Characterization of Human Engraftment and Hemophagocytic Lymphohistiocytosis in NSG-SGM3 Neonate Mice Engrafted with Purified CD34+ Hematopoietic Stem Cells.

Immunodeficient mice bearing human immune systems, or "humanized" chimeric mice, are widely used in basic research, along with the preclinical stages of drug development. Nonobese diabetic-severe combined immunodeficiency (NOD-SCID) IL2Rγnull (NSG) mice expressing human stem cell factor, granulocyte-macrophage colony stimulating factor, and interleukin-3 (NSG-SGM3) support robust development of human myeloid cells and T cells but have reduced longevity due to the development of fatal hemophagocytic lymphohistiocytosis (HLH). Here, we describe an optimized protocol for development of human immune chimerism in NSG-SGM3 mice. We demonstrate that efficient human CD45+ reconstitution can be achieved and HLH delayed by engraftment of neonatal NSG-SGM3 with low numbers of human umbilical cord-derived CD34+ hematopoietic stem cells in the absence of preconditioning irradiation.

Wellness in Nursing Education to Promote Resilience and Reduce Burnout: Protocol for a Holistic Multidimensional Wellness Intervention and Longitudinal Research Study Design in Nursing Education.

BACKGROUND: The United States faces a nursing shortage driven by a burnout epidemic among nurses and nursing students. Nursing students are an integral population to fuel the nursing workforce at high risk of burnout and increased rates of perceived stress. OBJECTIVE: The aim of this paper is to describe WellNurse, a holistic, interdisciplinary, multidimensional longitudinal research study that examines evidence-based interventions intended to reduce burnout and increase resilience among graduate and undergraduate nursing students. METHODS: Graduate and undergraduate nursing students matriculated at a large public university in the northeastern United States are eligible to enroll in this ongoing, longitudinal cohort study beginning in March 2021. Participants complete a battery of health measurements twice each semester during the fourth week and the week before final examinations. The measures include the Perceived Stress Scale, the Satisfaction with Life Scale, the Oldenburg Burnout Inventory, the Brief Resilience Scale, and the Pittsburgh Sleep Quality Index. Participants are eligible to enroll in a variety of interventions, including mindfulness-based stress reduction, mindful eating, fitness training, and massage therapy. Those who enroll in specific, targeted interventions complete additional measures designed to target the aim of the intervention. All participants receive a free Fitbit device. Additional environmental changes are being implemented to further promote a culture that supports academic well-being, including recruiting a diverse student population through evidence-based holistic admissions, inclusive teaching design, targeted resilience and stress reduction workshops, and cultural shifts within classrooms and curricula. The study design protocol is registered at Open Science Framework (DOI 10.17605/OSF.IO/NCBPE). RESULTS: The project was funded on January 1, 2022. Data collection started in March 2022. A total of 267 participants have been recruited. Results will be published after each semester starting in December 2023. WellNurse evaluation follows the Rapid Cycle Quality Improvement framework to continuously monitor ongoing project processes, activity outcomes, and progress toward reducing burnout and increasing resilience. Rapid Cycle Quality Improvement promotes the ability to alter WellNurse interventions, examine multiple interventions, and test their effectiveness among the nursing education population to identify the most effective interventions. CONCLUSIONS: Academic nursing organizations must address student burnout risk and increase resilience to produce a future workforce that provides high-quality patient care to a diverse population. Findings from WellNurse will support evidence-based implementations for public baccalaureate and master's nursing programs in the United States. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/49020.

p53-Bad* Fusion Gene Therapy Induces Apoptosis In Vitro and Reduces Zebrafish Tumor Burden in Hepatocellular Carcinoma.

With few curative treatments and a global yearly death rate of over 800,000, hepatocellular carcinoma (HCC) desperately needs new therapies. Although wild-type p53 gene therapy has been shown to be safe in HCC patients, it has not shown enough efficacy to merit approval. This work aims to show how p53 can be re-engineered through fusion to the pro-apoptotic BH3 protein Bcl-2 antagonist of cell death (Bad) to improve anti-HCC activity and potentially lead to a novel HCC therapeutic, p53-Bad*. p53-Bad* is a fusion of p53 and Bad, with two mutations, S112A and S136A. We determined mitochondrial localization of p53-Bad* in liver cancer cell lines with varying p53 mutation statuses via fluorescence microscopy. We defined the apoptotic activity of p53-Bad* in four liver cancer cell lines using flow cytometry. To determine the effects of p53-Bad* in vivo, we generated and analyzed transgenic zebrafish expressing hepatocyte-specific p53-Bad*. p53-Bad* localized to the mitochondria regardless of the p53 mutation status and demonstrated superior apoptotic activity over WT p53 in early, middle, and late apoptosis assays. Tumor burden in zebrafish HCC was reduced by p53-Bad* as measured by the liver-to-body mass ratio and histopathology. p53-Bad* induced significant apoptosis in zebrafish HCC as measured by TUNEL staining but did not induce apoptosis in non-HCC fish. p53-Bad* can induce apoptosis in a panel of liver cancer cell lines with varying p53 mutation statuses and induce apoptosis/reduce HCC tumor burden in vivo in zebrafish. p53-Bad* warrants further investigation as a potential new HCC therapeutic.

Peripartum and Postpartum Analgesia and Pain in Women Prescribed Buprenorphine for Opioid Use Disorder Who Deliver by Cesarean Section.

Aim: Little is known about whether pain can be effectively managed in pregnant women with opioid use disorder (OUD) during delivery hospitalization, particularly those undergoing surgery and taking buprenorphine as medication for OUD (MOUD). To address this question, we compared pain scores and opioid analgesic utilization during delivery hospitalization in women taking their pre-hospital dose of buprenorphine who delivered by cesarean section to matched controls. To inform future research efforts, we also began to explore opioid analgesic utilization and pain scores by type of anesthesia as this variable is often not included in related literature. Methods: Retrospective matched cohort study of 46 women prescribed buprenorphine during pregnancy who delivered by cesarean section during a 7-year period. Results: When compared to matched controls, women taking their pre-hospital dose of buprenorphine undergoing cesarean section utilized more opioid analgesics as measured by morphine milligram equivalents (MME) (mean MME first 48 hours 153.0 mg vs 175.1 mg, respectively, P < .01) but had similar pain scores during delivery hospitalization. There was no difference in MME utilization by maternal dose of buprenorphine though sample sizes were small. Women on buprenorphine who received spinal anesthesia with morphine had mean pain scores that were 1.4 points lower (P = .01) during the first 48 hours than women on buprenorphine receiving other methods of anesthesia. Discussion And Conclusions: Pregnant women taking their pre-hospital dose of buprenorphine throughout their surgical delivery hospitalization were able to achieve pain relief similar to women not on MOUD but had higher MME requirements. Our results add to the emerging body of evidence suggesting that individuals on MOUD can achieve adequate post-surgical pain management without adjusting their pre-hospital dose of buprenorphine. Further research is required to fully understand the optimal buprenorphine dosing regimen during surgical hospitalizations. Our results also provide important preliminary evidence that spinal anesthesia containing opioids can be used effectively in individuals with OUD requiring surgical intervention.

Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model.

BACKGROUND: The conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma. METHODS: We developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1). RESULTS: Blood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment. CONCLUSIONS: These data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Implications of perinatal buprenorphine exposure on infant head circumference at birth.

Objective: To determine the potential impact of prenatal buprenorphine exposure on head circumference at birth and analyze whether head circumference may be related to maternal buprenorphine dose at delivery, delayed maternal entry into buprenorphine treatment or exposure to a variety of other medications and substances.Methods: A retrospective cohort study was performed of 137 full-term infants exposed to buprenorphine during pregnancy from January 2013 to December 2017. Pearson's correlation was calculated to investigate the potential relationship between head circumference and maternal dose of buprenorphine at delivery. t-tests were conducted to analyze head circumference in relationship to dichotomous variables.Results: Head circumference in infants exposed to buprenorphine during pregnancy was not significantly different from national norms for either male infants (95% CI 28.2-33.5 cm, norm 31.5 cm, and 28.5-34.9 cm, norm 33.1 cm, for the 3rd and 10th percentile, respectively) or female infants (95% CI 28.7-32.8 cm, norm 31.9 cm, and 29.1-34.3, norm 32.8 cm for the 3rd and 10th percentile, respectively). Head circumference was not associated with delayed maternal entry into buprenorphine treatment (t = -1.0715, p = .287) or exposure to psychotropic medications during pregnancy (t = 0.4194, p = .677). There was no relationship between infant head circumference and maternal buprenorphine dose at delivery (r = 0.004, p = .967). Head circumference was not associated with maternal smoking (t = 0.003, p = .998) or exposure to marijuana (t = 0.7277, p = .468), illicit opioids (t = -0.6701, p = .504), illicit amphetamines (t = -0.4062, p = .687) or illicit benzodiazepines (t = -0.6288, p = .535) during pregnancy.Conclusions: Exposure to buprenorphine prenatally does not appear to be associated with reduced head circumference at birth. Head circumference at birth also does not appear related to either maternal buprenorphine dose at delivery or delayed entry into treatment. As previous literature suggests that high dose methadone exposure during pregnancy may be associated with smaller head circumference and that smaller head circumference may be associated with risk of neurocognitive disorders, our results further support the use of buprenorphine as a first line treatment for opioid use disorders during pregnancy.

Human CLEC9A antibodies deliver NY-ESO-1 antigen to CD141+ dendritic cells to activate naïve and memory NY-ESO-1-specific CD8+ T cells.

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Linear mixed-effects models for estimation of pulmonary metastasis growth rate: implications for CT surveillance in patients with sarcoma.

OBJECTIVES: Sarcoma patients often undergo surveillance chest CT for detection of pulmonary metastases. No data exist on the optimal surveillance interval for chest CT. The aim of this study was to estimate pulmonary metastasis growth rate in sarcoma patients. METHODS: This was a retrospective review of 95 patients with pulmonary metastases (43 patients with histologically confirmed metastases and 52 with clinically diagnosed metastases) from sarcoma treated at an academic tertiary-care center between 01 January 2000 and 01 June 2019. Age, sex, primary tumor size, grade, subtype, size and volume of the pulmonary metastasis over successive chest CT scans were recorded. Two metastases per patient were chosen if possible. Multivariate linear mixed-effects models with random effects for each pulmonary metastasis and each patient were used to estimate pulmonary metastasis growth rate, evaluating the impact of patient age, tumor size, tumor grade, chemotherapy and tumor subtype. We estimated the pulmonary metastasis volume doubling time using these analyses. RESULTS: Maximal primary tumor size at diagnosis (LRT statistic = 2.58, df = 2, p = 0.275), tumor grade (LRT statistic = 1.13, df = 2, p = 0.567), tumor type (LRT statistic = 7.59, df = 6, p = 0.269), and patient age at diagnosis (LRT statistic = 0.735, df = 2, p = 0.736) were not statistically significant predictors of pulmonary nodule growth from baseline values. Chemotherapy decreased the rate of pulmonary nodule growth from baseline (LRT statistic = 7.96, df = 2, p = 0.0187). 95% of untreated pulmonary metastases are expected to grow less than 6 mm in 6.4 months. There was significant intrapatient and interpatient variation in pulmonary metastasis growth rate. Pulmonary metastasis volume growth rate was best fit with an exponential model in time. The volume doubling time for pulmonary metastases assuming an exponential model in time was 143 days (95% CI (104, 231) days). CONCLUSIONS: Assuming a 2 mm nodule is the smallest reliably detectable nodule by CT, the data suggest that an untreated pulmonary metastasis is expected to grow to 8 mm in 8.4 months (95% CI (4.9, 10.2) months). Tumor size, grade and sarcoma subtype did not significantly alter pulmonary metastasis growth rate. However, chemotherapy slowed the pulmonary metastasis growth rate. ADVANCES IN KNOWLEDGE: CT surveillance intervals for pulmonary metastases can be estimated based on metastasis growth rate. There was significant variation in the pulmonary metastasis growth rate between metastases within patient and between patients. Pulmonary nodule volume growth followed an exponential model, linear in time.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Can Dendritic Cell Vaccination Prevent Leukemia Relapse?

Leukemias are clonal proliferative disorders arising from immature leukocytes in the bone marrow. While the advent of targeted therapies has improved survival in certain subtypes, relapse after initial therapy is a major problem. Dendritic cell (DC) vaccination has the potential to induce tumor-specific T cells providing long-lasting, anti-tumor immunity. This approach has demonstrated safety but limited clinical success until recently, as DC vaccination faces several barriers in both solid and hematological malignancies. Importantly, vaccine-mediated stimulation of protective immune responses is hindered by the aberrant production of immunosuppressive factors by cancer cells which impede both DC and T cell function. Leukemias present the additional challenge of severely disrupted hematopoiesis owing to both cytogenic defects in hematopoietic progenitors and an abnormal hematopoietic stem cell niche in the bone marrow; these factors accentuate systemic immunosuppression and DC malfunction. Despite these obstacles, several recent clinical trials have caused great excitement by extending survival in Acute Myeloid Leukemia (AML) patients through DC vaccination. Here, we review the phenotype and functional capacity of DCs in leukemia and approaches to harness DCs in leukemia patients. We describe the recent clinical successes in AML and detail the multiple new strategies that might enhance prognosis in AML and other leukemias.

Maternal and infant outcomes following third trimester exposure to marijuana in opioid dependent pregnant women maintained on buprenorphine.

BACKGROUND: To determine whether maternal and infant outcomes are associated with exposure to marijuana during the third trimester in a population of opioid dependent pregnant women maintained on buprenorphine. METHODS: This retrospective cohort study of 191 maternal-infant dyads exposed to buprenorphine during pregnancy examines a variety of variables including gestational age, birthweight, method of delivery, Apgar scores at one and five minutes, duration of infant hospital stay, peak neonatal abstinence syndrome (NAS) score, duration of NAS and incidence of pharmacologic treatment of NAS in infants exposed to marijuana during the third trimester as compared to infants not exposed to marijuana during the third trimester. RESULTS: Analyses failed to support any significant relationship between marijuana use in the third trimester and a variety of maternal and infant outcomes. Two important variables - the likelihood of requiring pharmacologic treatment for NAS (27.6% in marijuana exposed infants vs. 15.7% in non-marijuana exposed infants, p=0.066) and the duration of infant hospital stay (7.7days in marijuana exposed infants vs. 6.6days in non-exposed infants, p=0.053) trended toward significance. CONCLUSIONS: Preliminary results indicate that marijuana exposure in the third trimester does not complicate the pregnancy or the delivery process. However, the severity of the infant withdrawal syndrome in the immediate postnatal period may be impacted by marijuana exposure. Because previous study of prenatal marijuana exposure has yielded mixed results, further analysis is needed to determine whether these findings are indeed significant.

Assessment of trunk muscle density using CT and its association with degenerative disc and facet joint disease of the lumbar spine.

OBJECTIVE: The purpose of this study was (1) to evaluate the association of trunk muscle density assessed by computed tomography (CT) with age, gender, and BMI and (2) to evaluate the association between trunk muscle CT density and degenerative disc and facet joint disease of the lumbar spine. MATERIAL AND METHODS: The study was IRB approved and HIPAA compliant. The study group comprised 100 subjects (mean age 44.4 ± 22.2 years, 51 % male) who underwent CT of the abdomen and pelvis without intravenous contrast. Exclusion criteria included prior abdominal or spine surgery, active malignancy and scoliosis. CTs were reviewed and the attenuation of the rectus abdominis, transverse abdominis, internal and external obliques, psoas, multifidus, longissimus and gluteus maximus were measured bilaterally at consistent levels. Degenerative disc and bilateral facet joint disease were scored using established methods. Univariate analyses were performed using linear regression. Multivariate linear regression was performed to adjust for age, gender and BMI. RESULTS: CT density of each trunk muscle correlated inversely with age (p < 0.001) and BMI (p < 0.001). CT density of each trunk muscle correlated inversely with degenerative disc and facet joint disease in the univariate analyses (p < 0.001); however, only the gluteus maximus and the transverse abdominis remained significant predictors of degenerative disc and facet joint disease respectively in the multivariate analysis. CONCLUSION: Fatty infiltration of trunk musculature increases with age and BMI. Fatty infiltration of the gluteus maximus and transverse abdominis are associated with degenerative disc and facet joint disease, independent of age, gender and BMI.

Impact of a school health coordinator intervention on health-related school policies and student behavior.

BACKGROUND: Health-related, school-based interventions may serve to prevent disease and improve academic performance. The Healthy Maine Partnerships (HMP) initiative funded local school health coordinators (SHCs) as a part of Maine's Coordinated School Health Program (CSHP) beginning in January 2001. SHCs established school health leadership teams and implemented annual work plans to address health risk behaviors. This study evaluates the impact of the Healthy Maine Partnerships SHC (HMPSHC) intervention on school policies and student risk behaviors after its first 5 years. METHODS: Data sources include the Maine School Health Profiles Survey and the Maine Youth Drug and Alcohol Use Survey/Youth Tobacco Survey. Cross-sectional analyses were performed on 2006 data to assess physical activity, nutrition, and tobacco-related policy associations with the HMPSHC intervention. Finally, policy and student behavior analyses were conducted to assess associations. RESULTS: Intervention schools were more likely to be associated with physical activity intramural offerings, improved nutritional offerings, and tobacco cessation programs. In intervention schools, supportive school policies were associated with decreased soda consumption, decreased inactivity, and decreased tobacco use. Required school health education curricula were more predictive of decreased risk behavior in intervention schools than in nonintervention schools. CONCLUSIONS: In schools with SHCs, there exists a stronger association with improved school programs. Improved policies and programs were associated with decreases in risk behavior among students in intervention schools. The HMPSHC intervention may be a viable CSHP model to replicate and evaluate in other settings.