Emmanuel Sampene

Acute Fetal Demise with First Trimester Maternal Infection Resulting from Listeria monocytogenes in a Nonhuman Primate Model

ABSTRACT Infection with Listeria monocytogenes during pregnancy is associated with miscarriage, preterm birth, and neonatal complications, including sepsis and meningitis. While the risk of these conditions is thought to be greatest during the third trimester of pregnancy, the determinants of fetoplacental susceptibility to infection, the contribution of gestational age, and the in vivo progression of disease at the maternal-fetal interface are poorly understood. We developed a nonhuman primate model of listeriosis to better understand antecedents of adverse pregnancy outcomes in early pregnancy. Four pregnant cynomolgus macaques (Macaca fascicularis) received a single intragastric inoculation between days 36 and 46 of gestation with 107 CFU of an L. monocytogenes strain isolated from a previous cluster of human listeriosis cases that resulted in adverse pregnancy outcomes. Fecal shedding, maternal bacteremia, and fetal demise were consistently noted within 7 to 13 days. Biopsy specimens of maternal liver, spleen, and lymph node displayed variable inflammation and relatively low bacterial burden. In comparison, we observed greater bacterial burden in the decidua and placenta and the highest burden in fetal tissues. Histopathology indicated vasculitis, fibrinoid necrosis, and thrombosis of the decidual spiral arteries, acute chorioamnionitis and villitis in the placenta, and hematogenous infection of the fetus. Vascular pathology suggests early impact of L. monocytogenes infection on spiral arteries in the decidua, which we hypothesize precipitates subsequent placentitis and fetal demise. These results demonstrate that L. monocytogenes tropism for the maternal reproductive tract results in infection of the decidua, placenta, and the fetus itself during the first trimester of pregnancy. IMPORTANCE Although listeriosis is known to cause significant fetal morbidity and mortality, it is typically recognized in the third trimester of human pregnancy. Its impact on early pregnancy is poorly defined. Here we provide evidence that exposure to L. monocytogenes in the first trimester poses a greater risk of fetal loss than currently appreciated. Similarities in human and nonhuman primate placentation, physiology, and reproductive immunology make this work highly relevant to human pregnancy. We highlight the concept that the maternal immune response that protects the mother from serious disease is unable to protect the fetus, a concept relevant to classic TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes) infections and newly illuminated by current Zika virus outbreaks. Studies with this model, using the well-understood organism L. monocytogenes, will permit precise analysis of host-pathogen interactions at the maternal-fetal interface and have broad significance to both recognized and emerging infections in the setting of pregnancy. Although listeriosis is known to cause significant fetal morbidity and mortality, it is typically recognized in the third trimester of human pregnancy. Its impact on early pregnancy is poorly defined. Here we provide evidence that exposure to L. monocytogenes in the first trimester poses a greater risk of fetal loss than currently appreciated. Similarities in human and nonhuman primate placentation, physiology, and reproductive immunology make this work highly relevant to human pregnancy. We highlight the concept that the maternal immune response that protects the mother from serious disease is unable to protect the fetus, a concept relevant to classic TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes) infections and newly illuminated by current Zika virus outbreaks. Studies with this model, using the well-understood organism L. monocytogenes, will permit precise analysis of host-pathogen interactions at the maternal-fetal interface and have broad significance to both recognized and emerging infections in the setting of pregnancy.

Defining the boundaries and expanding the utility of head and neck cancer patient derived xenografts

BACKGROUND Patient derived xenografts (PDXs) represent an essential tool in oncologic research, and we sought to further expand our repertoire of head and neck squamous cell carcinoma (HNSCC) while determining potential boundaries for this system. METHODS We consented new patients for PDX development and determined if a 24-h time delay from tumor excision to xenograft implantation affected PDX establishment. We developed a tissue microarray (TMA) from formalin fixed, paraffin embedded PDXs and their subsequent passages and carried out quantitative immunohistochemistry for EGFR, pEGFR, pAkt, pERK and ERCC1. First and last passaged PDXs were compared via a paired t-test to examine for the stability of protein expression across passages. We performed a similar comparison of the mutational profile of the patient tumor and resulting xenografts using a targeted sequencing approach. RESULTS No patient/tumor characteristics influenced PDX take rate and the 24-h time delay from tumor excision to xenograft implantation did not affect PDX establishment, growth or histology. There was no significant difference in biomarker expression between the first and last passaged PDXs for EGFR, pEGFR, pAkt, and ERCC1. For pERK there was a significant difference (p=0.002), but further analysis demonstrated this only arose in three of 15 PDXs. Targeted sequencing revealed striking stability of passenger and likely driver mutations from patient to xenograft. CONCLUSIONS The stability of protein expression across PDX passages will hopefully allow greater investigation of predictive biomarkers in order to identify ones for further pre-clinical and clinical investigation.

Renin-angiotensin system transgenic mouse model recapitulates pathophysiology similar to human preeclampsia with renal injury that may be mediated through VEGF

Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild-type (WT) C57Bl/6 mice were cross-bred: female hAGT × male hREN for preeclampsia (PRE) model and female WT × male WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1, and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; P = 0.02), and placental necrosis (60% vs. 0%; P < 0.01). PRE group demonstrated declining BPs with advancing gestation. Plasma sFlt1 increased across pregnancy in PRE; VEGF did not vary. IHC demonstrated the presence of sFlt1 in glomeruli, lymphatics, and collecting tubules of PRE kidneys, suggesting excretion. VEGF immunostaining was increased specifically in the glomeruli of PRE kidneys. Placenta in PRE showed marked immunostaining for sFlt1. We conclude that this transgenic model of preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to acetylcholine. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling, and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF.

Cotargeting mTORC and EGFR Signaling as a Therapeutic Strategy in HNSCC.

Head and neck squamous cell carcinomas (HNSCC) are frequently altered along the PI3K/AKT/mTORC signaling axis. Despite excellent preclinical data, the use of compounds targeting this pathway as monotherapy has been underwhelming in initial clinical trials, and identification of predictive biomarkers remains challenging. To investigate mTORC-specific inhibition, we tested catalytic mTORC (AZD8055) and PI3K/mTORC (NVP-BEZ-235) inhibitors ± cetuximab in a panel of HNSCC cell lines and patient-derived xenografts (PDX). Cell lines were assayed for response to all agents and siRNA knockdown of targets by multiple approaches. All cell lines showed similar response to both drug and siRNA inhibition of both PI3K and mTORC pathways, with anti-EGFR combination producing modest additive effect. Five PDX models that presented PIK3CA mutation or intrinsic cetuximab resistance were treated with a combination of cetuximab and AZD8055. In vivo single-agent mTORC inhibition inhibited growth of one PIK3CA-mutant cancer, but had little effect on any PIK3CAWT or a second PIK3CA-mutant model. In all models, the combination therapy showed greater growth delay than monotherapy. The uniform ability of PI3K and mTORC inhibition to suppress the growth of HNSCC cells highlights the pathways role in driving proliferation. Although single-agent therapy was largely ineffective in vivo, improved response of combination treatment in an array of PDXs suggests the potential for adding a catalytic mTORC inhibitor to cetuximab therapy. Overall, these results add to a growing body of evidence, suggesting that approaches that attempt to match biomarkers to the optimal therapy in HNSCC remain complex and challenging. Mol Cancer Ther; 16(7); 1257–68. ©2017 AACR.

Folic Acid Modulates Matrix Metalloproteinase-2 Expression, Alleviates Neuropathic Pain, and Improves Functional Recovery in Spinal Cord-Injured Rats

Background: The molecular underpinnings of spinal cord injury (SCI) associated with neuropathic pain (NP) are unknown. Recent studies have demonstrated that matrix metalloproteinases (MMPs) such as MMP2 play a critical role in inducing NP following SCI. Promoter methylation of MMPs is known to suppress their transcription and reduce NP. In this context, it has been shown in rodents that folic acid (FA), an FDA approved dietary supplement and key methyl donor in the central nervous system (CNS), increases axonal regeneration and repair of injured CNS in part via methylation. Purpose: Based on above observations, in this study, we test whether FA could decrease MMP2 expression and thereby decrease SCI-induced NP. Methods: Sprague-Dawley male rats weighing 250-270 g received contusion spinal cord injuries (cSCIs) with a custom spinal cord impactor device that drops a 10 g weight from a height of 12.5 mm. The injured rats received either i.p. injections of FA (80 µg/kg) or water (control) 3 days prior and 17 days post-cSCI (mid phase) or for 3 days pre-cSCI and 14 days post-cSCI ending on the 42nd day of cSCI (late phase). The functional neurological deficits due to cSCI were then assessed by Basso, Beattie, and Bresnahan (BBB) scores either on post-impaction days 0 through 18 post-cSCI (mid phase) or on days 0, 2, 7, 14, 21, 28, 35, and 42 (late phase). Baseline measurements were taken the day before starting treatments. Thermal hyperalgesia (TH) testing for pain was performed on 4 days pre-cSCI (baseline data) and on days 18, 21, 28, 35, and 42 post-cSCI. Following TH testing, animals were euthanized and spinal cords harvested for MMP-2 expression analysis. Result: The FA-treated groups showed higher BBB scores during mid phase (day 18) and in late phase (day 42) of injury compared to controls, suggesting enhanced functional recovery. There is a transient decline in TH in animals from the FA-treated group compared to controls when tested on days 18, 21, 28, and 35, indicative of a decrease in NP. However, when tested 25 days after stopping FA administration on day 42 of cSCI, no significant difference in TH was observed between FA-treated and control animals. Western blot analysis of the injured spinal cord from FA-treated animals showed significant decline in MMP2 expression compared to spinal cord samples from water-treated controls. Conclusion: Together, these data suggest that FA could alleviate NP and improve functional recovery post-SCI, possibly by reducing the expression of MMP2. Further studies will open up a novel and easy natural therapy, ideal for clinical translation with minimal side effects, for managing SCI-induced NP. Such studies might also throw light on a possible epigenetic mechanism in FA-induced recovery after SCI.

Oxidative stress via inhibition of the mitochondrial electron transport and Nrf-2-mediated anti-oxidative response regulate the cytotoxic activity of plumbagin

Plumbagin, an anti-cancer agent, is toxic to cells of multiple species. We investigated if plumbagin targets conserved biochemical processes. Plumbagin induced DNA damage and apoptosis in cells of diverse mutational background with comparable potency. A 3–5 fold increase in intracellular oxygen radicals occurred in response to plumbagin. Neutralization of the reactive oxygen species by N-acetylcysteine blocked apoptosis, indicating a central role for oxidative stress in plumbagin-mediated cell death. Plumbagin docks in the ubiquinone binding sites (Q0 and Qi) of mitochondrial complexes I–III, the major sites for oxygen radicals. Plumbagin decreased oxygen consumption rate, ATP production and optical redox ratio (NAD(P)H/FAD) indicating interference with electron transport downstream of mitochondrial Complex II. Oxidative stress induced by plumbagin triggered an anti-oxidative response via activation of Nrf2. Plumbagin and the Nrf2 inhibitor, brusatol, synergized to inhibit cell proliferation. These data indicate that while inhibition of electron transport is the conserved mechanism responsible for plumbagin’s chemotoxicity, activation of Nrf2 is the resulting anti-oxidative response that allows plumbagin to serve as a chemopreventive agent. This study provides the basis for designing potent and selective plumbagin analogs that can be coupled with suitable Nrf2 inhibitors for chemotherapy or administered as single agents to induce Nrf2-mediated chemoprevention.

Indeterminate Adnexal Cysts at US: Prevalence and Characteristics of Ovarian Cancer

Purpose To assess the prevalence of indeterminate adnexal cysts in women presenting to academic medical centers for pelvic ultrasonography (US), determine the incidence of malignancy, and identify cyst and patient characteristics that are predictive of malignancy. Materials and Methods A multicenter study of US-detected adnexal cysts with appropriate follow-up (surgical pathologic examination, imaging and/or clinical examination) was conducted from January 2008 to June 2012. Indeterminate cysts were classified as category 1 (typical benign appearing cysts >5 cm) or category 2 (cysts with avascular solid components) on the basis of a combination of definitions in the existing literature. The incidence of neoplasms and malignant tumors was calculated. Patient and cyst characteristics associated with neoplasm and malignant tumors were evaluated with the χ2 test or Fisher exact test for categorical variables and the t test for continuous variables. A backward stepwise logistic regression model was performed for two outcomes: (a) the presence of any neoplasm (benign or malignant) and (b) the presence of a malignant tumor. Results There were 1637 women with an adnexal cyst at US; 391 (mean age = 41.8 years ± 13.5.1; range = 17-91 years) had an indeterminate adnexal cyst at US. The prevalence of indeterminate adnexal cysts was 23.9% (391 of 1637; 95% confidence interval [CI]: 0.22, 0.26). Three hundred three indeterminate cysts in 280 women (mean age = 42.9 years ± 14.1; range = 17-88 years) had adequate follow-up. The incidence of ovarian neoplasms (benign and malignant) was 24.8% (75 of 303 cysts; 95% CI: 0.20, 0.30), and the incidence of malignant tumors was 3.6% (11 of 303 cysts; 95% CI: 0.02, 0.06). The proportion of ovarian neoplasms differed between category 1 and category 2 cysts (17.5% [25 of 143 cysts; 95% CI: 0.12, 0.25] vs 31.3% [50 of 160 cysts; 95% CI: 0.24, 0.39], respectively; P = .001). The proportion of malignant tumors differed between categories 1 and 2 cysts (0% [0 of 143 cysts] vs 6.9% [11 of 160 cysts; 95% CI: 0.03, 0.12]; P < .001). The presence of an avascular nodular component was a significant predictor of malignancy at stepwise logistic regression analysis (odds ratio = 2.83; P ≤ .0001; 95% CI: 1.69, 4.70). Conclusion The presence of an avascular nodular component was the most significant predictor of the presence of malignancy in indeterminate adnexal cysts. The risk of malignancy is higher with category 2 cysts than with category 1 cysts.

Seasonal and Geographic Patterns in Seeking Cardiovascular Health Information: An Analysis of the Online Search Trends

Objective: To ascertain whether temporal and geographic interest in seeking cardiovascular disease (CVD) information online follows seasonal and geographic patterns similar to those observed in real‐world data. Methods: We searched Google Trends for popular search terms relating to CVD. Relative search volumes (RSVs) were obtained for the period January 4, 2004, to April 19, 2014, for the United States and Australia. We compared average RSVs by month and season and used cosinor analysis to test for seasonal variation in RSVs. We also assessed correlations between state‐level RSVs and CVD burden using an ecological correlational design. Results: RSVs were 15% higher in the United States and 45% higher in Australia for winter compared with summer (P<.001 for difference for both). In the United States, RSVs were 36% higher in February compared with August, while in Australia, RSVs were 75% higher in August compared with January. On cosinor analysis, we found a significant seasonal variability in RSVs, with winter peaks and summer troughs for both the United States and Australia (P<.001 for zero amplitude test for both). We found a significant correlation between state‐level RSVs and mortality from CVD (r=0.62; P<.001), heart disease (r=0.58; P<.001), coronary heart disease (r=0.48; P<.001), heart failure (r=0.51; P<.001), and stroke (r=0.60; P<.001). Conclusion: Google search query volumes related to CVD follow strong seasonal patterns with winter peaks and summer troughs. There is moderate to strong positive correlation between state‐level search query volumes and burden of CVD mortality.

Neoadjuvant chemotherapy is associated with more anemia and perioperative blood transfusions than primary debulking surgery in women with advanced stage ovarian cancer

OBJECTIVE The purpose of this case-controlled study was to determine the prevalence of anemia and incidence of perioperative blood transfusions in patients undergoing treatment for advanced ovarian cancer with neoadjuvant chemotherapy (NACT) or primary debulking surgery (PDS). METHODS We performed a single institution review of patients diagnosed with stage IIIB-IVB epithelial ovarian cancer between 2010 and 2013 undergoing either NACT or PDS. Anemia was defined as a hemoglobin (Hgb) concentration of ≤11.5 g/dL. Continuous variables were compared by student t-test and binary variables compared via chi square analysis. RESULTS One hundred thirty-one women were included, 66 treated with NACT and 65 treated with PDS. Average Hgb prior to surgery was lower in women who received NACT (10.7 g/dL vs 12.8 g/dL, p < 0.0001). Women treated with NACT had a decrease in mean Hgb during chemotherapy treatment (11.8 g/dL at diagnosis to 10.7 g/dL preoperatively). Seventy-seven percent of NACT patients were anemic prior to surgery compared to 15% of patients prior to PDS (p < 0.001). Mean EBL at debulking was higher in patients selected for PDS (871 mL) than NACT (544 mL); however, the perioperative transfusion rate was higher during interval debulking surgeries (NACT 77% vs PDS 56%, p = 0.01). CONCLUSION Women selected for NACT were more likely to be anemic at diagnosis and became progressively anemic during NACT. Despite less blood loss during debulking surgery, NACT patients receive more blood transfusions perioperatively than patients undergoing PDS. This represents a potential opportunity for therapeutic intervention during NACT to correct anemia prior to interval debulking surgery.

Maternal obesity is not associated with increased crown-rump length in first-trimester ultrasounds of singleton gestations

Abstract Objective: To evaluate whether or not obesity affects fetal growth in the first trimester of pregnancy. Study design: A retrospective cohort study of obese versus non-obese women in our ultrasound database was performed to compare crown-rump length (CRL), a surrogate of fetal growth, at the first-trimester genetic screening. Results: A total of 50 obese and 50 non-obese women were included. CRL for both groups was performed at an average of 12wk5d ± 3 d. A linear regression analysis demonstrated that there was no difference between the cohorts in respect to CRL in the first trimester (p = .482). However, the estimated fetal weight at second-trimester anatomy ultrasound and the neonatal birth weight were increased in obese women (p < .001 for both analyses). Conclusion: Maternal obesity does not significantly alter the fetal CRL. However, maternal obesity appears to be associated with increased fetal growth as early as the second trimester.