dimensional crystals produced in this trap undergo in-plane potential-energy fluctuations of the order of 10mK. Our study therefore motivates the need for designing improved techniques to cool the in-plane degrees of freedom.

Unique osmoregulatory morphology in primitive sharks: an intermediate state between holocephalan and derived shark secretory morphology.

Discovery of an unusual rectal gland in the Atlantic sixgill shark Hexanchus vitulus led us to examine the rectal glands of 31 species of sharks to study diversity in rectal-gland morphology. Twenty-four of 31 species of sharks had digitiform glands (mean width-length ratio ± SD = 0.17 ± 0.04) previously assumed to be characteristic of all elasmobranchs regardless of habitat depth or phylogenetic age. Rectal glands from the family Somniosidae were kidney bean-shaped (mean width: length ± SD = 0.46 ± 0.05); whereas those from families Echinorhinidae and Hexanchidae were lobulate (mean width: length ± SD = 0.55 ± 0.06). Rectal gland width: length were different among species with digitiform morphology and lobulate morphology (ANOVA; R2 = 0.9; df = 15, 386; 401, F = 219.24; P < 0.001). Histological and morphological characteristics of the digitiform morphology from deep-sea sharks were similar to those from shallow-water sharks. Histology of lobulate rectal glands from hexanchids were characterised by tubule bundles separated by smooth muscle around a central lumen. Additionally, we examined plasma chemistry of four species of sharks with digitiform rectal glands and two species with lobulate rectal-gland morphology to see if there were differences between morphologies. Plasma chemistry analysis showed that urea and trimethylamine N-oxide (TMAO) followed the piezolyte hypothesis, with TMAO being highest and urea being lowest in deep-sea sharks. Among electrolytes, Na+ was highest in species with lobulate rectal glands. Hexanchids and echinorhinids both have lobulate rectal glands similar to those of holocephalans, despite the more than 400 million years separating these two groups. The morphological similarities between the lobulate rectal-gland anatomy of primitive sharks and the secretory morphology of holocephalans may represent an intermediate state between Holocephali and derived shark species.

Is placebo analgesia for heat pain a sensory effect? An exploratory study on minimizing the influence of response bias.

We explored the ongoing question of whether placebo analgesia alters afferent nociceptive processing in a novel paradigm designed to minimize the role of response bias in placebo measurement. First, healthy adult participants received a standard heat placebo induction and conditioning procedure using a topical "analgesic" cream applied to one arm. During a subsequent placebo testing procedure, participants rated stimuli on the placebo-treated arm and untreated arm, using a task that minimized subjects' ability to guess the expected response, thus reducing experimenter demand. Retrospectively participants reported moderate analgesia effectiveness (mean=5.3/10), but for individual temperature ratings, only 2 subjects exhibited a perceptual placebo response >5 points. Next, these subjects completed a novel, exploratory task designed to measure changes in inter-arm in discriminative accuracy that would be expected from changes in afferent nociception. Both placebo responders (but no non-responders) showed reduced discriminative ability when the hotter stimulus occurred on the placebo arm, an effect consistent with alterations in nociceptive afferent flow and unlikely to be caused by response bias.

Improved efficacy against malignant brain tumors with EGFRwt/EGFRvIII targeting immunotoxin and checkpoint inhibitor combinations.

BACKGROUND: D2C7-IT is a novel immunotoxin (IT) targeting wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins in glioblastoma. In addition to inherent tumoricidal activity, immunotoxins induce secondary immune responses through the activation of T cells. However, glioblastoma-induced immune suppression is a major obstacle to an effective and durable immunotoxin-mediated antitumor response. We hypothesized that D2C7-IT-induced immune response could be effectively augmented in combination with αCTLA-4/αPD-1/αPD-L1 therapies in murine models of glioma. METHODS: To study this, we overexpressed the D2C7-IT antigen, murine EGFRvIII (dmEGFRvIII), in established glioma lines, CT-2A and SMA560. The reactivity and therapeutic efficacy of D2C7-IT against CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII cells was determined by flow cytometry and in vitro cytotoxicity assays, respectively. Antitumor efficacy of D2C7-IT was examined in immunocompetent, intracranial murine glioma models and the role of T cells was assessed by CD4+ and CD8+ T cell depletion. In vivo efficacy of D2C7-IT/αCTLA-4/αPD-1 monotherapy or D2C7-IT+αCTLA-4/αPD-1 combination therapy was evaluated in subcutaneous unilateral and bilateral CT-2A-dmEGFRvIII glioma-bearing immunocompetent mice. Further, antitumor efficacy of D2C7-IT+αCTLA-4/αPD-1/αPD-L1/αTim-3/αLag-3/αCD73 combination therapy was evaluated in intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII glioma-bearing mice. Pairwise differences in survival curves were assessed using the generalized Wilcoxon test. RESULTS: D2C7-IT effectively killed CT-2A-dmEGFRvIII (IC50 = 0.47 ng/mL) and SMA560-dmEGFRvIII (IC50 = 1.05 ng/mL) cells in vitro. Treatment of intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII tumors with D2C7-IT prolonged survival (P = 0.0188 and P = 0.0057, respectively), which was significantly reduced by the depletion of CD4+ and CD8+ T cells. To augment antitumor immune responses, we combined D2C7-IT with αCTLA-4/αPD-1 in an in vivo subcutaneous CT-2A-dmEGFRvIII model. Tumor-bearing mice exhibited complete tumor regressions (4/10 in D2C7-IT+αCTLA-4 and 5/10 in D2C7-IT+αPD-1 treatment groups), and combination therapy-induced systemic antitumor response was effective against both dmEGFRvIII-positive and dmEGFRvIII-negative CT-2A tumors. In a subcutaneous bilateral CT-2A-dmEGFRvIII model, D2C7-IT+αCTLA-4/αPD-1 combination therapies showed dramatic regression of the treated tumors and measurable regression of untreated tumors. Notably, in CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII intracranial glioma models, D2C7-IT+αPD-1/αPD-L1 combinations improved survival, and in selected cases generated cures and protection against tumor re-challenge. CONCLUSIONS: These data support the development of D2C7-IT and immune checkpoint blockade combinations for patients with malignant glioma.

Role of Family Obligation Stress on Ugandan Women's Participation in Preventive Breast Health.

BACKGROUND: The purpose of this study is to determine the role of family obligation stress on Ugandan women's participation in preventive breast health through the receipt of breast cancer education and health check-ups. MATERIALS AND METHODS: A validated survey was conducted on a community sample of Ugandan women, providing a multi-item scale to assess preventive breast-health-seeking behaviors and measure family obligation stress (FO; range 6-18). Univariate and multivariate linear regression was used to assess associations between sociodemographic factors and FO. Univariate and multivariate linear regression (used in conjunction with the robust sandwich estimator for standard errors) and probability differences (PDs) were used to evaluate associations between preventive breast-health-seeking behaviors, sociodemographic factors, and FO. RESULTS: A total of 401 Ugandan women ages 25-74 participated in the survey. Most had three or more children in the home (60%) and were employed full time (69%). Higher FO was associated with increasing number of children and/or adults in the household (p < .05), full-time employment (p < .001), and being single (p = .003). Women with higher FO were less likely to participate in breast cancer education (PD = -0.02 per 1-point increase, p = .008) and preventive health check-ups (PD = -0.02, p = .018), associations that persisted on multivariate analysis controlling for sociodemographic factors. CONCLUSION: Ugandan women with high FO are less likely to participate in preventive breast cancer detection efforts including breast cancer education and preventive health check-ups. Special efforts should be made to reach women with elevated FO, because it may be a risk factor for late-stage presentation among women who develop breast cancer. IMPLICATIONS FOR PRACTICE: High family obligation stress (FO) significantly reduces women's participation in preventive health check-ups and breast cancer education. These findings support research in U.S. Latinas showing high FO negatively affects women's health, suggesting that FO is an important factor in women's health-seeking behavior in other cultures. Addressing family obligation stress by including family members involved in decision-making is essential for improving breast cancer outcomes in low- and middle-income countries, such as Uganda.