Addressing the Need For Establishment of School-Based Health Centers to Provide Reproductive Health Care in Philadelphia: A Meta-Analysis

OBJECTIVE: Currently, the communities of Philadelphia, which have predominantly African American residents, suffer from massive racial and ethnic health disparities. Children in these communities are at high risk of dropping out of school while also lacking access to reproductive health care. This myriad of unfortunate circumstances perpetuates the cycle of poverty that residents in these lowincome communities have been experiencing for decades. School-based health centers are a potential answer to this problem. The objective of this meta-analysis is to review published studies and carry out a meta-analysis to assess potential benefits and feasibility of SBHCs in the Philadelphia public school system as well any concerns that may arise with their provision. METHOD: A literature review and analysis was conducted through an online search of databases and government websites including Medline, PubMed, National Institute of Health, Google Scholar, National Center for Biotechnology Information, Center for Disease Control, and Google Scholar. A Google search for local credible news sources was also performed. The database searches were completed using the following search terms: SBHCs,”; “school-based health center”; “health inequity in schools”; “reproductive health care in schools . Abstracts were reviewed for relevance, and those remaining were critically reviewed. EXPECTED OUTCOME: The review of the relevant literature should reveal several benefits to implementing SBHCs as well as whether there is feasibility in establishing SBHCs in Philadelphia’s public school system. These findings can have an important implication for addressing the reproductive health care needs of underserved youth in low-income areas

The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Pretreatment with Naltrindole Generates Dose Dependent Effects on Reduction of Infarct Size During Myocardial Ischemia Reperfusion in An In-Vivo Model

Over one million individuals experience a myocardial infarction (MI) every year. Currently, no therapeutic is available to salvage necrotic cardiac tissue following an MI. The use of a prophylactic agent to reduce infarct size in patients at high risk for an MI would attenuate the development of heart failure. Interventional cardiologists state that a 25% reduction in infarct size is clinically meaningful to reduce heart failure. Previously, a 5 min pretreatment with high dose (HD) naltrindole (NTI) (7.5 mg/kg x 5 min = 37.5 mg/kg, n=3) resulted in a robust 74% (15±6%) reduction in infarct size compared to control in an in-vivo model of ischemia reperfusion injury (IRI). This effect was accompanied by a reduction in irreversible hypercontracture known as rigor. However, it caused a transient reduction in cardiac function during the infusion period. In this study, we aim to determine the effect of intermediate (ID; 3.75 mg/kg x 5 min = 18.75 mg/kg) and low dose (LD; 0.2-1.0 mg/kg x 5 min = 1.0-5.0 mg/kg) NTI pretreatment on infarct size and cardiac function in a rat in vivo model of IRI. Female Sprague-Dawley rats (~250g) were divided into four cohorts: non-treated control (n=15), LD (n=18), ID (n=5), and HD (n=5) NTI groups. All cohorts were anesthetized with Ketamine-xylazine (90 mg/kg, 9 mg/kg). Control rats received normal saline (0.9% NaCl), while treatment groups received either HD, ID, or LD NTI infused through the jugular vein (0.4mL/min) over 5 mins before inducing regional ischemia by occluding the left descending coronary artery for 30 min followed by 3 hr reperfusion. Cardiac function was measured through a pressure transducer catheter placed into the left ventricle (LV) in NTI treated groups. Planimetered photographs of heart slices were used to determine infarct size. Data were analyzed via ANOVA, Bonferroni-Dunn, and post-hoc analysis. Probability values \u3c0.05 were considered statistically significant. HD, ID, and LD NTI reduced infarct size by 81% (11±4%) p\u3c0.05, 55% (26±7%) p\u3c0.05, and 4% (55±4%) respectively compared to control (57±2%). Cardiac function parameters such as dP/dt min, the maximal rate of LV pressure relaxation, was not significantly affected by ID NTI compared to LD NTI in contrast to the cardiac depression elicited by HD NTI. These results indicate that the cardiac depressive effects of NTI are dose dependent and the infarct reducing effects of NTI can be achieved through doses that spare cardiac function. Therefore, ID NTI prophylaxis may be an effective strategy to limit cardiac tissue death in populations at high risk of an MI. Future studies will be conducted on porcine cardiac IRI models

Dual conjugation of protein kinase C epsilon peptide inhibitor with myristic acid and trans-activator of transcription mitigates myocardial ischemia-reperfusion injury in an in vivo porcine model

Abstract Protein Kinase C epsilon (PKCε) signaling is known to mediate superoxide production from mitochondrial and uncoupled endothelial nitric oxide synthase sources in myocardial ischemia-reperfusion (I/R) injury. Previously, PKCε peptide inhibitor conjugated with myristic acid and trans-activator of transcription (N-Myr-Tat-CC-EAVSLKPT [PKCε-]; Myr-Tat-PKCε-) exhibited profound reduction in infarct size compared to Myr-PKCε- or Tat-PKCε- in ex vivo rat hearts♰. This study aims to evaluate the effects of Myr-Tat-PKCε- in porcine myocardial I/R in vivo compared to a scrambled control peptide. Male Yorkshire castrated pigs (38-54kg) were subjected to regional I(1hr)/R(3hrs) via catheter-balloon in the left anterior descending coronary artery (LAD) at the location of the second LAD branch. Myr-Tat-PKCε- or Myr-Tat-PKCε-scrambled control peptide (N-Myr-Tat-CC-LSETKPAV [PKCε-scram]; Myr-Tat-PKCε-scram) bolus (0.2 mg/kg) was administered into the LAD at reperfusion. Echocardiography was used to determine ejection fraction (EF). Following reperfusion, hearts were excised and stained. The area at risk (AR) and area of necrosis (AN) were identified with 1% Evans Blue dye and 1% triphenyltetrazolium chloride respectively. Infarct size (AN/AR) and EF were analyzed with unpaired Student’s t-test. Myr-Tat-PKCε-scram exhibited a reduced final EF compared to baseline (551 vs 621%, n=3). Myr-Tat-PKCε- significantly increased final EF back to baseline (591 vs 591%, n=5; p\u3c0.05). Myr-Tat-PKCε- exhibited a reduction in infarct size (102%, n=4; p\u3c0.01) compared to Myr-Tat-PKCε-scram (297%, n=3). Results suggest that Myr-Tat-PKCε- mitigates myocardial I/R injury when administered during reperfusion. Future studies will test the effects of Myr-Tat-PKCε- in an 8-week porcine myocardial I/R survival study to determine its therapeutic potential for heart attack patients

Pretreatment with Naltrindole Demonstrates Cardioprotection in an Acute In Vivo Model of Ischemia/Reperfusion Injury

Myocardial infarctions (MI) remain prevalent in the U.S. with over one million cardiac events every year. Previous research indicates delta opioid receptor activation mimics pharmacologic preconditioning by reducing infarct size post-MI. However, our results have shown that naltrindole (NTI), a selective delta opioid receptor antagonist, elicits unexpected cardioprotective effects ex-vivo when given prior to 30min global ischemia (I) followed by 45min reperfusion (R). We aim to determine the cardioprotective effects of NTI in an in-vivo model. Female Sprague-Dawley rats (~250g) were assigned to control (0.9% NaCl, n=15) or (0.2mg/kg NTI, n=15). Thereafter, LD NTI (0.4-1.0 mg/kg, n=3) and high dose (HD, 4.0-8.0 mg/kg, n=5) were added to the study to measure cardiac function via a pressure transducer. Rats were anesthetized with ketamine-xylazine (90 mg/kg, 9mg/kg). NTI was infused (0.4mL/min) through the jugular vein over 5 mins (pretreatment) before I(30min)/R(3hrs) via proximal left anterior descending coronary artery. Hearts were stained with Superimperse blue dye and 1% triphenyltetrazolium chloride to determine infarct size. Data were analyzed via ANOVA Student Newman Keuls post-hoc analysis and unpaired t-tests, p\u3c0.05 was considered statistically significant. HD NTI (n=5) significantly depressed dP/dt max (6167±489 mmHg/s vs. 3714±645 mmHg/s), dP/dt min (-7015±942 mmHg/s vs. -4034±776 mmHg/s), and heart rate (261±20 BPM vs. 208±3 BPM) at the end of pretreatment compared to LD NTI (0.4-1.0 mg/kg, n=3 all p\u3c0.05). This preconditioning effect correlated with decreased infarct size. HD NTI exhibited ~60% reduction in infarct size (21±7 %; n=4, p\u3c0.01) vs. control (57±2 %; n=15) and LD NTI (55±4 %; n=18). These results mirrored ex-vivo studies, although a 20x dose was needed in-vivo to presumably overcome compensatory neurohormonal responses. Results suggest that NTI pretreatment can protect against I/R injury. Future studies will further examine 15 and 30 mg/kg dosage of NTI to examine potential effects on infarct size reduction

Myristic Acid-Trans-Activator of Transcription Dual Conjugation Enhances Intracellular delivery of protein kinase C beta II peptide inhibitor for concentration-dependent attenuation of superoxide release in isolated rat polymorphonuclear leukocytes

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX-2). Previously, myristic acid conjugated (or myristoylated) PKCβII inhibitor (myr-PKCβII-) significantly attenuated PMN SO release when dual conjugated to myristic acid and Trans-activator of transcription (myr-Tat-PKCβII-; N-myr-Tat-CC-SLNPEWNET) compared to myr-conjugation alone. However, the optimal concentration of myr-Tat-PKCβII- has yet to be determined. We hypothesized that myr-Tat conjugation would enhance the intracellular delivery of PKCβII- cargo and attenuate SO release in a concentration-dependent manner while retaining greater cell viability at lower concentrations. This study tested the concentration-dependent effects of myr-Tat-PKCβII- on SO release and cell viability compared to myr-Tat-PKCβII scrambled (myr-Tat-PKCβII- scram; N-myr-Tat-CC-WNPESLNTE), unconjugated PKCβII-, and untreated control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII- (20μM), myr-Tat-PKCβII- (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr-Tat-PKCβII-scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after phorbol-12-myristate-13 acetate (PMA) stimulation (100nM). Intracellular delivery was evaluated by the magnitude of PMA-induced PMN SO release attenuation with the PKCβII- cargo. Data were analyzed with ANOVA Fisher’s PLSD post-hoc analysis. Myr-Tat toxicity occurs at concentrations higher than 2 μM. Myr-Tat PKCβII- significantly decreased SO release compared to control while retaining similar cell viability at 5 μM (n=15, 0.384±0.03) and 7.5μM (n=11, 0.391±0.05) concentrations. Myr-Tat PKCβII-scram 5μM (n=7, 0.44±0.07) and 7.5μM (n=5, 0.409±0.11) were not different from control. Results suggest that Myr-Tat-PKCβII- 5μM significantly attenuates PMA-induced PMA SO release while retaining cell viability at \u3e 80%. Future studies will assess the effects of myr- or myr-Tat-PKCβII- peptides on PKCβII- translocation activity

Infarct-sparing effects of naltrindole and analogues through inhibition of myocardial hypercontracture during ischemia/reperfusion injury

Coronary artery disease remains the leading cause of death worldwide. Reduction in infarct size may be a potential preventive strategy for cardiovascular complications. Increased left ventricular end-diastolic pressure (LVEDP) or Ischemic Peak Pressure (IPP) during ischemia, is associated with infarct size. Previously, we showed that naltrindole (NTI, selective delta opioid receptor antagonist), reduced LVEDP and infarct size in ex vivo rat hearts. This study examined the effects of NTI and analogues naltriben (NTB, delta opioid receptor antagonist) and guanidinonaltrindole (GNTI, kappa opioid receptor antagonist) (all 5 μM) compared to control hearts. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to 30-min global ischemia (I)/45-min reperfusion (R) with treatments infused for 5 min before I and during the first 5 min of R. After R, 1% triphenyltetrazolium chloride staining assessed infarct size. Data were evaluated using ANOVA Student-Neuman-Keuls post-hoc analysis. NTI (n=8) and NTB (n=6) elicited a transient decrease in the maximal rate in the rise of LV pressure (dP/dt max) to 1581±379 mmHg/s and 929±243 mmHg/s, respectively when given prior to I compared to control (2471±72 mmHg/s, n=12, p These results suggest that NTI, NTB and GNTI are cardioprotective against myocardial I/R injury. The negative inotropic effects of NTI and NTB were associated with ~75% reduction and GNTI with ~50% reduction in infarct size compared to control. These results suggest that NTI and analogues exert tissue-salvaging effects. Future studies will examine the cardioprotective effects of NTI and analogues given at different ischemic times