Intracardiac Route Research Articles

Re-evaluating intra-cardiac arrest adjunctive medications and routes of drug administration.

This narrative review summarizes the evidence for the most commonly used intra-cardiac arrest adjunctive medications and routes of administration and discusses promising new therapies from preclinical animal models. Large trials on the administration of calcium as well as the combination of vasopressin and glucocorticoids during cardiac arrest have been published. Calcium administration during cardiopulmonary resuscitation does not improve outcomes and might cause harm. Vasopressin and glucocorticoid administration during cardiopulmonary resuscitation improve the chance of return of spontaneous circulation but has uncertain effects on survival. We identified a total of seven ongoing clinical trials investigating the potential role of bicarbonate, of vasopressin and glucocorticoids, and of intravenous versus intraosseous vascular access. Several medications such as levosimendan and inhaled nitric oxide show promise in preclinical studies, and clinical trials are either planned or actively recruiting. Large trials on intra-cardiac arrest administration of calcium and vasopressin with glucocorticoids have been performed. Several trials are ongoing that will provide valuable insights into the potential benefit of other intra-cardiac arrest medications such as bicarbonate as well as the potential benefit of intravenous or intraosseous vascular access.

Sildenafil as a Salvage Treatment for Ovarian Torsion: A Rat Model

OBJECTIVE: In this study, we aimed to investigate the effect of sildenafil, acting as an anti-oxidant on the ischemic rat ovarian torsion model by pointing out ovarian reserve via AMH levels which is the best marker for oocyte pool.STUDY DESIGN: This study is an experimental animal study conducted according to the principles of the Declaration of Helsinki. Twenty-seven rats were assigned into three groups, each consisting of nine individuals. Group 1: Sham operation (laparotomy only) was performed. Group 2: The torsion group had bilateral torsion and detorsion of the ovaries. Group 3: In Sildenafil and torsion group the rats received 0.7 mg/kg sildenafil (Viagra, Pfizer, New York, ABD) intraperitoneally one hour before torsion and detorsion operation. One month after the initial operation, blood was drawn via the intra-cardiac route, and serum antimullerian hormone (AMH) concentrations were studied.RESULTS: The mean weights of the rats were similar 224 g, 229 g, and 222 g for groups 1, 2, and 3 respectively. Only one rat from Group 2(T/D) died during the experiment leaving eight rats for analysis. The mean value for AMH was 24.9227±12.19124 ng/ml and the mean value for weight was 226.22±19.200. The differences in AMH groups were not statistically significant (p=0.873)CONCLUSIONS: As a result, as is shown in our study, the ovarian reserve may not be influenced by ischemia developed by ovarian torsion or any influences could not change AMH levels. Sildenafil which was given to the rats before detorsion to protect against reperfusion ischemia was found to be ineffective on ovary reserve according to AMH assessment.

Carboxylesterase-overexpressing hTERT-immortalized human adipose stem cells in prostate tumor growth inhibition by irinotecan.

Effective chemotherapy has not yet to be developed for castration-resistant prostate cancer (CRPC). Cell-mediated enzyme prodrug therapy (EPT), including a combination of carboxylesterase (CE) and irinotecan (CPT-11), could be a possible treatment option. This study explored a cell-mediated EPT, including a combination of CE and irinotecan (CPT-11), to inhibit CRPC tumor growth using rabbit CE-overexpressing human TERT-immortalized adipose-derived stem cells (hTERT-ADSC.CE). An hTERT ADSC.CE cell line was established by transfection with a lentiviral vector (CLV-Ubic) encoding the rabbit CE gene. To determine the in vitro suicide effects of hTERT-ADSC.CE, cell cultures were performed using various concentrations of CPT-11 (0.01-5 μM), and to determine the in vitro cytotoxic effects of hTERT-ADSC.CE cells, PC3 and hTERT-ADSC.CE cells were co-cultured. For the in vivo model, PC3 cells (1 × 106 cells) were injected subcutaneously into the flanks of nude mice and hTERT-ADSC.CE cells were injected via an intracardiac route, followed by the continuous treatment using CPT-11 for 2 weeks. The final change in tumor volume was measured and immunohistochemical analysis was performed. The directional and selective migration of hTERT-ADSC.CE cells toward PC3 cells was significantly stimulated by PC3 cells in vitro. The number of apoptotic PC3 cells significantly increased in the presence of hTERT-ADSC.CE and CPT-11 compared to CPT-11 alone. In the in vivo study, the inhibitory effects of hTERT-ADSC.CE combined with CPT-11 were higher than those of CPT-11 monotherapy. After treatment with CPT-11 alone or ADSC.CE in combination with CPT-11, the removed tumor tissues showed hyperchromatic nuclei and apoptotic bodies. CE-overexpressing ADSCs potentiated the inhibition of tumor growth in CRPC-bearing mice in the presence of CPT-11 prodrugs. This report suggests that cell-mediated EPT including CE and CPT-11 may be efficacious in treating CRPC.

The CDK4/6 Inhibitor Palbociclib Inhibits Estrogen-Positive and Triple Negative Breast Cancer Bone Metastasis In Vivo

CDK 4/6 inhibitors have demonstrated significant improved survival for patients with estrogen receptor (ER) positive breast cancer (BC). However, the ability of these promising agents to inhibit bone metastasis from either ER+ve or triple negative BC (TNBC) remains to be established. We therefore investigated the effects of the CDK 4/6 inhibitor, palbociclib, using in vivo models of breast cancer bone metastasis. In an ER+ve T47D model of spontaneous breast cancer metastasis from the mammary fat pad to bone, primary tumour growth and the number of hind limb skeletal tumours were significantly lower in palbociclib treated animals compared to vehicle controls. In the TNBC MDA-MB-231 model of metastatic outgrowth in bone (intracardiac route), continuous palbociclib treatment significantly inhibited tumour growth in bone compared to vehicle. When a 7-day break was introduced after 28 days (mimicking the clinical schedule), tumour growth resumed and was not inhibited by a second cycle of palbociclib, either alone or when combined with the bone-targeted agent, zoledronic acid (Zol), or a CDK7 inhibitor. Downstream phosphoprotein analysis of the MAPK pathway identified a number of phosphoproteins, such as p38, that may contribute to drug-insensitive tumour growth. These data encourage further investigation of targeting alternative pathways in CDK 4/6-insensitive tumour growth.

Investigation of Potassium Chloride for Euthanasia of Anesthetized Marine Toads (Rhinella marina)

Euthanasia techniques in amphibians are poorly described and sparsely validated. This study investigated potassium chloride (KCl) for euthanasia of anesthetized marine toads (Rhinella marina). Twenty-three toads were immersed in buffered MS-222 (2 g/L) for 5 min beyond loss of righting reflex, manually removed, and randomly administered KCl (n = 6/group) via one of three routes: intracardiac at 10 mEq/kg (IC), intracoelomic at 100 mEq/kg (ICe), or immersion at 4,500 mEq/L (IMS) or no treatment (C) (n = 5/group). Doppler sounds were assessed continuously from prior to treatment until 5 min posttreatment and every 5 min thereafter until sound cessation or resumption of spontaneous movement. Plasma potassium concentration (K+) was measured at the time of Doppler sound cessation in ICe and IMS. In IC, ICe, IMS, and C, Doppler sound cessation occurred in 4/6, 6/6, 6/6, and 1/5 toads with median (range) or mean ± SD times of 0.23 (0–4.65), 17.5 ± 9.0, 40.6 ± 10.9, and >420 min, respectively. Nonsuccess in 2/6 toads in IC was suspected due to technique failure. Plasma K+ exceeded the limits of detection (>9 mmol/L) in 12/12 toads in ICe and IMS. Five of six toads in C resumed spontaneous movement at median (range) times of 327 (300–367) min. KCl delivered via an intracardiac, intracoelomic, or immersion route resulted in Doppler sound cessation in 16 of 18 toads and may be appropriate for euthanasia of anesthetized marine toads.

Investigation of Potassium Chloride for Euthanasia of Anesthetized Marine Toads (Rhinella marina)

Euthanasia techniques in amphibians are poorly described and sparsely validated. This study investigated potassium chloride (KCl) for euthanasia of anesthetized marine toads ( Rhinella marina ). Twenty three toads were immersed in buffered MS-222 (2 g/L) for five minutes (min) beyond loss of righting reflex, manually removed, and randomly administered KCl (n = 6/group) via one of three routes: intracardiac at 10 mEq/kg (IC), intracoelomic at 100 mEq/kg (ICe), or immersion at 4500 mEq/L (IMS) or no treatment (C) (n = 5/group). Doppler sounds were assessed continuously from prior to treatment until two min post-treatment and every five min thereafter until sound cessation or resumption of spontaneous movement. Plasma potassium concentration (K+) was measured at the time of Doppler sound cessation in ICe and IMS. In IC, ICe, IMS, and C, Doppler sound cessation occurred in 4/6, 6/6, 6/6, and 1/5 toads with median (range) or mean + SD times of 0.23 (0-4.65), 17.5 + 9.0, 40.6 + 10.9, and >420 min, respectively. Nonsuccess in 2/6 toads in IC was suspected due to technique failure. Plasma K+ exceeded the limits of detection (>9 mmol/L) in 12/12 toads in ICe and IMS. Five of six toads in C resumed spontaneous movement at median (range) times of 327 (300-367) min. KCl delivered via an intracardiac, intracoelomic, or immersion routes resulted in Doppler sound cessation in 16 of 18 toads and may be appropriate for euthanasia of anesthetized marine toads.

MP22-16 DEVELOPMENT OF NON-INVASIVE CLINICALLY APPLICABLE IN VIVO TRACKING OF EXTRACELLULAR VESICLES USING MAGNETIC RESONANCE IMAGING (MRI)

You have accessJournal of UrologyImaging/Radiology: Uroradiology II (MP22)1 Sep 2021MP22-16 DEVELOPMENT OF NON-INVASIVE CLINICALLY APPLICABLE IN VIVO TRACKING OF EXTRACELLULAR VESICLES USING MAGNETIC RESONANCE IMAGING (MRI) Johnny Akers, Paola Aguiari, Hasmik Soloyan, Seda Mkhitaryan, Gevorg Karaptyan, Roger De Filippo, Mya Thu, Laura Perin, and Sargis Sedrakyan Johnny AkersJohnny Akers More articles by this author , Paola AguiariPaola Aguiari More articles by this author , Hasmik SoloyanHasmik Soloyan More articles by this author , Seda MkhitaryanSeda Mkhitaryan More articles by this author , Gevorg KaraptyanGevorg Karaptyan More articles by this author , Roger De FilippoRoger De Filippo More articles by this author , Mya ThuMya Thu More articles by this author , Laura PerinLaura Perin More articles by this author , and Sargis SedrakyanSargis Sedrakyan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002013.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSC) hold great potential for the treatment of chronic kidney diseases (CKD). We have already shown that AFSC-EVs are renoprotective in a mouse model of CKD, Alport syndrome. However, there is an important unmet need for real-time in vivo monitoring of these therapeutic EVs after they are injected into a subject to understand their safety, targeting, and effectiveness. While current optical imaging solutions like bioluminescence and fluorescence are useful for EV tracking studies in animal models, there is limited utility in clinical applications. Here we present a novel in vivo tracking solution for our therapeutic EVs in Alport mice, utilizing clinically applicable MRI technology. METHODS: To generate trackable EVs, AFSC were labeled with a novel magnetic agent (VSCM). EVs secreted by the labeled AFSC were isolated by ultracentrifugation. The viability and morphology of labeled-cells were evaluated, and the in vitro MR properties of EVs were analyzed by magnetometer. Purity, potency and identity of labeled EV was compared to non-labeled EVs. In vivo biodistribution of labeled EVs was evaluated in WT and Alport mice by MRI at 10 min and 3 hr post injection, and retro-orbital and intra-cardiac routes of delivery were compared. RESULTS: The magnetic label did not affect the physiological characteristics of the cells and did not change identity, purity and potency (therapeutic effect in vivo) of EVs. MRI phantom studies confirmed the in vitro/ex vivo detectability of labeled-EVs. Importantly, as expected MRI studies showed that EV homing to the kidney injected intra-cardiacally into Alport mice was more efficient vs the retro-orbital route, and Prussian blue staining of sections confirmed EV homing to the kidney. CONCLUSIONS: We have developed a clinically applicable novel magnetic nanoparticle agent that can be used to label and track the biodistribution of EVs in the kidney and other organs using non-invasive, safe, and effective MRI technology that’s widely available. This technology is highly adaptable and can be deployed in both preclinical and clinical settings. Source of Funding: The GOFARR Fund; Research Career Development Award, TSRI, CHLA © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e397-e398 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnny Akers More articles by this author Paola Aguiari More articles by this author Hasmik Soloyan More articles by this author Seda Mkhitaryan More articles by this author Gevorg Karaptyan More articles by this author Roger De Filippo More articles by this author Mya Thu More articles by this author Laura Perin More articles by this author Sargis Sedrakyan More articles by this author Expand All Advertisement Loading ...

Abstract PS17-09: Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant breast cancer

Abstract Background: ESR1 mutations are acquired following ERα targeted therapies and are a major determinant of therapy-resistance. These ESR1 mutations maintain ESR1 signaling, albeit in a ligand-independent manner. Effective drugs targeting these mutant (MT) ERα proteins represent a significant unmet clinical need. We had previously shown that ERX-11, an ESR1-coregulator binding inhibitor, could block the function of these MT ERα proteins. In this study, we sought to leverage recently published structures of MT ERα to develop more potent analogues of ERX-11. Methods: Virtual screening of >250,000 derivatives of ERX-11 was performed with simulated docking on the MT ERα to identify and design analogues of ERX-11. Several hundred analogues were synthesized and tested in vitro using multiple BC model cells that express wild type (WT) ESR1 or mutant (MT) ESR1 (Y537S or D538G). Mechanistic studies were performed using RNA-Seq, Western blotting, qRT-PCR and reporter gene assays. The in vivo efficacy of the most potent ERX-11 analogue ERX-245 was examined using xenograft, PDX and metastatic models of MT-ER driven BC. Results: From our virtual and functional screen, we identified an ERX-11 analogue, ERX-245 as the most potent hit to target MT-ERα. Docking studies modeled a better fit of ERX-245 into the ligand binding domain of both the Y537S and D538G MT-ERα. ERX-245 potently reduced (IC50 ~250 nM) the cell viability of both WT-ERα and MT-ERα driven BC cells but not ERα negative BC cells. ERX-245 significantly reduced the growth (colony formation, clonogenic and mammosphere assays) of MT-ERα BC cells. ERX-245 exhibited synergistic activity in combination with CDK4/6 inhibitors. In distinction to classic SERDs like fulvestrant (which degrade ERα with in 4h), ERX-245 treatment decreased MT-ERα protein levels over 24 hours. PK studies indicated that ERX-245 is more polar and has better solubility and pharmacokinetic properties than ERX-11. ERX-245 reduced tumor growth of subcutaneous xenograft and PDX models driven by MT-ERα as well as the proliferation of xenograft derived MT-ERα explant models. ERX-245 significantly reduced the invasive capability of MT-ERα BC cells in vitro and inhibited both the metastatic capability and growth of metastatic tumors derived from MT-ERα BC cells injected by intracardiac or intratibial routes. Conclusions: Taken together, these results indicate that ERX-245 is a potent and pharmacologically translatable analog of ERX-11, with activity against both primary and metastatic tumors driven by MT-ERα. Citation Format: Ganesh V Raj, Suryavathi Viswanadhapalli, Karla Parra, Shihong Ma, Tae-Kyung Lee, Xihui Liu, Kara Kassees, Weiwei Tang, Junhao Liu, Zexuan Liu, Uday P Pratap, Behnam Ebrahimi, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ratna K Vadlamudi. Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-09.

Abstract PD13-03: Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets

Abstract Background: Leptomeningeal metastasis remains a devastating development in breast cancer, with a median survival of 3-4 months, no widely accepted standard treatment, and limited access to trials of novel therapies. In addition, lack of access to leptomeningeal metastatic material hampers the pre-clinical investigation of the disease process and molecular drivers. This project uses CSF as a liquid biopsy to characterise BCLM, through genomic analysis of the cell-free DNA (cfDNA), and the development of pre-clinical BCLM models by the expansion of CSF disseminated tumour cells. Methods: CSF (surplus to clinical requirements) and blood were collected from patients undergoing evaluation of leptomeningeal metastasis. cfDNA was extracted from CSF and plasma, and subjected to ultra-low pass whole genome sequencing (ulpWGS) to assess tumour-derived cfDNA fraction. Samples with >10% tumour fraction underwent whole exome sequencing, along with matched archival primary tumour, archival extra-cranial metastatic site(s) and germline DNA. CSF cells were expanded in vitro (to establish 3D patient-derived organoids (PDOs). Results: Cohort demographics are shown in Table 1. Whole exome sequencing (WES) in 21 patients reveals that 65.2% of variants found in CSF cfDNA were not shared with the primary tumour or other matched samples. Phylogenetic analysis shows a divergent evolution from extra-cranial metastatic sites, represented by plasma cfDNA (n = 12) and/or metastatic site tissue DNA (n = 7). The most frequently mutated cancer-associated genes in CSF were MUC16 (12/21), TP53 (11/21), CDH1 (10/21), and KMT2D (7/21). The common occurrence of CDH1 loss-of-function mutations was in keeping with the large number of lobular cases, however were also discovered in CSF of two cases with E-cadherin positive ductal primary tumours. Furthermore, mutations (including frameshift indels) in JAK family proteins (JAK1, JAK3 and TYK2) were present in 5/21 cases, and were private to CSF in 4/21. Potential actionable gene alterations private to CSF include; IDH2 (3/21), GLI1 (3/21), PIK3CA (2/21) and PTCH1 (2/21). Further, there was an enrichment for somatic BRCA1/2 mutations (5/21, 2 private to CSF) indicating potential for platinum and/or PARP inhibitor therapy in these individuals. Patient-derived organoids (PDOs) were established using CSF tumour cells from 3 ER+/HER2- and 2 TNBC BCLM cases. WES of PDOs revealed high concordance with genomic variants identified in the matched CSF cfDNA. Therapeutic compound testing revealed 3/5 PDOs did not display sensitivity to methotrexate, the most commonly used BCLM intrathecal treatment. Patient-derived xenograft (PDX) models have been established by mammary fat pad, intraductal, intracardiac and intracerebroventricular injection routes. Conclusion/future plans: WES of CSF cfDNA provides insight into genomic changes in BCLM, including the divergent evolution, and BCLM specific alterations, some of which are potentially targetable. Parallel PDO and PDX models are being used to validate potential drivers and therapeutic targets. Treatment options beyond intrathecal methotrexate are urgently needed and in future might be molecularly tailored based on alterations discovered by CSF cfDNA sequencing. Table 1. Clinical demographicsDemographicsMedian, years (range)Age at BC diagnosis45 (24 – 66)Time from primary BC to BCLM4.7 (0.7 – 14.7)Time from first metastasis to BCLM1.2 (0.0 – 6.5)Histological Typen (%)Lobular10 (48)Ductal8 (38)Mixed ductal/lobular3 (14)Immuno-histochemical phenotypen (%)ER+ HER2-13 (62)TNBC5 (24)ER+ HER2+2 (9)ER- HER2+1 (5)Metastatic sitesn (%)No other metastatic sites (BCLM only)3 (14)Bone12 (57)Serosal8 (38)Brain6 (29)Liver4 (19)Ovary4 (19) Citation Format: Amanda Fitzpatrick, Marjan Iravani, Adam Mills, Eleanor Knight, Thanussuyah Alaguthurai, Vandna Shah, Alicia Okines, Nick Turner, Mark Harries, Syed Haider, Andrew Tutt, Clare Isacke. Genomic profiling of breast cancer leptomeningeal metastasis (BCLM) reveals a divergent evolution and therapeutic targets [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD13-03.

Intrauterine Fetal Blood Transfusion (IUBT) for Rh Incompatibility – 12 Years’ Experience from Pakistan

To determine the perinatal outcome of pregnancies complicated by Rh-alloimmunisation, requiring intrauterine blood transfusion. Observational study. Feto-maternal Unit of Gene Tech Laboratory, Lahore, from 2007 to 2019. A retrospective analysis was done on the data of cases of intrauterine, intravascular blood transfusion given to at-risk foetuses to correct foetal anaemia due to Rh-alloimmunisation or parvovirus B19. All cases, who were eligible to receive IUBT were included in the study. Cases where historic data was not available have been excluded. A total of 305 intrauterine blood transfusion (IUBT) procedures were performed on 127 foetuses. The gestational age ranged from 18-32 weeks at the time of referral. Infra-hepatic part of umbilical vein was preferred for transfusion, but in some cases of anterior placenta, the cord insertion was approached with exception of only two cases where intra-cardiac route was employed. In this study, 71.6% of the babies survived, 14.2% were loss to follow and 14.2% died. IUBT is a safe procedure, especially when performed by experienced hands, and helps save the foetuses at risk. Mothers with Rh-alloimmunisation should be referred before developing hydrops fetalis for better outcome. Key Words: Red cell alloimmunisation, Intrauterine intravascular blood transfusion, Foetal anaemia.

Diazepam Alters the Ultrastructure of Fetal Atrium in Rat

Diazepam (DZ) is a synthetic minor tranquilizer, used in patients with psychological and psychiatric disorders. It is a relaxing sedative, anticonvulsant and antipsychotic. DZ crosses the human placental barrier in mouse. Young women who are addicted to the drug, if they become pregnant and continue to use it, particularly during the first trimester, expose their children to psychomotor disorders. The purpose of this study was to investigate whether DZ administered during pregnancy induces ultrastructural alterations of fetal mouse myocardium. The group (DZ) of pregnant female mice of the CD-1 strain was treated with a single daily dose of 1.0 mg / kg / pc / sc of day 6 to 17 and a group (C) that received saline solution. On day 18 females of both groups were anesthetized, the fetuses were perfused by intracardiac route with 1 % paraformaldehyde and 2.5 % glutaraldehyde, the heart was removed, the atrium was dissected, fixed in 1 % OsO4, it was immersed in epoxy resin. The fine sections were contrasted with uranyl acetate and lead citrate and observed in a transmission electron microscope. In the myocytes of the fetuses of the DZ group, the sarcomers of the compact myocardium were shorter than those of the C group. Areas with disorganized myofibrils were observed. The sarcoplasmic reticulum of some myocytes had distended and fragmented cisterns, altered mitochondria, and abundant polyribosomes were observed. The changes may be due to the effect of DZ on the synthesis of actin and heavy myosin and on cytoplasmic organelles mediated by peripheral benzodiazepine receptors present on the outer membrane of the mitochondria and associated with voltage-dependent calcium channels. Ultrastructural alterations of the atrial myocardium of fetuses of mice exposed to DZ in utero may have postnatal effects.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.

Systemic Gene Delivery by Single-Dose Intracardiac Administration of scAAV2/9 and scAAV2/rh10 Variants in Newborn Rats.

Recombinant adeno-associated virus serotype 9 (rAAV2/9) and pseudotype rhesus-10 (rAAV2/rh10) are used for gene delivery, especially into the central nervous system. Both serotypes cross the blood-brain barrier and mediate stable long-term transduction in dividing and nondividing cells. Among possible routes of administration, intracardiac injection holds the potential for widespread vector diffusion associated with a relatively simple approach. In this study adopting the intracardiac route, we compare the cell-specific tropism and transfection efficacy of a panel of engineered rAAV2/9 and rAAV2/rh10 vectors encoding the enhanced green fluorescent protein. We observed transduction in the brain and peripherally, with a predominant neuronal tropism while the various serotypes achieved different expression patterns.

Abstract 44: Characterization and targeting of a temporal micro-metastatic signature in human brain metastases

Abstract Metastases to the brain (BM) are the most common neoplasms to affect the adult central nervous system, arising in 40% of cancer patients and at a rate 10 times greater than primary brain tumors. Despite the prevalence and poor survival rates, therapeutic strategies for BM remain limited, and a substantial cause is the lack of proper preclinical models available to interrogate the intricacies of BM development. Previous work in our lab utilized BM samples from patient-derived lung-to-brain metastases to successfully establish clinically relevant in vivo models of BM. Here we further characterized the cells responsible for BM, termed brain metastasis initiation cells (BMIC). Patient-derived BMICs were injected via intracranial (BT), intracardiac (IC) and intrathoracic (IT) routes into NOD/SCID mice and re-isolated at different stages of metastatic progression. We isolated cells from primary lung tumors (LT), cells injected via intra-thoracic route that crossed the blood brain barrier to seed the brain forming micro-metastases (BMIT), and cells injected via intracardiac route that seeded large macro-metastases (BMIC). Through RNA sequencing we determined cells from BMIT (micro-metastasis stage) to retain a vastly different genetic profile compared to BMICs isolated at other stages of metastasis. Several of these genes belonged to pathways implicated in autonomic central nervous system neoplasms and neural development. Through connectivity mapping of BMIT profiles we discovered drugs that could inhibit the micro-metatsasis signature, and further in vitro validation revealed apomorphine to reduce BMIC sphere formation and proliferation. In vivo treatment with apomorphine blocked both micro- and macro-metastatic stages of our BMIC model. Of 5 BMIT genes identified to be specifically targeted by apomorphine, KIF16B, TESK2 and SEPW1 were shown to have significant value when applied as an independent prognostic signature in a cohort of lung adenocarcinoma patients. Future work will further validate the efficacy of apomorphine in targeting BMICs in primary lung cancer patient samples. With this work we present a possible new avenue for therapeutic targeting toward the prevention of BM development, where it is anticipated to transform a uniformly fatal disease into one that is eminently more treatable. Citation Format: Mohini Singh, Chitra Venugopal, Tomas Tokar, Nicole McFarlane, Minomi Subhapanditha, David Bakhshinyan, Maleeha Qazi, Parvez Vora, Neil Savage, Naresh K. Murty, Igor Jurisica, Sheila K. Singh. Characterization and targeting of a temporal micro-metastatic signature in human brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 44.

Abstract 1085: Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors

Abstract BACKGROUND: Bone metastasis is a complex process involving reciprocal interplay between cancer and bone cells. Metastatic prostate cancer (PCA) cells secrete extracellular vesicles (EVs), that contain microRNAs (miRNAs), and these can be found in blood. We hypothesize that precursor osteoclasts scavenge PCA EVs, leading to a manipulation of their behavior thereby driving metastasis. METHODS: To test how EV-miRNAs affect metastasis, we stably overexpressed two metastasis-related miRNAs in PCA cells. Bone metastases were induced by inoculating miRNA overexpressing PCA cells into nude mice via intracardiac route with or without the presence of subcutaneous tumors. Tumor progression was monitored by F-luciferase signal (IVIS) while bones were analyzed by microCT ex vivo. miRNAs expression was analyzed by qRT-PCR on precursor osteoclasts isolated from mice blood. Osteoclast differentiation assays were performed using human peripheral mononucleated blood cells on bone chips together with RANKL and M-CSF to drive differentiation. RESULTS: In vivo, intracardiac injection of PC3 cells that overexpress selected miRNAs accelerated the formation of bone metastases (0-85%) compared to control PC3 cells (43%). To determine whether EVs secreted by PCA cells affect osteoclasts, human monocytes were allowed to differentiate to osteoclasts while exposing them to PCA EVs. EVs secreted by control PCA cell lines increased osteoclast differentiation by 22.5% (p<0.01), compared to EVs secreted by RWPE1 noncancerous prostate cells. Exposure to EVs secreted by the miRNA overexpressing PCA cells further enhanced osteoclast formation by an additional 10% (p<0.05). To test whether pro-metastatic miRNAs enhance metastases when mouse osteoclast precursors are pre-stimulated (via EVs or soluble factors), subcutaneous tumours of pro-metastatic cells were grown for 2 weeks. In circulating osteoclast precursors of mice that were pre-stimulated with PCA factors that were secreted by subcutaneous tumors (2 weeks), the miRNA levels were increased by 1.5 - 4 fold (p<0.05), demonstrating that these cells scavenge the secreted tumor miRNAs. Subsequently, mice received an intracardiac injection with matching cells to induce metastases. Trabecular bone volume of >15% was used as indicator for metastasis induced bone loss. Mice that grew both a subcutaneous tumor and also received an intracardiac injection to drive metastasis formation led to an ~30% increased number of mice with >15% bone loss (p<0.01). This indicates that osteoclasts may be manipulated by the cancer cell-secreted factors to form a pro-metastatic niche. CONCLUSION: PCA miRNAs secreted via EVs stimulate osteoclast formation, potentially increasing the formation of overt metastases. Further investigation is needed to unravel novel pathways that can be targeted to prevent bone metastasis. Citation Format: L. Ayse Erozenci, Ning Wang, Albert A. Geldof, Jorn Mulder, Connie R. Jimenez, R. Jeroen van Moorselaar, Allison Gartland, Teun J. de Vries, Irene V. Bijnsdorp. Prostate cancer secreted microRNAs influence early steps in bone metastasis via osteoclast precursors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1085.

The effects of CAPE on oxidative stress and histopathological values in rats treated with subacute dichlorvos

This study was designed to investigate the protective effect of CAPE against dichlorvos induced hepatotoxicity in rats. 40 rats were used in the study, including 10 in each group. Corn oil of 5 mg/kg (solvent) was administered intragastrically to rats in the group 1, dichlorvos of 5 mg/kg was dissolved in corn oil and administered to rats in the group 2 via gavage, dichlorvos (5 mg/kg, via gavage route) and CAPE (10 μM/kg, intraperitoneally) were administered to rats of the group 3 with one-hour interval providing that CAPE treatment was started three days earlier than dichlorvos administration. The rats in the group 4 were administered with 10% solvent ethanol via intraperitoneally. All administrations continued for 15 days and blood of animals was drawn via intracardiac route after they were sacrificed by cervical dislocation under ketamine/xylazine anesthesia. Total antioxidant, total oxidant, and paraoxonase levels were measured from the obtained plasma samples. As a result of dichlorvos administration, paraoxonase activity and total antioxidant levels decreased; whereas, total oxidant levels increased. Histopathological analysis revealed that liver tissue appeared normal in control groups (1 and 4); on the other hand, there were degeneration, congestion, cellular infiltration, and necrotic areas in the group administered with dichlorvos. Even though frequency of lesions decreased, similar lesions were observed in the group with Dichlorvos+CAPE. Consequently, it was determined that while dichlorvos administration increased oxidative stress, CAPE administration had a protective potential increasing antioxidant capacity.

Haemato-Biochemical Changes in Staphylococcal Species Induced Mastitis in Mouse Model

In this study, haemato-biochemical changes in mouse mastitis by inoculating Staphylococcus epidermidis, S. chromogenes, S. haemolyticus and S. Aureus isolated from bovine milk was studied. Blood was collected by intracardiac route at 6, 12, 24, 48, 72 and 96 h. Haematological changes noticed were: white blood cells (WBC) decreased in S. haemolyticus and S. chromogenes at 48 h; S. haemolyticus and S. aureus at 96 h. S. haemolyticus showed increased lymphocyte at 24 and 48 h and in S. aureus at 48 h. Neutrophil was decreased in S. haemolyticus and S. aureus group. Glucose increased at 6 h with three Coagulase Negative Staphylococcus (CNS) species and S. aureus. Glucose and total protein increased in S. chromogenes infected mice. Globulin levels showed decrease in S. epidermidis and S. aureus. Albumin globulin ratio showed decreasing trend in S. chromogenes and S. haemolyticus inoculated mice. Aspartate aminotransferase (AST) level increased at 6 and 12 h in all infected groups. Alanine aminotransferase increased at 12 and 48 h in S. chromogenes and S. aureus respectively. Lactate dehydrogenase (LDH) increased at 6 h in all the infected mice. Thus, three CNS species induced haemato-biochemical changes in haemoglobin, WBC, neutrophil, glucose, total protein, AST and LDH which may be used as indicators for diagnosis of CNS species mastitis in mice. Keywords: Staphylococcus species, mouse mastitis, haemato-biochemical changes Cite this Article Krishnamoorthy P, Satyanarayana ML, Shome BR, et al. Haemato-Biochemical Changes in Staphylococcal Species induced Mastitis in Mouse Model + . Research & Reviews: Journal of Veterinary Science and Technology . 2015; 4(3): 8–14p.

Transient iatrogenic heart block following foetal intracardiac transfusion for severe twin anaemia-polycythaemia sequence

Abstract Background: Intrauterine blood transfusion is an important treatment of foetal anaemia. Although the standard access to the foetal vasculature for transfusion is the umbilical vein, the intracardiac route is used when foetal or placental positions make other accesses technically challenging. Intrauterine, intracardiac blood transfusion is associated with complications including haemopericardium, damage to cardiac tissues and foetal bradycardia. Highlights of the present report: We report a case of monochorionic twins with twin anaemia-polycythaemia sequence (TAPS). Intracardiac, intrauterine blood transfusion of the donor twin was complicated by haemopericardium and sustained bradycardia which necessitated delivery by emergency caesarean section. Postnatally, the bradycardia was sustained and was diagnosed electrocardiographically as heart block, which spontaneously reversed on the second day after birth. The management of heart block in the neonatal period is discussed. Conclusion: Foetal intracardiac intrauterine blood transfusion can be associated with transient congenital heart block (CHB).

Clinical, hematological and biochemical alterations in hamster (Mesocricetus auratus) experimentally infected with Leishmania infantum through different routes of inoculation.

BackgroundLeishmaniasis remains among the most important parasitic diseases in the developing world and visceral leishmaniasis (VL) is the most fatal. The hamster Mesocricetus auratus is a susceptible model for the characterization of the disease, since infection of hamsters with L. infantum reproduces the clinical and pathological features of human VL. In this context, it provides a unique opportunity to study VL in its active form. The main goal of this study was to evaluate the clinical, biochemical, and hematological changes in male hamsters infected through different routes and strains of L. infantum.MethodsIn the current study, hamsters (Mesocricetus auratus) were infected with the L. infantum strains (WHO/MHOM/BR/74/PP75 and MCAN/BR/2008/OP46) by intradermal, intraperitoneal and intracardiac routes. The animals were monitored for a nine month follow-up period.ResultsThe hamsters showed clinical signs similar to those observed in classical canine and human symptomatic VL, including splenomegaly, severe weight loss, anemia, and leucopenia. Therefore the OP46 strain was more infective, clinical signs were more frequent and more exacerbated in IC group with 80 to 100 % of the animals showing splenomegaly, in the last month infection. Additionally, desquamation, hair loss and external mucocutaneous lesions and ulcers localized in the snout, accompanied by swelling of the paws in all animals, were observed. Consequently, the animals presented severe weight loss/cachexia, hunched posture, an inability to eat or drink, and non-responsiveness to external stimuli. Furthermore, regardless of strain, route of inoculum and time assessed, the animals showed renal and hepatic alterations, with increased serum levels of urea and creatinine as well as elevated serum levels of aspartate aminotransferase and alanine aminotransferase.ConclusionsThese results strongly suggest that the inoculation through the intracardiac route resulted in a higher severity among infections, especially in the sixth and ninth month after infection via intracardiac, exhibited clinical manifestations and biochemical/hematological findings similar to human visceral leishmaniasis. Therefore, we suggest that this route must be preferentially used in experimental infections for pathogenesis studies of VL in the hamster model.

Presence of 'ghosts' and mortality after transvenous lead extraction.

The number of cardiovascular implantable electronic devices has increased progressively, leading to an increased need for transvenous lead extraction (TLE) due to device infections. Previous studies described 'ghost' as a post-removal, new, tubular, mobile mass detected by echocardiography following the lead's intracardiac route in the right-sided heart chambers, associated with diagnosis of cardiac device-related infective endocarditis. We aimed to analyse the association between 'ghosts' assessed by transesophageal echocardiography (TEE) and intracardiac echocardiography (ICE) and mortality in patients undergoing TLE. We prospectively enrolled 217 patients (70 ± 13 years; 164 males) undergoing TLE for systemic infection (139), local device infection (67), and lead malfunction (11). All patients underwent TEE before and 48 h after TLE and ICE during TLE. Patients were allocated to two groups: either with (Group 1) or without (Group 2) post-procedural 'ghost'. Mid-term clinical follow-up was obtained in all patients (11 months, IQR 1-34 months). We identified 30 (14%) patients with 'ghost', after TLE. The significant predictors of 'ghost' were Charlson co-morbidity index (HR = 1.24, 95% CI 1.04-1.48, P = 0.03) and diagnosis of endocarditis assessed by ICE (HR = 1.82, 95% CI 1.01-3.29, P = 0.04). Mortality was higher in Group 1 than in Group 2 (28 vs. 5%; log-rank P < 0.001). Independent predictors of mid-term mortality were the presence of 'ghost' and systemic infection as the clinical presentation of device infection (HR = 3.47, 95% CI 1.18-10.18, P = 0.002; HR = 3.39, 95% CI 1.15-9.95, P = 0.001, respectively). The presence of 'ghost' could be an independent predictor of mortality after TLE, thus identifying a subgroup of patients who need closer clinical surveillance to promptly detect any complications.