Patient-Derived Orthotopic Xenograft Models for High-Grade Pediatric Brain Cancers.

Patient-derived orthotopic xenograft (PDOX) mouse models are considered the gold standard for evidence-based preclinical research in pediatric neuro-oncology. This protocol describes the generation of PDOX models by intracranial implantation of human pediatric brain cancer cells into immune-deficient mice, and their continued propagation to establish cohorts of animals for preclinical research.

PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens.

The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.

Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αv ß3 -targeted iron oxide microparticles.

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly antiangiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a Arg-Gly-Asp peptide (RGD) targeting integrin αv ß3 . The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastases in vivo. A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T2 *-weighted imaging was used to visualise MPIO-induced hypointense voxels in vivo, and Prussian blue staining was used to visualise MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualising angiogenic vessels per se was reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. While pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our preliminary findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.

Veliparib Is an Effective Radiosensitizing Agent in a Preclinical Model of Medulloblastoma.

Medulloblastoma is the most common malignant childhood brain tumor, and 5-year overall survival rates are as low as 40% depending on molecular subtype, with new therapies critically important. As radiotherapy and chemotherapy act through the induction of DNA damage, the sensitization of cancer cells through the inhibition of DNA damage repair pathways is a potential therapeutic strategy. The poly-(ADP-ribose) polymerase (PARP) inhibitor veliparib was assessed for its ability to augment the cellular response to radiation-induced DNA damage in human medulloblastoma cells. DNA repair following irradiation was assessed using the alkaline comet assay, with veliparib inhibiting the rate of DNA repair. Veliparib treatment also increased the number of γH2AX foci in cells treated with radiation, and analysis of downstream pathways indicated persistent activation of the DNA damage response pathway. Clonogenicity assays demonstrated that veliparib effectively inhibited the colony-forming capacity of medulloblastoma cells, both as a single agent and in combination with irradiation. These data were then validated in vivo using an orthotopic implant model of medulloblastoma. Mice harboring intracranial D425 medulloblastoma xenografts were treated with vehicle, veliparib, 18 Gy multifractionated craniospinal irradiation (CSI), or veliparib combined with 18 Gy CSI. Animals treated with combination therapy exhibited reduced tumor growth rates concomitant with increased intra-tumoral apoptosis observed by immunohistochemistry. Kaplan-Meier analyses revealed a statistically significant increase in survival with combination therapy compared to CSI alone. In summary, PARP inhibition enhanced radiation-induced cytotoxicity of medulloblastoma cells; thus, veliparib or other brain-penetrant PARP inhibitors are potential radiosensitizing agents for the treatment of medulloblastoma.

PD-1 restraint of regulatory T cell suppressive activity is critical for immune tolerance.

Inhibitory signals through the PD-1 pathway regulate T cell activation, T cell tolerance, and T cell exhaustion. Studies of PD-1 function have focused primarily on effector T cells. Far less is known about PD-1 function in regulatory T (T reg) cells. To study the role of PD-1 in T reg cells, we generated mice that selectively lack PD-1 in T reg cells. PD-1-deficient T reg cells exhibit an activated phenotype and enhanced immunosuppressive function. The in vivo significance of the potent suppressive capacity of PD-1-deficient T reg cells is illustrated by ameliorated experimental autoimmune encephalomyelitis (EAE) and protection from diabetes in nonobese diabetic (NOD) mice lacking PD-1 selectively in T reg cells. We identified reduced signaling through the PI3K-AKT pathway as a mechanism underlying the enhanced suppressive capacity of PD-1-deficient T reg cells. Our findings demonstrate that cell-intrinsic PD-1 restraint of T reg cells is a significant mechanism by which PD-1 inhibitory signals regulate T cell tolerance and autoimmunity.

The Role of Executive Functions in Socioeconomic Attainment Gaps: Results From a Randomized Controlled Trial.

The socioeconomic attainment gap in mathematics starts early and increases over time. This study aimed to examine why this gap exists. Four-year-olds from diverse backgrounds were randomly allocated to a brief intervention designed to improve executive functions (N = 87) or to an active control group (N = 88). The study was preregistered and followed CONSORT guidelines. Executive functions and mathematical skills were measured at baseline, 1 week, 3 months, 6 months, and 1 year posttraining. Executive functions mediated the relation between socioeconomic status and mathematical skills. Children improved over training, but this did not transfer to untrained executive functions or mathematics. Executive functions may explain socioeconomic attainment gaps, but cognitive training directly targeting executive functions is not an effective way to narrow this gap.

Sensitivity of Multiphase Pseudocontinuous Arterial Spin Labelling (MP pCASL) Magnetic Resonance Imaging for Measuring Brain and Tumour Blood Flow in Mice.

Brain and tumour blood flow can be measured noninvasively using arterial spin labelling (ASL) magnetic resonance imaging (MRI), but reliable quantification in mouse models remains difficult. Pseudocontinuous ASL (pCASL) is recommended as the clinical standard for ASL and can be improved using multiphase labelling (MP pCASL). The aim of this study was to optimise and validate MP pCASL MRI for cerebral blood flow (CBF) measurement in mice and to assess its sensitivity to tumour perfusion. Following optimization of the MP pCASL sequence, CBF data were compared with gold-standard autoradiography, showing close agreement. Subsequently, MP pCASL data were acquired at weekly intervals in models of primary and secondary brain tumours, and tumour microvessel density was determined histologically. MP pCASL measurements in a secondary brain tumour model revealed a significant reduction in blood flow at day 35 after induction, despite a higher density of blood vessels. Tumour core regions also showed reduced blood flow compared with the tumour rim. Similarly, significant reductions in CBF were found in a model of glioma 28 days after tumour induction, together with an increased density of blood vessels. These findings indicate that MP pCASL MRI provides accurate and robust measurements of cerebral blood flow in naïve mice and is sensitive to changes in tumour perfusion.

Cervical length in adolescents versus adults.

PURPOSE: Two of the known risk factors for spontaneous preterm birth (sPTB) are short cervical length (CL) ≤ 25 mm and adolescence (≤19 years). Our objective was to evaluate whether adolescent women have a higher incidence of short CL compared to their 20-24 year old counterparts. MATERIALS AND METHODS: Retrospective cohort of nulliparous singleton gestations undergoing universal second trimester transvaginal ultrasound (TVU) CL screening between January 2012 and June 2013. Adolescent women ≤19 years of age were compared to women 20-24 years of age. Primary outcomes were mean CL and incidence of CL ≤25 mm. Secondary outcomes were incidence of PTB <37 weeks, delivery mode, birth weight, and NICU admission. RESULTS: One hundred and five adolescents and 236 women 20-24 years underwent TVU CL screening. There was no difference in mean CL (40.6 mm vs. 40.6 mm, p = 0.51) or incidence of CL ≤25 mm (1.0% vs. 1.7%; OR 0.56 [0.06-5.1]). After controlling for maternal differences, there still was no significant correlation between maternal age and CL. There was no significant difference in PTB, birth weight, or NICU admission between the groups. CL measurements did not significantly differ across all maternal ages (14-42 years). CONCLUSIONS: There is no difference in mean CL or incidence of CL ≤25 mm among adolescents compared to women 20-24 years.

Racial disparities in cervical length for prediction of preterm birth in a low risk population.

PURPOSE: Racial minorities experience higher rates of spontaneous preterm birth (sPTB). Our objective was to evaluate whether there are racial discrepancies in the incidence of second trimester short cervical length (≤25 mm). MATERIALS AND METHODS: Retrospective cohort of women with singleton gestations without prior sPTB undergoing universal second trimester transvaginal ultrasound cervical length (CL) screening between January 2012 and December 2013. Black women were compared to non-Hispanic white women. Our primary outcome was the incidence of CL ≤25 mm. Secondary outcomes were incidence of PTB ≤37 weeks, delivery mode, birth weight and neonatal intensive care unit (NICU) admission. RESULTS: Black women (n = 1092) differed from non-Hispanic white women (n = 659) with respect to maternal age (26.0 versus 30.7 years), gravidity (3.1 versus 2.1), prepregnancy BMI (29.6 versus 25.0 kg/m2), and smoking status (9.8% versus 16%), respectively (p < 0.001). Black women had higher incidence of CL ≤25 mm (1.9% versus 0.6%; OR: 3.21 [1.1-9.4]), rates of sPTB (8.5% versus 4.4%; aOR: 1.95 [1.1-3.4]), incidence of low birthweight infants (<2500 g, 8.3% versus 5.6%; aOR 1.80 [1.1-3.0]) and were more likely to have their infants admitted to the NICU (16% versus 11%; OR: 1.52 [1.0-2.3]). CONCLUSIONS: Black women had a 2.8-fold increased risk of CL ≤25 mm compared non-Hispanic white women in a low-risk population.

The concomitant use of fesoterodine and topical vaginal estrogen in the management of overactive bladder and sexual dysfunction in postmenopausal women.

OBJECTIVE: The objective of this study is to investigate the combination effect of anti-muscarinic medication and topical vaginal estrogen in the treatment of overactive bladder (OAB) and female sexual dysfunction in postmenopausal women. STUDY DESIGN: After IRB approval, 23 female subjects who met the entry criteria were randomized into two groups: (1) fesoterodine (Toviaz®, Pfizer, NY) with topical vaginal estrogen (Premarin®, Pfizer, NY) once daily or (2) fesoterodine once daily alone. If 4 mg fesoterodine was tolerated at 1-week, the dose was increased to 8 mg. MAIN OUTCOME MEASURES: Primary endpoints were improvement in OAB symptom severity (Overactive Bladder Questionnaire, OAB-Q SF), improvement in OAB health-related quality of life (HRQL) (OAB-Q SF), and sexual function (Sexual Quality of Life-Female, SQOL-F) after 12 weeks. Secondary endpoint was change in total number of micturitions. RESULTS: After 12-weeks, the combination group had a significant improvement in OAB symptom severity (p = 0.006), HRQL (p = 0.029), and SQOL-F (0.0003). The fesoterodine alone group also had significant improvement in OAB symptom severity (p < 0.0001), HRQL (p = 0.0002), and SQOL-F (p = 0.02). When compared directly to the fesoterodine alone group, the combination group after 12-weeks had a reduced OAB symptom severity (10 versus 23.3; p = 0.35), higher HRQL (96.9 versus 84.6; p = 0.75), and higher SQOL-F (99 versus 81; p = 0.098). The total number of micturitions over 3 d was significantly reduced in the combination group (45-26, p = 0.03) between baseline and 12-weeks. CONCLUSIONS: The combined effect of fesoterodine and topical vaginal estrogen improved OAB symptoms and sexual function in postmenopausal women.

Trends and Reinterventions in the Surgical Management of Stress Urinary Incontinence among Female Medicare Beneficiaries.

INTRODUCTION: We compare the use of bulking agents and slings for the treatment of stress urinary incontinence among female Medicare beneficiaries. METHODS: We analyzed data from a 5% national random sample of Medicare claims from 2000 to 2011. Female beneficiaries who underwent a sling or bulking agent procedure were identified based on CPT-4 and ICD-9 procedure codes. Statistical analysis for categorical data determined differences in the distribution of patient demographics and comorbidities. The 90-day adverse events and reinterventions were compared between treatment groups. Time to event analysis was used to determine freedom from reintervention after therapy. RESULTS: We identified 21,134 and 3,475 patients treated with sling and bulking procedures, respectively. There was a 29.7% increase in the number of sling procedures and a 59.5% decrease in bulking procedures from 2001 to 2011. Patients treated with bulking agents had higher rates of diabetes, cardiovascular disease, heart failure and renal failure (p <0.01). The 90-day adverse events after both procedures were rare, with the exception of urinary retention, which was increased in women treated with a sling but frequent in both groups (sling 11.3%, bulking agent 8.4%; p <0.01). A smaller proportion of patients who underwent sling surgery had reinterventions (repeat sling 7.4%, bulking agent 38.2%; p <0.01). Overall 53.2% of the patients treated with a sling and 76.3% treated with bulking agents who underwent subsequent procedures were treated with the same procedure at the first intervention. CONCLUSIONS: Sling and bulking procedures are safe in terms of short-term performance, although the rates of retention were high in both groups. Patients treated with reinterventions tend to repeat the same therapy instead of converting to another procedure.

Use and risks of surgical mesh for pelvic organ prolapse surgery in women in New York state: population based cohort study.

OBJECTIVE: To assess the use of mesh in pelvic organ prolapse surgery, and compare short term outcomes between procedures using and not using mesh. DESIGN: All inclusive, population based cohort study. SETTING: Statewide surgical care captured in the New York Statewide Planning and Research Cooperative System. PARTICIPANTS: Women who underwent prolapse repair procedures in New York state from 2008 to 2011. MAIN OUTCOMES MEASURES: 90 day safety events and reinterventions within one year, after propensity score matching. Categorical, time to event, and subgroup analyses (<65 and ≥ 65 year age groups) were conducted. RESULTS: Of 27,991 patients in total, 7338 and 20, 653 underwent prolapse repair procedures with and without mesh, respectively. Mesh use increased by 44.7%, from 1461 procedures in 2008 to 2114 procedures in 2011. Most patients in the cohort were younger than 65 years (62.3% (n=17,424/27, 991)). However, more patients were aged 65 years and older in the mesh group than in the non-mesh group (44.3% (n=3249) v 35.4% (n=7318)). Complications after surgery were not common, irrespective of the use or non-use of mesh. After propensity score matching, patients who received the surgery with mesh had a higher chance of having a reintervention within one year (mesh 3.3% v no mesh 2.2%, hazard ratio 1.47 (95% confidence interval 1.21 to 1.79)) and were more likely to have urinary retention within 90 days (mesh 7.5% v no mesh 5.6%, risk ratio 1.33 (95% confidence interval 1.18 to 1.51)), compared with those who received surgery without mesh. In subgroup analyses based on age, mesh use was associated with an increased risk of reintervention within one year in patients under age 65 years, and increased risk of urinary retention in patients aged 65 years and over. CONCLUSIONS: Despite multiple warnings released by the US Food and Drug Administration since 2008, use of mesh in pelvic organ prolapse surgery continues to grow. In this statewide comprehensive study, mesh procedures were associated with an increased risk of reinterventions within one year and urinary retention after surgery.

Lipid and Metabolite Deregulation in the Breast Tissue of Women Carrying BRCA1 and BRCA2 Genetic Mutations.

PURPOSE: To use localized correlated spectroscopy (COSY) to assess for an altered biochemical state or states in breast tissue of women with BRCA gene mutations that potentially constitute preinvasive conditions. MATERIALS AND METHODS: Institutional review board approval was obtained. Participants provided written informed consent. In vivo localized COSY images were recorded at 3 T in the breast tissue of women carrying BRCA1 (n = 9) or BRCA2 (n = 14) gene mutations and were compared with images in healthy control subjects with no family history of breast cancer (n = 10). All participants underwent contrast material-enhanced MR imaging and ultrasonography (US). Statistical significance was calculated with the Mann-Whitney two-sided nonparametric test. RESULTS: No abnormality was recorded with MR imaging or US. Metabolite levels in the BRCA1 cohort were reduced by 79% (P = .014) when compared with triglycerides level, and there was a 19% increase in lipid unsaturation and triglyceride levels (P = .027 and P = .086, respectively) when compared with cellular cholesterol level. Cholesterol level was reduced by 47% (P = .027) when compared with diallylic lipid level. Metabolite levels in the BRCA2 cohort showed increased unsaturation of 21% (P = .030) relative to triglycerides level. Comparison of the BRCA1 and BRCA2 cohorts showed a 47% (P = .002) increase in cholesterol level in the BRCA2 cohort when compared with diallylic lipid level and a 52% (P = .003) increase when compared with triglycerides level. Levels of diallylic lipid, unsaturated lipid, triglycerides, and terminal methyl on the acyl chain are reduced by 46% (P = .002), 57% (P = .003), 66% (P = .003), and 29% (P = .010), respectively, when compared with cholesterol level. CONCLUSION: Localized COSY recorded significant changes in women with BRCA1 and BRCA2 gene mutations when compared with control subjects. If these changes are ultimately proven to be a premalignant stage, this method may prove useful in screening.

Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model.

RATIONALE: Yes-associated protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. OBJECTIVE: We hypothesized that YAP activation after myocardial infarction (MI) would preserve cardiac function and improve survival. METHODS AND RESULTS: We used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after MI preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP (hYAP) in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated expression of cell cycle genes and promoted a less mature cardiac gene expression signature. CONCLUSIONS: Cardiac-specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.

Prostate cancer: predicting high-risk prostate cancer-a novel stratification tool.

Currently, numerous systems exist for the identification of high-risk prostate cancer, but few of these systems can guide treatment strategies. A new stratification tool that uses common diagnostic factors can help to predict outcomes after radical prostatectomy. The tool aids physicians in the identification of appropriate candidates for aggressive, local treatment.

YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy.

Heart growth is tightly controlled so that the heart reaches a predetermined size. Fetal heart growth occurs through cardiomyocyte proliferation, whereas postnatal heart growth involves primarily physiological cardiomyocyte hypertrophy. The Hippo kinase cascade is an important regulator of organ growth. A major target of this kinase cascade is YAP1, a transcriptional coactivator that is inactivated by Hippo kinase activity. Here, we used both genetic gain and loss of Yap1 function to investigate its role in regulating proliferative and physiologic hypertrophic heart growth. Fetal Yap1 inactivation caused marked, lethal myocardial hypoplasia and decreased cardiomyocyte proliferation, whereas fetal activation of YAP1 stimulated cardiomyocyte proliferation. Enhanced proliferation was particularly dramatic in trabecular cardiomyocytes that normally exit from the cell cycle. Remarkably, YAP1 activation was sufficient to stimulate proliferation of postnatal cardiomyocytes, both in culture and in the intact heart. A dominant negative peptide that blocked YAP1 binding to TEAD transcription factors inhibited YAP1 proliferative activity, indicating that this activity requires YAP1-TEAD interaction. Although Yap1 was a critical regulator of cardiomyocyte proliferation, it did not influence physiological hypertrophic growth of cardiomyocytes, because postnatal Yap1 gain or loss of function did not significantly alter cardiomyocyte size. These studies demonstrate that Yap1 is a crucial regulator of cardiomyocyte proliferation, cardiac morphogenesis, and myocardial trabeculation. Activation of Yap1 in postnatal cardiomyocytes may be a useful strategy to stimulate cardiomyocyte expansion in therapeutic myocardial regeneration.

Community-associated methicillin-resistant Staphylococcus aureus: trends in case and isolate characteristics from six years of prospective surveillance.

OBJECTIVES: In 2000, the Minnesota Department of Health (MDH) implemented active, sentinel site surveillance for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Data from 2000-2005 were analyzed to determine trends in case characteristics, pulsed-field types (PFTs), and antimicrobial susceptibilities including inducible clindamycin resistance (ICR). METHODS: Active sentinel site surveillance was initiated in 2000 at 12 hospital laboratories that served inpatients and outpatients. Patient medical records were reviewed to determine if they met the epidemiologic case criteria for CA-MRSA; isolates were obtained from patients meeting these criteria. The MDH Public Health Laboratory performed pulsed-field gel electrophoresis subtyping and antimicrobial susceptibility testing, including ICR. RESULTS: The proportion of MRSA cases classified as CA increased from 11% to 33% (p<0.01). The proportion of cases with skin or soft tissue infections also increased compared with other infection types from 75% to 87% (p<0.01). During the surveillance period, USA300 replaced USA400 as the dominant PFT. With the change in dominant PFT, the proportion of isolates susceptible to erythromycin (45% to 13%, p<0.01) and ciprofloxacin (80% to 59%, p<0.01) decreased. The proportion of erythromycin-resistant/clindamycin-susceptible isolates with ICR (93% to 14%, p<0.01) decreased. The proportion of susceptible isolates also changed within the USA300 PFT; the proportion of isolates susceptible to erythromycin (33% vs. 3%) and the proportion susceptible to ciprofloxacin (67% to 62%) decreased significantly. CONCLUSION: CA-MRSA increased dramatically from 2000 to 2005. Changes in the predominant PFT have impacted susceptibility profiles of CA-MRSA, including ICR. Continued surveillance is needed to monitor the changing epidemiology of CA-MRSA and to inform clinical decisions.

HIV and obstetric complications and fetal outcomes in Vellore, India.

Antenatal prevalence is more than 1% in parts of India, yet little is known about the complications and fetal outcomes in this region. We reviewed the records of 23,386 women who delivered at the Christian Medical College Hospital in Vellore, India from 2000 through 2002. HIV-infected women were more likely than HIV-uninfected women to have pregnancy-induced hypertension, anaemia, breech presentations, stillborn babies and fetal deaths. HIV-infected women who did not receive mother-to-child transmission prophylaxis or had breech fetal presentation were more likely to have fetal deaths (P = 0.001). HIV prophylaxis and optimal prenatal care should be a priority for HIV-infected pregnant women in resource-limited countries.