Limitations of Noninvasive Tests-Based Population-Level Risk Stratification Strategy for Nonalcoholic Fatty Liver Disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are highly prevalent but underdiagnosed. AIMS: We used an electronic health record data network to test a population-level risk stratification strategy using noninvasive tests (NITs) of liver fibrosis. METHODS: Data were obtained from PCORnet® sites in the East, Midwest, Southwest, and Southeast United States from patients aged [Formula: see text] 18 with or without ICD-10-CM diagnosis codes for NAFLD, NASH, and NASH-cirrhosis between 9/1/2017 and 8/31/2020. Average and standard deviations (SD) for Fibrosis-4 index (FIB-4), NAFLD fibrosis score (NFS), and Hepatic Steatosis Index (HSI) were estimated by site for each patient cohort. Sample-wide estimates were calculated as weighted averages across study sites. RESULTS: Of 11,875,959 patients, 0.8% and 0.1% were coded with NAFLD and NASH, respectively. NAFLD diagnosis rates in White, Black, and Hispanic patients were 0.93%, 0.50%, and 1.25%, respectively, and for NASH 0.19%, 0.04%, and 0.16%, respectively. Among undiagnosed patients, insufficient EHR data for estimating NITs ranged from 68% (FIB-4) to 76% (NFS). Predicted prevalence of NAFLD by HSI was 60%, with estimated prevalence of advanced fibrosis of 13% by NFS and 7% by FIB-4. Approximately, 15% and 23% of patients were classified in the intermediate range by FIB-4 and NFS, respectively. Among NAFLD-cirrhosis patients, a third had FIB-4 scores in the low or intermediate range. CONCLUSIONS: We identified several potential barriers to a population-level NIT-based screening strategy. HSI-based NAFLD screening appears unrealistic. Further research is needed to define merits of NFS- versus FIB-4-based strategies, which may identify different high-risk groups.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo.

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

Functional Comparison of HBZ and the Related APH-2 Protein Provides Insight into Human T-Cell Leukemia Virus Type 1 Pathogenesis.

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that transform T cells in vitro but have distinct pathological outcomes in vivo. HTLV-1 encodes a protein from the antisense strand of its proviral genome, the HTLV-1 basic leucine zipper factor (HBZ), which inhibits Tax-1-mediated viral transcription and promotes cell proliferation, a high proviral load, and persistence in vivo. In adult T-cell leukemia/lymphoma (ATL) cell lines and patient T cells, hbz is often the only viral gene expressed. The antisense strand of the HTLV-2 proviral genome also encodes a protein termed APH-2. Like HBZ, APH-2 is able to inhibit Tax-2-mediated viral transcription and is detectable in most primary lymphocytes from HTLV-2-infected patients. However, unlike HBZ, the loss of APH-2 in vivo results in increased viral replication and proviral loads, suggesting that HBZ and APH-2 modulate the virus and cellular pathways differently. Herein, we examined the effect of APH-2 on several known HBZ-modulated pathways: NF-κB (p65) transactivation, transforming growth factor ß (TGF-ß) signaling, and interferon regulatory factor 1 (IRF-1) transactivation. Like HBZ, APH-2 has the ability to inhibit p65 transactivation. Conversely, HBZ and APH-2 have divergent effects on TGF-ß signaling and IRF-1 transactivation. Quantitative PCR and protein half-life experiments revealed a substantial disparity between HBZ and APH-2 transcript levels and protein stability, respectively. Taken together, our data further elucidate the functional differences between HBZ and APH-2 and how these differences can have profound effects on the survival of infected cells and, ultimately, pathogenesis. IMPORTANCE: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are highly related retroviruses that have distinct pathological outcomes in infected hosts. Functional comparisons of HTLV-1 and HTLV-2 proteins provide a better understanding about how HTLV-1 infection is associated with disease and HTLV-2 infection is not. The HTLV genome antisense-strand genes hbz and aph-2 are often the only viral genes expressed in HTLV-infected T cells. Previously, our group found that HTLV-1 HBZ and HTLV-2 APH-2 had distinct effects in vivo and hypothesized that the differences in the interactions of HBZ and APH-2 with important cell signaling pathways dictate whether cells undergo proliferation, apoptosis, or senescence. Ultimately, these functional differences may affect how HTLV-1 causes disease but HTLV-2 generally does not. In the current study, we compared the effects of HBZ and APH-2 on several HTLV-relevant cellular pathways, including the TGF-ß signaling, NF-κB activation, and IRF-1 transactivation pathways.

Pten in stromal fibroblasts suppresses mammary epithelial tumours.

The tumour stroma is believed to contribute to some of the most malignant characteristics of epithelial tumours. However, signalling between stromal and tumour cells is complex and remains poorly understood. Here we show that the genetic inactivation of Pten in stromal fibroblasts of mouse mammary glands accelerated the initiation, progression and malignant transformation of mammary epithelial tumours. This was associated with the massive remodelling of the extracellular matrix (ECM), innate immune cell infiltration and increased angiogenesis. Loss of Pten in stromal fibroblasts led to increased expression, phosphorylation (T72) and recruitment of Ets2 to target promoters known to be involved in these processes. Remarkably, Ets2 inactivation in Pten stroma-deleted tumours ameliorated disruption of the tumour microenvironment and was sufficient to decrease tumour growth and progression. Global gene expression profiling of mammary stromal cells identified a Pten-specific signature that was highly represented in the tumour stroma of patients with breast cancer. These findings identify the Pten-Ets2 axis as a critical stroma-specific signalling pathway that suppresses mammary epithelial tumours.

Mouse development with a single E2F activator.

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a(3bki) or E2f3a(1ki), respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Evaluating fentanyl test strips as a harm reduction strategy in rural and urban counties: study protocol for a randomized controlled trial.

BACKGROUND: Opioid-related fatalities are a leading cause of death in Ohio and nationally, with an increasing number of overdoses attributable to fentanyl. Rapid fentanyl test strips can identify fentanyl and some fentanyl analogs in urine samples and are increasingly being used to check illicit drugs for fentanyl before they are used. Fentanyl test strips are a promising harm reduction strategy; however, little is known about the real-world acceptability and impact of fentanyl test strip use. This study investigates fentanyl test strip distribution and education as a harm reduction strategy to prevent overdoses among people who use drugs. METHODS: The research team will recruit 2400 individuals ≥ 18 years with self-reported use of illicit drugs or drugs purchased on the street within the past 6 months. Recruitment will occur at opioid overdose education and naloxone distribution programs in 16 urban and 12 rural Ohio counties. Participating sites will be randomized at the county level to the intervention or non-intervention study arm. A brief fentanyl test strip educational intervention and fentanyl test strips will be provided to participants recruited from sites in the intervention arm. These participants will be eligible to receive additional fentanyl test strips for 2 years post-enrollment. Participants recruited from sites in the non-intervention arm will not receive fentanyl test strip education or fentanyl test strips. All participants will be followed for 2 years post-enrollment using biweekly, quarterly, and 6-month surveys. Primary outcomes include (1) identification of perceived barriers and facilitating factors associated with incorporating fentanyl test strip education and distribution into opioid overdose education and naloxone distribution programs; (2) differences in knowledge and self-efficacy regarding how to test drugs for fentanyl and strategies for reducing overdose risk between the intervention and non-intervention groups; and (3) differences in non-fatal and fatal overdose rates between the intervention and non-intervention groups. DISCUSSION: Findings from this cluster randomized controlled trial will contribute valuable information about the feasibility, acceptability, and impact of integrating fentanyl test strip drug checking in rural and urban communities in Ohio and help guide future overdose prevention interventions. TRIAL REGISTRATION: ClinicalTrials.gov NCT05463341. Registered on July 19, 2022. https://clinicaltrials.gov/study/NCT05463341.

The histone deacetylase inhibitor valproic acid lessens NK cell action against oncolytic virus-infected glioblastoma cells by inhibition of STAT5/T-BET signaling and generation of gamma interferon.

Tumor virotherapy has been and continues to be used in clinical trials. One barrier to effective viral oncolysis, consisting of the interferon (IFN) response induced by viral infection, is inhibited by valproic acid (VPA) and other histone deacetylase inhibitors (HDACi). Innate immune cell recruitment and activation have been shown to be deleterious to the efficacy of oncolytic herpes simplex virus (oHSV) infection, and in this report we demonstrate that VPA limits this deleterious response. VPA, administered prior to oHSV inoculation in an orthotopic glioblastoma mouse model, resulted in a decline in NK and macrophage recruitment into tumor-bearing brains at 6 and 24 h post-oHSV infection. Interestingly, there was a robust rebound of recruitment of these cells at 72 h post-oHSV infection. The observed initial decline in immune cell recruitment was accompanied by a reduction in their activation status. VPA was also found to have a profound immunosuppressive effect on human NK cells in vitro. NK cytotoxicity was abrogated following exposure to VPA, consistent with downmodulation of cytotoxic gene expression of granzyme B and perforin at the mRNA and protein levels. In addition, suppression of gamma IFN (IFN-γ) production by VPA was associated with decreased STAT5 phosphorylation and dampened T-BET expression. Despite VPA-mediated immune suppression, mice were not at significantly increased risk for HSV encephalitis. These findings indicate that one of the avenues by which VPA enhances oHSV efficacy is through initial suppression of immune cell recruitment and inhibition of inflammatory cell pathways within NK cells.

Allele-specific tumor spectrum in pten knockin mice.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

Analysis of Urine Drug Test Results From Substance Use Disorder Treatment Practices and Overdose Mortality Rates, 2013-2020.

Importance: Drug overdose deaths in the US are currently the highest ever recorded; data collected from public health surveillance sources can help to identify emerging drug use patterns associated with overdose mortality rates, but the time lag in results often limits utility. Urine drug testing (UDT) is one potentially underused source that could augment surveillance efforts through timely data collection. Objective: To evaluate the correlation between real-time UDT results from a proprietary national database and overdose mortality data from the National Vital Statistics System. Design, Setting, and Participants: This retrospective cross-sectional study included 500 000 urine specimens submitted for UDT by substance use disorder (SUD) treatment health care practices and collected between January 1, 2013, and December 31, 2020. Real-time UDT data were obtained from the Millennium Health proprietary national database, and overdose mortality data were obtained from the National Vital Statistics System of the Centers for Disease Control and Prevention (CDC WONDER). Specimens were analyzed for specific drugs in 5 categories (cocaine, heroin, methamphetamine, synthetic opioids, and other opioids) using liquid chromatography-tandem mass spectrometry. Participants were adults aged 18 years and older who provided urine specimens at SUD treatment practices. Exposures: Urine drug testing. Main Outcomes and Measures: The primary outcome was the correlation between UDT positivity rates and overdose mortality rates at national, state, and county levels. Univariate and multivariate regression models were also used to evaluate the association between state- and county-level overdose mortality and standardized UDT positivity rates. Results: Among 500 000 unique patient specimens collected from SUD treatment practices between 2013 and 2020, 288 534 specimens (57.7%) were from men, and the median age of the study population was 34 years (IQR, 17-51 years). On a national level, synthetic opioids and methamphetamine were highly correlated with overdose mortality (Spearman ρ = 0.96 for both). When synthetic opioids were coinvolved, methamphetamine (ρ = 0.98), heroin (ρ = 0.78), cocaine (ρ = 0.94), and other opioids (ρ = 0.83) were also highly correlated with overdose mortality. In the absence of synthetic opioids, all drug categories were highly correlated (ρ = 0.75 for other opioids, 0.81 for heroin, and 0.88 for methamphetamine), with the exception of cocaine (ρ = -0.37). Synthetic opioids (ρ = 0.77) and methamphetamine (ρ = 0.80) had the strongest state-level correlations over time, whereas other opioids had the lowest correlation for both total positivity (ρ = 0.31) and positivity in the absence of synthetic opioids (ρ = 0.23). In Ohio, county-level correlation was strongest for synthetic opioids (ρ = 0.71), followed by heroin (ρ = 0.69) and methamphetamine (ρ = 0.67). At the state level, the multivariate incidence rate ratio (IRR) for synthetic opioids was 1.16 (95% CI, 1.14-1.19; P < .001), and at the county level, the IRR was 1.13 (95% CI, 1.09-1.17; P < .001), suggesting that for every 1-SD increase in the UDT positivity rate, there were 16.2% and 12.8% increases, respectively, in monthly overdose deaths. Both methamphetamine (11.7% increase per 1-SD increase in UDT positivity rate; IRR, 1.12; 95% CI, 1.09-1.14; P < .001) and cocaine (5.1% increase per 1-SD increase in UDT positivity rate; IRR, 1.05; 95% CI, 1.03-1.07; P < .001) also had significant positive associations with mortality rates, but the effect sizes were smaller than that of synthetic opioids (IRR, 1.16). Conclusions and Relevance: In this study, UDT results were highly correlated with mortality rates at national, state, and county levels. These findings suggest that real-time UDT surveillance can help to quickly identify changes in drug use patterns that might inform targeted harm reduction strategies designed to prevent overdose deaths.

Performance of model-based vs. permutation tests in the HEALing (Helping to End Addiction Long-termSM) Communities Study, a covariate-constrained cluster randomized trial.

BACKGROUND: The HEALing (Helping to End Addiction Long-termSM) Communities Study (HCS) is a multi-site parallel group cluster randomized wait-list comparison trial designed to evaluate the effect of the Communities That Heal (CTH) intervention compared to usual care on opioid overdose deaths. Covariate-constrained randomization (CCR) was applied to balance the community-level baseline covariates in the HCS. The purpose of this paper is to evaluate the performance of model-based tests and permutation tests in the HCS setting. We conducted a simulation study to evaluate type I error rates and power for model-based and permutation tests for the multi-site HCS as well as for a subgroup analysis of a single state (Massachusetts). We also investigated whether the maximum degree of imbalance in the CCR design has an impact on the performance of the tests. METHODS: The primary outcome, the number of opioid overdose deaths, is count data assessed at the community level that will be analyzed using a negative binomial regression model. We conducted a simulation study to evaluate the type I error rates and power for 3 tests: (1) Wald-type t-test with small-sample corrected empirical standard error estimates, (2) Wald-type z-test with model-based standard error estimates, and (3) permutation test with test statistics calculated by the difference in average residuals for the two groups. RESULTS: Our simulation results demonstrated that Wald-type t-tests with small-sample corrected empirical standard error estimates from the negative binomial regression model maintained proper type I error. Wald-type z-tests with model-based standard error estimates were anti-conservative. Permutation tests preserved type I error rates if the constrained space was not too small. For all tests, the power was high to detect the hypothesized 40% reduction in opioid overdose deaths for the intervention vs. comparison group both for the overall HCS and the subgroup analysis of Massachusetts (MA). CONCLUSIONS: Based on the results of our simulation study, the Wald-type t-test with small-sample corrected empirical standard error estimates from a negative binomial regression model is a valid and appropriate approach for analyzing cluster-level count data from the HEALing Communities Study. TRIAL REGISTRATION: ClinicalTrials.gov http://www. CLINICALTRIALS: gov ; Identifier: NCT04111939.

Stromal p53 Regulates Breast Cancer Development, the Immune Landscape, and Survival in an Oncogene-Specific Manner.

Coevolution of tumor cells and adjacent stromal elements is a key feature during tumor progression; however, the precise regulatory mechanisms during this process remain unknown. Here, we show stromal p53 loss enhances oncogenic KrasG12D, but not ErbB2, driven tumorigenesis in murine mammary epithelia. Stroma-specific p53 deletion increases both epithelial and fibroblast proliferation in mammary glands bearing the KrasG12D oncogene in epithelia, while concurrently increasing DNA damage and/or DNA replication stress and decreasing apoptosis in the tumor cells proper. Normal epithelia was not affected by stromal p53 deletion. Tumors with p53-null stroma had a significant decrease in total, cytotoxic, and regulatory T cells; however, there was a significant increase in myeloid-derived suppressor cells, total macrophages, and M2-polarized tumor-associated macrophages, with no impact on angiogenesis or connective tissue deposition. Stroma-specific p53 deletion reprogrammed gene expression in both fibroblasts and adjacent epithelium, with p53 targets and chemokine receptors/chemokine signaling pathways in fibroblasts and DNA replication, DNA damage repair, and apoptosis in epithelia being the most significantly impacted biological processes. A gene cluster in p53-deficient mouse fibroblasts was negatively associated with patient survival when compared with two independent datasets. In summary, stroma-specific p53 loss promotes mammary tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival. IMPLICATIONS: Expression of the p53 tumor suppressor in breast cancer tumor stroma regulates tumorigenesis in an oncogene-specific manner, influences the tumor immune landscape, and ultimately impacts patient survival.

Development of a distributed international patient data registry for hairy cell leukemia.

Hairy cell leukemia (HCL) is a rare lymphoproliferative disorder, comprising only 2% of all leukemias. The Hairy Cell Leukemia Foundation (HCLF) has developed a patient data registry to enable investigators to better study the clinical features, treatment outcomes, and complications of patients with HCL. This system utilizes a centralized registry architecture. Patients are enrolled at HCL Centers of Excellence (COE) or via a web-based portal. All data are de-identified, which reduces regulatory burden and increases opportunities for data access and re-use. To date, 579 patients have been enrolled in the registry. Efforts are underway to engage additional COE's to expand access to patients across the globe. This international PDR will enable researchers to study outcomes in HCL in ways not previously possible due to the rarity of the disease and will serve as a platform for future prospective research.

Measles virotherapy in a mouse model of adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) is a highly aggressive CD4(+) T-cell malignancy caused by human T-cell leukaemia virus type 1. Measles virus (MV) oncolytic therapy has been reported to be efficient in reducing tumour burden in subcutaneous xenograft models of lymphoproliferative disorders such as myeloma, B-cell lymphoma and cutaneous T-cell lymphoma, but its potential to reduce tumour burden in disseminated lymphoproliferative disorders such as ATL remains to be determined. In this study, MV oncolytic therapy was evaluated in the MET-1/NOD/SCID xenograft mouse model of ATL. Treatment with the vaccine-related strain MV-NSE led to a significant reduction in tumour burden. In mice with a high tumour burden, therapy with MV-NSE significantly increased survival beyond any other single treatment tested previously using this model. Interestingly, signs of morbidity (cachexia) in mice treated with MV were not directly associated with tumour burden, but were correlated with the secretion of interleukin-6 by MET-1 cells and host cells. The results suggest that MV therapy could be a promising therapy for generalized lymphoproliferative disease.

Effects of short-term zoledronic acid treatment on bone remodeling and healing at surgical sites in the maxilla and mandible of aged dogs.

PURPOSE: It is unknown whether zoledronic acid (ZA) interferes with initial bone healing at extraction and implant sites. The goal of this study was to examine the effect of short-duration ZA on bone remodeling and healing after surgical insult in an aged dog model. MATERIALS AND METHODS: Four 2- to 3-year-old male dogs were administered ZA (0.1 mg/kg per month for 4 months), and 3 age-matched untreated dogs received no drug. In both groups, after the ZA-treated group had completed receiving the drug, the third premolar was extracted unilaterally and 2 orthodontic mini-implants per jaw per dog were placed on the ipsilateral side. After a 6-week healing period, a pair of calcein bone labels were administered. Bone sections from the mandible, maxilla, rib, and femur were obtained. The percent necrosis in the alveolar and basal regions of tooth-supporting bone was assayed by lactate dehydrogenase, and dynamic histomorphometric parameters were quantified and analyzed by use of mixed models. RESULTS: All extraction sites healed uneventfully, and no lesions resembling osteonecrosis were detected. The total percent necrosis was limited to less than 1% for all the bone sites examined. The ZA reduced bone remodeling at both surgical sites (extraction sites and mini-implant site) and nonsurgical sites. Although there was a significant (P < .05) increase in bone formation rate at the surgical sites in the untreated group, this increase was not significant (P = .3) in the ZA-treated group. CONCLUSIONS: Bone remodeling occurs in ZA-treated animals at surgical sites. ZA dramatically reduced bone turnover, but no exposed lesions resembling osteonecrosis developed at extraction and mini-implant sites after the 4-month drug duration.

Lower esophageal sphincter relaxation reflex kinetics: effects of peristaltic reflexes and maturation in human premature neonates.

We defined the sensory-motor characteristics of the lower esophageal sphincter relaxation (LESR) (stimulus threshold volume, response onset, and relaxation period, relaxation magnitude, nadir) during maturation in human neonates. We hypothesized that LESR kinetics differs during maturation and with peristaltic reflex type. Basal and adaptive esophageal motility testing was performed (N = 20 premature neonates) at 34.7 and 39.1 wk (time 1 and time 2). Effects of midesophageal provocation with graded stimuli (N = 1,267 stimuli, air and liquids) on LESR kinetics during esophagodeglutition response (EDR) and secondary peristalsis (SP) were analyzed by mixed models. Frequency of LESR with basal primary peristalsis were different during maturation (P = 0.03). During adaptive responses with maturation, 1) the frequencies of peristaltic reflexes and LESR were similar; 2) liquid stimuli resulted in a shorter LESR response latency and LESR nadir and greater LESR magnitude (all P < 0.05); 3) media differences were noted with LESR response latency (air vs. liquids, P < 0.02); and 4) infusion flow rate-LESR were different (P < 0.01 for air and liquids). Mechanistically, 1) frequency of LESR was greater during peristaltic reflexes at both times (vs. none, P < 0.0001); 2) LESR response latency, duration, and time to complete LESR were longer with EDR (all P < 0.05, vs. SP at time 2); and 3) graded stimulus volume LESR were different for air and liquids (P < 0.01). In conclusion, sensory-motor characteristics of LESR depend on the mechanosensitive properties of the stimulus (media, volume, flow), type of peristaltic reflex, and postnatal maturation. Maturation modulates an increased recruitment of inhibitory pathways that favor LESR.

Delayed administration of filgrastim (G-CSF) following autologous peripheral blood stem cell transplantation (APBSCT) in pediatric patients does not change time to neutrophil engraftment and reduces use of G-CSF.

BACKGROUND: Delayed initiation of granulocyte colony stimulating factor (G-CSF) after high-dose chemotherapy and autologous bone marrow or peripheral blood stem cell (APBSCT) in adult patients does not affect time to neutrophil or platelet engraftment, duration of fever, incidence of bacteremia, duration of non-prophylactic antibiotic therapy, and length of hospitalization when compared to early initiation. This study compares the effect of delayed (day +6) versus early (day +1) administration of G-CSF in pediatric patients on time to neutrophil engraftment (TNE), duration and cost of G-CSF therapy, incidence of blood stream infections, duration of febrile-neutropenia, duration of non-prophylactic antibiotic therapy, and duration of hospitalization due to febrile-neutropenia. METHODS: This is a retrospective review of 65 patients who engrafted after receiving APBSCT and G-CSF between 1993 and 2006. They were divided into the delayed group (day +6) (n = 46) and the early group (day +1) (n = 19). RESULTS: The median ages were 4.7 and 5.3 years in the early and delayed groups, respectively. There was no significant difference in TNE (P = 0.06) between the two groups. The duration of G-CSF administration was significantly less in the delayed group (P = 0.003). No significant differences were observed in the duration of neutropenia, time to platelet engraftment, the incidence of blood stream infections, and duration of fevers. Duration of hospitalization due to febrile-neutropenia was significantly lower in the delayed group (P = 0.01). Significant cost savings were observed by delaying G-CSF administration. CONCLUSION: Delayed administration of G-CSF after APBSCT in children has no adverse effect on TNE or other clinical outcomes when compared to early administration and may incur substantial cost savings.

Pediatric pedestrian injuries and associated hospital resource utilization in the United States, 2003.

BACKGROUND: The objective was to calculate national estimates of pedestrian-related hospitalizations and associated use of healthcare resources among children or=16. A traffic-related event was the cause of 92.7% of hospitalizations. Older age, higher injury severity, urban and Western hospital location, children's hospital type, and longer LOS were significantly associated with higher total hospital charges. Older age and higher injury severity were significantly associated with longer LOS. CONCLUSION: Pediatric pedestrian injuries contribute substantially to the healthcare resource burden in the United States, accounting for approximately 45,000 days of hospitalization and >USD 290 million in inpatient charges annually.

Preclinical Toxicology of rQNestin34.5v.2: An Oncolytic Herpes Virus with Transcriptional Regulation of the ICP34.5 Neurovirulence Gene.

rQNestin34.5v.2 is an oncolytic herpes simplex virus 1 (oHSV) that retains expression of the neurovirulent ICP34.5 gene under glioma-selective transcriptional regulation. To prepare an investigational new drug (IND) application, we performed toxicology and efficacy studies of rQNestin34.5v.2 in mice in the presence or absence of the immunomodulating drug cyclophosphamide (CPA). ICP34.5 allows HSV1 to survive interferon and improves viral replication by dephosphorylation of the eIF-2α translation factor. rQNestin34.5v.2 dephosphorylated eIF-2α in human glioma cells, but not in human normal cells, resulting in significantly higher cytotoxicity and viral replication in the former compared to the latter. In vivo toxicity of rQNestin34.5v.2 was compared with that of wild-type F strain in immunocompetent BALB/c mice and athymic mice by multiple routes of administration in the presence or absence of CPA. A likely no observed adverse effect level (NOAEL) dose for intracranial rQNestin34.5v.2 was estimated, justifying a phase 1 clinical trial in recurrent glioma patients (ClinicalTrials.gov: NCT03152318), after successful submission of an IND.

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: implications for transformation.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. RESULTS: We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1alpha (MIP-1alpha), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection. CONCLUSION: These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.