Publications by authors named "Kathleen Gabrielson"

101 Publications

Transport-driven engineering of liposomes for delivery of α-particle radiotherapy to solid tumors: effect on inhibition of tumor progression and onset delay of spontaneous metastases.

Eur J Nucl Med Mol Imaging 2021 Jun 12. Epub 2021 Jun 12.

Chemical and Biomolecular Engineering (ChemBE), Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA.

Purpose: Highly cytotoxic α-particle radiotherapy delivered by tumor-selective nanocarriers is evaluated on metastatic Triple Negative Breast Cancer (TNBC). On vascularized tumors, the limited penetration of nanocarriers (<50-80 μm) combined with the short range of α-particles (40-100 μm) may, however, result in only partial tumor irradiation, compromising efficacy. Utilizing the α-particle emitter Actinium-225 (Ac), we studied how the therapeutic potential of a general delivery strategy using nanometer-sized engineered liposomes was affected by two key transport-driven properties: (1) the release from liposomes, when in the tumor interstitium, of the highly diffusing Ac-DOTA that improves the uniformity of tumor irradiation by α-particles and (2) the adhesion of liposomes on the tumors' ECM that increases liposomes' time-integrated concentrations within tumors and, therefore, the tumor-delivered radioactivities.

Methods: On an orthotopic MDA-MB-231 TNBC murine model forming spontaneous metastases, we evaluated the maximum tolerated dose (MTD), biodistributions, and control of tumor growth and/or spreading after administration of Ac-DOTA-encapsulating liposomes, with different combinations of the two transport-driven properties.

Results: At 83% of MTD, Ac-DOTA-encapsulating liposomes with both properties (1) eliminated formation of spontaneous metastases and (2) best inhibited the progression of orthotopic xenografts, compared to liposomes lacking one or both properties. These findings were primarily affected by the extent of uniformity of the intratumoral microdistributions of Ac followed by the overall tumor uptake of radioactivity. At the MTD, long-term toxicities were not detected 9.5 months post administration.

Conclusion: Our findings demonstrate the potential of a general, transport-driven strategy enabling more uniform and prolonged solid tumor irradiation by α-particles without cell-specific targeting.
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http://dx.doi.org/10.1007/s00259-021-05406-zDOI Listing
June 2021

An improved At-labeled agent for PSMA-targeted alpha therapy.

J Nucl Med 2021 Jun 4. Epub 2021 Jun 4.

Johns Hopkins Medical Institutions, United States.

α-Particle emitters targeting the prostate-specific membrane antigen (PSMA) proved effective in treating patients with prostate cancer who were unresponsive to the corresponding β-particle therapy. Astatine-211 is an α-emitter that may engender less toxicity than other α-emitting agents. We synthesized a new At-labeled radiotracer targeting PSMA that resulted from the search for a pharmacokinetically optimized agent. A small series of I-labeled compounds were synthesized from their tin precursors to evaluate the effect of location of radiohalogen within the molecule and the presence of lutetium in the chelate on biodistribution. On that basis, At-VK-02-90-Lu was selected and evaluated in cell uptake and internalization studies, biodistribution and PSMA+ PC3 PIP tumor growth control in experimental flank and metastatic (PC3-ML-Luc) models. A long-term (13-month) toxicity study was performed for At-VK-02-90-Lu, including tissue chemistries and histopathology. The radiochemical yield of At-VK-02-90-Lu was 17.8 ± 8.2%. Lead compound At-VK-02-90-Lu demonstrated total uptake within PSMA+ PC3 PIP cells of 13.4 ± 0.5% of the input dose after 4 h of incubation with little uptake in control cells. In SCID mice, At-VK-02-90-Lu provided 30.6 ± 4.8 percentage of injected dose per gram (%ID/g) of uptake in PSMA+ PC3 PIP tumor at 1 h post-injection that decreased to 9.46 ± 0.96 %ID/g by 24 h. Tumor-to-salivary gland and tumor-to-kidney ratios were 129 ± 99 at 4 h and 130 ± 113 at 24 h, respectively. De-astatination was not significant (stomach 0.34 ± 0.20%ID/g at 4 h). Dose-dependent survival was demonstrated at higher doses (>1.48 MBq) in both flank and metastatic models. There was little off-target toxicity as demonstrated by hematopoietic stability, unchanged tissue chemistries, weight gain rather than loss throughout treatment, and favorable histopathology. Compound At-VK-02-90-Lu or close analogs may provide limited and acceptable toxicity while retaining efficacy in management of prostate cancer.
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http://dx.doi.org/10.2967/jnumed.121.262098DOI Listing
June 2021

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate.

J Vis Exp 2021 May 3(171). Epub 2021 May 3.

Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine; Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University;

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.
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http://dx.doi.org/10.3791/62139DOI Listing
May 2021

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile.

Sci Rep 2021 Mar 29;11(1):7114. Epub 2021 Mar 29.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical Institutions, Baltimore, MD, USA.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (K = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.
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http://dx.doi.org/10.1038/s41598-021-86551-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007718PMC
March 2021

A Procarcinogenic Colon Microbe Promotes Breast Tumorigenesis and Metastatic Progression and Concomitantly Activates Notch and β-Catenin Axes.

Cancer Discov 2021 May 6;11(5):1138-1157. Epub 2021 Jan 6.

Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.

The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic (ETBF), which secretes toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit "BFT memory" from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic . This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the β-catenin and Notch1 axis as its functional mediators, and proposes the concept of "BFT memory" that can have far-reaching biological implications after initial exposure to ETBF. SIGNIFICANCE: is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a "BFT memory" with long-term implications, functionally mediated by the β-catenin and Notch1 pathways..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0537DOI Listing
May 2021

Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon honokiol treatment.

Cell Death Discov 2020 6;6:81. Epub 2020 Sep 6.

Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231 USA.

Cancer cells hijack autophagy pathway to evade anti-cancer therapeutics. Many molecular signaling pathways associated with drug-resistance converge on autophagy induction. Honokiol (HNK), a natural phenolic compound purified from , has recently been shown to impede breast tumorigenesis and, in the present study, we investigated whether breast cancer cells evoke autophagy to modulate therapeutic efficacy and functional networks of HNK. Indeed, breast cancer cells exhibit increased autophagosomes-accumulation, MAP1LC3B-II/LC3B-II-conversion, expression of ATG proteins as well as elevated fusion of autophagosomes and lysosomes upon HNK treatment. Breast cancer cells treated with HNK demonstrate significant growth inhibition and apoptotic induction, and these biological processes are blunted by macroautophagy/autophagy. Consequently, inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of and effectively increase HNK-mediated apoptotic induction and growth inhibition. Next, we explored the functional impact of tumor suppressor STK11 in autophagy induction in HNK-treated cells. STK11-silencing abrogates LC3B-II-conversion, and blocks autophagosome/lysosome fusion and lysosomal activity as illustrated by LC3B-Rab7 co-staining and DQ-BSA assay. Our results exemplify the cytoprotective nature of autophagy invoked in HNK-treated breast cancer cells and put forth the notion that a combined strategy of autophagy inhibition with HNK would be more effective. Indeed, HNK and chloroquine (CQ) show synergistic inhibition of breast cancer cells and HNK-CQ combination treatment effectively inhibits breast tumorigenesis and metastatic progression. Tumor-dissociated cells from HNK-CQ treated tumors exhibit abrogated invasion and migration potential. Together, these results implicate that breast cancer cells undergo cytoprotective autophagy to circumvent HNK and a combined treatment with HNK and CQ can be a promising therapeutic strategy for breast cancer.
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http://dx.doi.org/10.1038/s41420-020-00315-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7475061PMC
September 2020

Development of 5D3-DM1: A Novel Anti-Prostate-Specific Membrane Antigen Antibody-Drug Conjugate for PSMA-Positive Prostate Cancer Therapy.

Mol Pharm 2020 09 17;17(9):3392-3402. Epub 2020 Aug 17.

The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205, United States.

Prostate cancer (PC) is a potentially high-risk disease and the most common cancer in American men. It is a leading cause of cancer-related deaths in men in the US, second only to lung and bronchus cancer. Advanced and metastatic PC is initially treated with androgen deprivation therapy (ADT), but nearly all cases eventually progress to castrate-resistant prostate cancer (CRPC). CRPC is incurable in the metastatic stage but can be slowed by some conventional chemotherapeutics and second-generation ADT, such as enzalutamide and abiraterone. Therefore, novel therapeutic strategies are urgently needed. Prostate-specific membrane antigen (PSMA) is overexpressed in almost all aggressive PCs. PSMA is widely used as a target for PC imaging and drug delivery. Anti-PSMA monoclonal antibodies (mAbs) have been developed as bioligands for diagnostic imaging and targeted PC therapy. However, these mAbs are successfully used in PC imaging and only a few have gone beyond phase-I for targeted therapy. The 5D3 mAb is a novel, high-affinity, and fast-internalizing anti-PSMA antibody. Importantly, 5D3 mAb demonstrates a unique pattern of cellular localization to the centrosome after internalization in PSMA(+) PC3-PIP cells. These characteristics make 5D3 mAb an ideal bioligand to deliver tubulin inhibitors, such as mertansine, to the cell centrosome, leading to mitotic arrest and elimination of dividing PC cells. We have successfully developed a 5D3 mAb- and mertansine (DM1)-based antibody-drug conjugate (ADC) and evaluated it for binding affinity, internalization, and cytotoxicity. The therapeutic efficacy of 5D3-DM1 ADC was evaluated in PSMA(+) PC3-PIP and PSMA(-) PC3-Flu mouse models of human PC. This therapeutic study has revealed that this new anti-PSMA ADC can successfully control the growth of PSMA(+) tumors without inducing systemic toxicity.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957835PMC
September 2020

Prenatal stress enhances NNK-induced lung tumors in A/J mice.

Carcinogenesis 2020 12;41(12):1713-1723

Sidney Kimmel Cancer Center, Department of Oncology, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA.

Children born to women who experience stress during pregnancy have an increased risk of cancer in later life, but no previous animal studies have tested such a link. We questioned whether prenatal stress (PS) in A/J mice affected the development of lung tumors after postnatal response to tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Timed-bred A/J mice were randomly assigned on gestation day 12.5 to PS by restraint for 5 consecutive days or control (no restraint). Adult offspring of control and stressed pregnancies were all treated with three NNK injections (50 mg/kg every other day) and euthanized 16 weeks later to examine their lungs. Compared with controls, PS dams exhibited significantly increased levels of plasma corticosterone, increased adrenal weights and decreased fetus weights without fetal loss. Prenatally stressed litters had a significantly higher neonatal death rate within first week of life, and surviving male and female offspring developed lung epithelial proliferations with increase multiplicity, increased area and aggressive morphology. PS also induced more advanced atypical adenomatous hyperplasia lesions. We found no difference in lung NNK-derived methyl DNA adducts, but PS did significantly enhance CD3+ T cell and Foxp3+ T cell tumor infiltration. PS significantly increases multiplicity, area of NNK-induced lung tumors and advanced morphology. PS did not affect production of NNK-derived methyl DNA adducts but did increase lymphocytic infiltration of lung tumors. To our knowledge, this is the first animal model of PS with evaluation of cancer development in offspring.
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http://dx.doi.org/10.1093/carcin/bgaa033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947993PMC
December 2020

(,)-1,12-Dimethylspermine can mitigate abnormal spermidine accumulation in Snyder-Robinson syndrome.

J Biol Chem 2020 03 29;295(10):3247-3256. Epub 2020 Jan 29.

Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, Maryland 21287. Electronic address:

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase () gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (,)-1,12-dimethylspermine (MeSPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. MeSPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate MeSPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. MeSPM administration to mice revealed that MeSPM significantly decreases spermidine levels in multiple tissues. Importantly, MeSPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (,)-diastereomer of 1,12-MeSPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.
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http://dx.doi.org/10.1074/jbc.RA119.011572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062180PMC
March 2020

Enhancing the abscopal effect of radiation and immune checkpoint inhibitor therapies with magnetic nanoparticle hyperthermia in a model of metastatic breast cancer.

Int J Hyperthermia 2019 11;36(sup1):47-63

Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Enhancing immune responses in triple negative breast cancers (TNBCs) remains a challenge. Our study aimed to determine whether magnetic iron oxide nanoparticle (MION) hyperthermia (HT) can enhance abscopal effects with radiotherapy (RT) and immune checkpoint inhibitors (IT) in a metastatic TNBC model. One week after implanting 4T1-luc cells into the mammary glands of BALB/c mice, tumors were treated with RT (3 × 8 Gy)±local HT, mild (HT, 43 °C/20 min) or partially ablative (HT, 45 °C/5 min plus 43 °C/15 min),±IT with anti-PD-1 and anti-CTLA-4 antibodies (both 4 × 10 mg/kg, i.p.). Tumor growth was measured daily. Two weeks after treatment, lungs and livers were harvested for histopathology evaluation of metastases. Compared to untreated controls, all treatment groups demonstrated a decreased tumor volume; however, when compared against surgical resection, only RT + HT+IT, RT + HT+IT and RT + HT had similar or smaller tumors. These cohorts showed more infiltration of CD3 T-lymphocytes into the primary tumor. Tumor growth effects were partially reversed with T-cell depletion. Combinations that proved most effective for primary tumors generated modest reductions in numbers of lung metastases. Conversely, numbers of lung metastases showed potential to increase following HT + IT treatment, particularly when compared to RT. Compared to untreated controls, there was no improvement in survival with any treatment. Single-fraction MION HT added to RT + IT improved local tumor control and recruitment of CD3 T-lymphocytes, with only a modest effect to reduce lung metastases and no improvement in overall survival. HT + IT showed potential to increase metastatic dissemination to lungs.
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http://dx.doi.org/10.1080/02656736.2019.1685686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017719PMC
November 2019

Engineered nanoparticles for systemic siRNA delivery to malignant brain tumours.

Nanoscale 2019 Nov 15;11(42):20045-20057. Epub 2019 Oct 15.

Department of Biomedical Engineering and the Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA. and Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD 21218, USA. and Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA and Departments of Neurosurgery, Oncology, and Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA and Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21231, USA and Sidney Kimmel Comprehensive Cancer Center and the Bloomberg∼Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Improved delivery materials are needed to enable siRNA transport across biological barriers, including the blood-brain barrier (BBB), to treat diseases like brain cancer. We engineered bioreducible nanoparticles for systemic siRNA delivery to patient-derived glioblastoma cells in an orthotopic mouse tumor model. We first utilized a newly developed biomimetic in vitro model to evaluate and optimize the performance of the engineered bioreducible nanoparticles at crossing the brain microvascular endothelium. We performed transmission electron microscopy imaging which indicated that the engineered nanoparticles are able to cross the BBB endothelium via a vesicular mechanism. The nanoparticle formulation engineered to best cross the BBB model in vitro led to safe delivery across the BBB to the brain in vivo. The nanoparticles were internalized by human brain cancer cells, released siRNA to the cytosol via environmentally-triggered degradation, and gene silencing was obtained both in vitro and in vivo. This study opens new frontiers for the in vitro evaluation and engineering of nanomedicines for delivery to the brain, and reports a systemically administered biodegradable nanocarrier for oligonucleotide delivery to treat glioma.
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http://dx.doi.org/10.1039/c9nr04795fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924015PMC
November 2019

FATAL RANAVIRUS INFECTION IN A GROUP OF ZOO-HOUSED MELLER'S CHAMELEONS ().

J Zoo Wildl Med 2019 Sep;50(3):696-705

Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA,

A group of five juvenile Meller's chameleons () experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle () group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.
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http://dx.doi.org/10.1638/2018-0044DOI Listing
September 2019

Fropofol prevents disease progression in mice with hypertrophic cardiomyopathy.

Cardiovasc Res 2020 05;116(6):1175-1185

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, 1800 Orleans Street, Zayed Tower 6208, Baltimore, MD 21287, USA.

Aims: Increased myofilament contractility is recognized as a crucial factor in the pathogenesis of hypertrophic cardiomyopathy (HCM). Direct myofilament desensitization might be beneficial in preventing HCM disease progression. Here, we tested whether the small molecule fropofol prevents HCM phenotype expression and disease progression by directly depressing myofilament force development.

Methods And Results: Force, intracellular Ca2+, and steady-state activation were determined in isolated trabecular muscles from wild-type (WT) and transgenic HCM mice with heterozygous human α-myosin heavy chain R403Q mutation (αMHC 403/+). αMHC 403/+ HCM mice were treated continuously with fropofol by intraperitoneal infusion for 12 weeks. Heart tissue was analysed with histology and real-time PCR of prohypertrophic and profibrotic genes. Fropofol decreased force in a concentration-dependent manner without significantly altering [Ca2+]i in isolated muscles from both WT and αMHC 403/+ HCM mouse hearts. Fropofol also depressed maximal Ca2+-activated force and increased the [Ca2+]i required for 50% activation during steady-state activation. In whole-animal studies, chronic intra-abdominal administration of fropofol prevented hypertrophy development and diastolic dysfunction. Chronic fropofol treatment also led to attenuation of prohypertrophic and profibrotic gene expression, reductions in cell size, and decreases in tissue fibrosis.

Conclusions: Direct inhibition of myofilament contraction by fropofol prevents HCM disease phenotypic expression and progression, suggesting that increased myofilament contractile force is the primary trigger for hypertrophy development and HCM disease progression.
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http://dx.doi.org/10.1093/cvr/cvz218DOI Listing
May 2020

Noninvasive Monitoring of Allogeneic Stem Cell Delivery with Dual-Modality Imaging-Visible Microcapsules in a Rabbit Model of Peripheral Arterial Disease.

Stem Cells Int 2019 14;2019:9732319. Epub 2019 Mar 14.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, MD, USA.

Stem cell therapies, although promising for treating peripheral arterial disease (PAD), often suffer from low engraftment rates and the inability to confirm the delivery success and track cell distribution and engraftment. Stem cell microencapsulation combined with imaging contrast agents may provide a means to simultaneously enhance cell survival and enable cell tracking with noninvasive imaging. Here, we have evaluated a novel MRI- and X-ray-visible microcapsule formulation for allogeneic mesenchymal stem cell (MSC) delivery and tracking in a large animal model. Bone marrow-derived MSCs from male New Zealand White rabbits were encapsulated using a modified cell encapsulation method to incorporate a dual-modality imaging contrast agent, perfluorooctyl bromide (PFOB). PFOB microcapsules (PFOBCaps) were then transplanted into the medial thigh of normal or PAD female rabbits. MSC viability remained high (79 ± 5% at 4 weeks of postencapsulation), and as few as two and ten PFOBCaps could be detected in phantoms using clinical C-arm CT and F MRI, respectively. Successful injections of PFOBCaps in the medial thigh of normal ( = 15) and PAD ( = 16) rabbits were demonstrated on C-arm CT at 1-14 days of postinjection. Using F MRI, transplanted PFOBCaps were clearly identified as "hot spots" and showed one-to-one correspondence to the radiopacities on C-arm CT. Concordance of F MRI and C-arm CT locations of PFOBCaps with postmortem locations was high (95%). Immunohistological analysis revealed high MSC survival in PFOBCaps (>56%) two weeks after transplantation while naked MSCs were no longer viable beyond three days after delivery. These findings demonstrate that PFOBCaps could maintain cell viability even in the ischemic tissue and provide a means to monitor cell delivery and track engraftment using clinical noninvasive imaging systems.
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http://dx.doi.org/10.1155/2019/9732319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437732PMC
March 2019

Stationary phase persister/biofilm microcolony of Borrelia burgdorferi causes more severe disease in a mouse model of Lyme arthritis: implications for understanding persistence, Post-treatment Lyme Disease Syndrome (PTLDS), and treatment failure.

Discov Med 2019 03;27(148):125-138

Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.

Although most patients with Lyme disease can be cured with a 2-4 week antibiotic therapy, about 10-20% of patients continue to suffer prolonged persistent symptoms, a condition called post-treatment Lyme disease syndrome (PTLDS). The cause for PTLDS is unclear and hotly debated. Borrelia burgdorferi develops morphological variants under stress conditions but their significance is not clear. Here we isolated the biofilm-like microcolony (MC) and planktonic (spirochetal form and round body) (SP) variant forms from the stationary phase culture of B. burgdorferi and showed that the MC and SP variant forms were not only more tolerant to the current Lyme antibiotics but also caused more severe arthritis in mice than the log phase spirochete form (LOG). We propose to divide the persistent Lyme disease into two categories: (1) early development of persistent disease from inoculation with persister/biofilm at the beginning of infection introduced by tick bites, or Type I persistent disease (i.e., PTLDS); and (2) late development of persistent disease due to initial infection not being diagnosed or treated in time such that the infection develops into late persistent disease, or Type II persistent disease. Importantly, we show that the murine infection caused by LOG could be eradicated by ceftriaxone whereas the persistent infection established with MC could not be eradicated by doxycycline (Doxy), ceftriaxone (CefT), or vancomycin (Van), or Doxy+CefT or Van+CefT, but could only be eradicated by the persister drug combination daptomycin+doxycycline+ceftriaxone. We conclude that varying levels of persistence and pathologies of Borrelia infection and the corresponding different treatment responses are mostly dictated by the heterogeneous B. burgdorferi variant forms inoculated at the time of tick bites. These findings may have broad implications for understanding pathogenesis and treatment of not only persistent Lyme disease but also other persistent infections in general and call for studies to evaluate if treatment of persistent infections with persister drug combination regimens is more effective than the current mostly single-antibiotic monotherapy.
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March 2019

BLT-Immune Humanized Mice as a Model for Nivolumab-Induced Immune-Mediated Adverse Events: Comparison of the NOG and NOG-EXL Strains.

Toxicol Sci 2019 05;169(1):194-208

Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, U.S. Food & Drug Administration, Silver Spring, Maryland.

Checkpoint inhibitors represent a new class of therapeutics in the treatment of cancer that has demonstrated remarkable clinical effectiveness. However, some patients have experienced serious immune-mediated adverse effects including pneumonitis, hepatitis, colitis, nephritis, dermatitis, encephalitis, and adrenal or pituitary insufficiency. These adverse events were not predicted by nonclinical studies. To determine if bone marrow-liver-thymus (BLT) immune humanized mice could demonstrate these adverse effects, we studied the effect of nivolumab on 2 strains of BLT-humanized mice, NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) and NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(SV40/HTLV-IL3, CSF2)10-7Jic/JicTac (NOG-EXL). Mice were treated with 2.5, 5.0, or 10.0 mg/kg nivolumab or saline twice weekly for 28 days. BLT-NOG mice had significantly reduced survival compared with BLT-NOG-EXL mice. In spite of the difference in survival, both BLT-humanized strains showed adverse reactions similar to those reported in humans, including pneumonitis and hepatitis, with nephritis, dermatitis and adrenalitis also noted in some individuals. Additional histopathologic findings included pancreatic atrophy, myositis, and osteomyelitis in some animals. T-cell activation increased with concomitant loss of PD-1 detection. These findings show that BLT immune humanized mice can demonstrate immune-mediated adverse effects of antiPD1 therapy, and may represent a model that can be used to better understand toxicity of this class of drugs.
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http://dx.doi.org/10.1093/toxsci/kfz045DOI Listing
May 2019

In Vivo Imaging With Confirmation by Histopathology for Increased Rigor and Reproducibility in Translational Research: A Review of Examples, Options, and Resources.

ILAR J 2018 12;59(1):80-98

Department of Radiology, Miller Research Building Molecular Imaging Service Center, Johns Hopkins University, Baltimore, Maryland.

Preclinical noninvasive imaging can be an indispensable tool for studying animal models of disease. In vivo imaging to assess anatomical, functional, and molecular features requires verification by a comparison to the macroscopic and microscopic morphological features, since all noninvasive in vivo imaging methods have much lower resolution than standard histopathology. Comprehensive pathological evaluation of the animal model is underutilized; yet, many institutions have veterinary or human pathologists with necessary comparative pathology expertise. By performing a rigorous comparison to gross or histopathology for image interpretation, these trained individuals can assist scientists with the development of the animal model, experimental design, and evaluation of the in vivo imaging data. These imaging and pathology corroboration studies undoubtedly increase scientific rigor and reproducibility in descriptive and hypothesis-driven research. A review of case examples including ultrasound, nuclear, optical, and MRI is provided to illustrate how a wide range of imaging modalities data can be confirmed by gross or microscopic pathology. This image confirmation and authentication will improve characterization of the model and may contribute to decreasing costs and number of animals used and to more rapid translation from preclinical animal model to the clinic.
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http://dx.doi.org/10.1093/ilar/ily010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6645176PMC
December 2018

Regulatory Forum Opinion Piece*: Imaging Applications in Toxicologic Pathology-Recommendations for Use in Regulated Nonclinical Toxicity Studies.

Toxicol Pathol 2017 06;45(4):444-471

8 Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Available imaging systems for use in preclinical toxicology studies increasingly show utility as important tools in the toxicologic pathologist's armamentarium, permit longitudinal evaluation of functional and morphological changes in tissues, and provide important information such as organ and lesion volume not obtained by conventional toxicology study parameters. Representative examples of practical imaging applications in toxicology research and preclinical studies are presented for ultrasound, positron emission tomography/single-photon emission computed tomography, optical, magnetic resonance imaging, and matrix-assisted laser desorption ionization-imaging mass spectrometry imaging. Some of the challenges for making imaging systems good laboratory practice-compliant for regulatory submission are presented. Use of imaging data on a case-by-case basis as part of safety evaluation in regulatory submissions is encouraged.
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http://dx.doi.org/10.1177/0192623317710014DOI Listing
June 2017

Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry.

PLoS One 2017 4;12(5):e0176958. Epub 2017 May 4.

Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417523PMC
September 2017

RK-33 Radiosensitizes Prostate Cancer Cells by Blocking the RNA Helicase DDX3.

Cancer Res 2016 11 12;76(21):6340-6350. Epub 2016 Sep 12.

Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland.

Despite advances in diagnosis and treatment, prostate cancer is the most prevalent cancer in males and the second highest cause of cancer-related mortality. We identified an RNA helicase gene, DDX3 (DDX3X), which is overexpressed in prostate cancers, and whose expression is directly correlated with high Gleason scores. Knockdown of DDX3 in the aggressive prostate cancer cell lines DU145 and 22Rv1 resulted in significantly reduced clonogenicity. To target DDX3, we rationally designed a small molecule, RK-33, which docks into the ATP-binding domain of DDX3. Functional studies indicated that RK-33 preferentially bound to DDX3 and perturbed its activity. RK-33 treatment of prostate cancer cell lines DU145, 22Rv1, and LNCaP (which have high DDX3 levels) decreased proliferation and induced a G phase cell-cycle arrest. Conversely, the low DDX3-expressing cell line, PC3, exhibited few changes following RK-33 treatment. Importantly, combination studies using RK-33 and radiation exhibited synergistic effects both in vitro and in a xenograft model of prostate cancer demonstrating the role of RK-33 as a radiosensitizer. Taken together, these results indicate that blocking DDX3 by RK-33 in combination with radiation treatment is a viable option for treating locally advanced prostate cancer. Cancer Res; 76(21); 6340-50. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576499PMC
November 2016

An Fc-Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor.

Chembiochem 2016 10 26;17(20):1951-1960. Epub 2016 Aug 26.

Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 302 Wood Basic Science Building, 725 N Wolfe St., Baltimore, MD, 21205, USA.

The adenosine A receptor (A R) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A R have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well-established potent and selective small molecule A R antagonist, ZM-241385 (ZM), has a short pharmacokinetic half-life and the potential for systemic toxicity due to A R effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein-small molecule conjugate, Fc-ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc-ZM was a potent A R antagonist, as measured by a cell-based cAMP assay. Cell-based assays also revealed that Fc-ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A R. We found that Fc-ZM, compared with the small molecule ZM, was a superior A R antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.
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http://dx.doi.org/10.1002/cbic.201600337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5292873PMC
October 2016

Echocardiographic Characterization of a Murine Model of Hypertrophic Obstructive Cardiomyopathy Induced by Cardiac-specific Overexpression of Epidermal Growth Factor Receptor 2.

Comp Med 2016 ;66(4):268-77

Department of Molecular and Comparative Pathobiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA.

Although rare, hypertrophic cardiomyopathy (HCM) with midventricular obstruction is often associated with severe symptoms and complications. None of the existing HCM animal models display this particular phenotype. Our group developed a mouse line that overexpresses the ErbB2 receptor (ErbB2(tg)) in cardiomyocytes; we previously showed that the ErbB2 receptor induces cardiomyocyte hypertrophy, myocyte disarray, and fibrosis compatible with HCM. In the current study, we sought to further echocardiographically characterize the ErbB2(tg) mouse line as a model of HCM. Compared with their wild-type littermates, ErbB2(tg) mice show increased left ventricular (LV) mass, concentric LV hypertrophy, and papillary muscle hypertrophy. This hypertrophy was accompanied by diastolic dysfunction, expressed as reduced E:A ratio, prolonged deceleration time, and elevated E:e' ratio. In addition, ErbB2(tg) mice consistently showed midcavity obstruction with elevated LV gradients, and the flow profile revealed a prolonged pressure increase and a delayed peak, indicating dynamic obstruction. The ejection fraction was increased in ErbB2(tg) mice, due to reduced end-diastolic and end-systolic LV volumes. Furthermore, systolic radial strain and systolic radial strain rate but not systolic circumferential strain and longitudinal strain were decreased in ErbB2(tg) compared with wild-type mice. In conclusion, the phenotype of the ErbB2(tg) mouse model is consistent with midventricular HCM in many important aspects, including massive LV hypertrophy, diastolic dysfunction, and midcavity obstruction. This pattern is unique for a small animal model, suggesting that ErbB2(tg) mice may be well suited for research into the hemodynamics and treatment of this rare form of HCM.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4983168PMC
October 2017

A preclinical murine model for the early detection of radiation-induced brain injury using magnetic resonance imaging and behavioral tests for learning and memory: with applications for the evaluation of possible stem cell imaging agents and therapies.

J Neurooncol 2016 06 28;128(2):225-33. Epub 2016 Mar 28.

Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Traylor Building 217, Baltimore, MD, 21205, USA.

Stem cell therapies are being developed for radiotherapy-induced brain injuries (RIBI). Magnetic resonance imaging (MRI) offers advantages for imaging transplanted stem cells. However, most MRI cell-tracking techniques employ superparamagnetic iron oxide particles (SPIOs), which are difficult to distinguish from hemorrhage. In current preclinical RIBI models, hemorrhage occurs concurrently with other injury markers. This makes the evaluation of the recruitment of transplanted SPIO-labeled stem cells to injury sites difficult. Here, we developed a RIBI model, with early injury markers reflective of hippocampal dysfunction, which can be detected noninvasively with MRI and behavioral tests. Lesions were generated by sub-hemispheric irradiation of mouse hippocampi with single X-ray beams of 80 Gy. Lesion formation was monitored with anatomical and contrast-enhanced MRI and changes in memory and learning were assessed with fear-conditioning tests. Early injury markers were detected 2 weeks after irradiation. These included an increase in the permeability of the blood-brain barrier, demonstrated by a 92 ± 20 % contrast enhancement of the irradiated versus the non-irradiated brain hemispheres, within 15 min of the administration of an MRI contrast agent. A change in short-term memory was also detected, as demonstrated by a 40.88 ± 5.03 % decrease in the freezing time measured during the short-term memory context test at this time point, compared to that before irradiation. SPIO-labeled stem cells transplanted contralateral to the lesion migrated toward the lesion at this time point. No hemorrhage was detected up to 10 weeks after irradiation. This model can be used to evaluate SPIO-based stem cell-tracking agents, short-term.
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http://dx.doi.org/10.1007/s11060-016-2111-3DOI Listing
June 2016

Bidirectional cross-regulation between ErbB2 and β-adrenergic signalling pathways.

Cardiovasc Res 2016 Mar 21;109(3):358-73. Epub 2015 Dec 21.

Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, MRB 807, 733 N. Broadway, Baltimore, MD 21205, USA

Aims: Despite the observation that ErbB2 regulates sensitivity of the heart to doxorubicin or ErbB2-targeted cancer therapies, mechanisms that regulate ErbB2 expression and activity have not been studied. Since isoproterenol up-regulates ErbB2 in kidney and salivary glands and β2AR and ErbB2 complex in brain and heart, we hypothesized that β-adrenergic receptors (AR) modulate ErbB2 signalling status.

Methods And Results: ErbB2 transfection of HEK293 cells up-regulates β2AR, and β2AR transfection of HEK293 up-regulates ErbB2. Interestingly, cardiomyocytes isolated from myocyte-specific ErbB2-overexpressing (ErbB2(tg)) mice have amplified response to selective β2-agonist zinterol, and right ventricular trabeculae baseline force generation is markedly reduced with β2-antagonist ICI-118 551. Consistently, receptor binding assays and western blotting demonstrate that β2ARs levels are markedly increased in ErbB2(tg) myocardium and reduced by EGFR/ErbB2 inhibitor, lapatinib. Intriguingly, acute treatment of mice with β1- and β2-AR agonist isoproterenol resulted in myocardial ErbB2 increase, while inhibition with either β1- or β2-AR antagonist did not completely prevent isoproterenol-induced ErbB2 expression. Furthermore, inhibition of ErbB2 kinase predisposed mice hearts to injury from chronic isoproterenol treatment while significantly reducing isoproterenol-induced pAKT and pERK levels, suggesting ErbB2's role in transactivation in the heart.

Conclusion: Our studies show that myocardial ErbB2 and βAR signalling are linked in a feedback loop with βAR activation leading to increased ErbB2 expression and activity, and increased ErbB2 activity regulating β2AR expression. Most importantly, ErbB2 kinase activity is crucial for cardioprotection in the setting of β-adrenergic stress, suggesting that this mechanism is important in the pathophysiology and treatment of cardiomyopathy induced by ErbB2-targeting antineoplastic drugs.
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http://dx.doi.org/10.1093/cvr/cvv274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752042PMC
March 2016

Imaging transplanted stem cells in real time using an MRI dual-contrast method.

Sci Rep 2015 Sep 2;5:13628. Epub 2015 Sep 2.

The In vivo Cellular and Molecular Imaging Center, Division of Cancer Imaging Research, The Russell H. Morgan Department of Radiology and Radiological Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD21205, USA.

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies.
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http://dx.doi.org/10.1038/srep13628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556978PMC
September 2015

Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase.

Comp Med 2015 Aug;65(4):295-307

Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2(tg)) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2(tg) mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2(tg) data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2(tg) from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4549675PMC
August 2015

ErbB2 overexpression upregulates antioxidant enzymes, reduces basal levels of reactive oxygen species, and protects against doxorubicin cardiotoxicity.

Am J Physiol Heart Circ Physiol 2015 Oct 7;309(8):H1271-80. Epub 2015 Aug 7.

Program in Molecular and Translational Toxicology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; Department of Molecular and Comparative Pathobiology, Johns Hopkins Medical Institutions, Baltimore, Maryland;

Levels of the HER2/ErbB2 protein in the heart are upregulated in some women during breast cancer therapy, and these women are at high risk for developing heart dysfunction after sequential treatment with anti-ErbB2/trastuzumab or doxorubicin. Doxorubicin is known to increase oxidative stress in the heart, and thus we considered the possibility that ErbB2 protein influences the status of cardiac antioxidant defenses in cardiomyocytes. In this study, we measured reactive oxygen species (ROS) in cardiac mitochondria and whole hearts from mice with cardiac-specific overexpression of ErbB2 (ErbB2(tg)) and found that, compared with control mice, high levels of ErbB2 in myocardium result in lower levels of ROS in mitochondria (P = 0.0075) and whole hearts (P = 0.0381). Neonatal cardiomyocytes isolated from ErbB2(tg) hearts have lower ROS levels and less cellular death (P < 0.0001) following doxorubicin treatment. Analyzing antioxidant enzyme levels and activities, we found that ErbB2(tg) hearts have increased levels of glutathione peroxidase 1 (GPx1) protein (P < 0.0001) and GPx activity (P = 0.0031) in addition to increased levels of two known GPx activators, c-Abl (P = 0.0284) and Arg (P < 0.0001). Interestingly, although mitochondrial ROS emission is reduced in the ErbB2(tg) hearts, oxygen consumption rates and complex I activity are similar to control littermates. Compared with these in vivo studies, H9c2 cells transfected with ErbB2 showed less cellular toxicity and produced less ROS (P < 0.0001) after doxorubicin treatment but upregulated GR activity (P = 0.0237) instead of GPx. Our study shows that ErbB2-dependent signaling contributes to antioxidant defenses and suggests a novel mechanism by which anticancer therapies involving ErbB2 antagonists can harm myocardial structure and function.
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http://dx.doi.org/10.1152/ajpheart.00517.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4666964PMC
October 2015

Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

EMBO Mol Med 2015 May;7(5):648-69

Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Lung cancer is the most common malignancy worldwide and is a focus for developing targeted therapies due to its refractory nature to current treatment. We identified a RNA helicase, DDX3, which is overexpressed in many cancer types including lung cancer and is associated with lower survival in lung cancer patients. We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity. Inhibition of DDX3 by RK-33 caused G1 cell cycle arrest, induced apoptosis, and promoted radiation sensitization in DDX3-overexpressing cells. Importantly, RK-33 in combination with radiation induced tumor regression in multiple mouse models of lung cancer. Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells. Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.
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http://dx.doi.org/10.15252/emmm.201404368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492822PMC
May 2015

Parkin-independent mitophagy requires Drp1 and maintains the integrity of mammalian heart and brain.

EMBO J 2014 Dec 27;33(23):2798-813. Epub 2014 Oct 27.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Mitochondrial dynamics and mitophagy have been linked to cardiovascular and neurodegenerative diseases. Here, we demonstrate that the mitochondrial division dynamin Drp1 and the Parkinson's disease-associated E3 ubiquitin ligase parkin synergistically maintain the integrity of mitochondrial structure and function in mouse heart and brain. Mice lacking cardiac Drp1 exhibited lethal heart defects. In Drp1KO cardiomyocytes, mitochondria increased their connectivity, accumulated ubiquitinated proteins, and decreased their respiration. In contrast to the current views of the role of parkin in ubiquitination of mitochondrial proteins, mitochondrial ubiquitination was independent of parkin in Drp1KO hearts, and simultaneous loss of Drp1 and parkin worsened cardiac defects. Drp1 and parkin also play synergistic roles in neuronal mitochondrial homeostasis and survival. Mitochondrial degradation was further decreased by combination of Drp1 and parkin deficiency, compared with their single loss. Thus, the physiological importance of parkin in mitochondrial homeostasis is revealed in the absence of mitochondrial division in mammals.
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http://dx.doi.org/10.15252/embj.201488658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282557PMC
December 2014

AEG-1 promoter-mediated imaging of prostate cancer.

Cancer Res 2014 Oct 21;74(20):5772-81. Epub 2014 Aug 21.

Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins Medical Institutions, Baltimore, Maryland.

We describe a new imaging method for detecting prostate cancer, whether localized or disseminated and metastatic to soft tissues and bone. The method relies on the use of imaging reporter genes under the control of the promoter of AEG-1 (MTDH), which is selectively active only in malignant cells. Through a systemic, nanoparticle-based delivery of the imaging construct, lesions can be identified through bioluminescence imaging and single-photon emission computed tomography in the PC3-ML murine model of prostate cancer at high sensitivity. This approach is applicable for the detection of prostate cancer metastases, including bone lesions for which there is no current reliable agent for noninvasive clinical imaging. Furthermore, the approach compares favorably with accepted and emerging clinical standards, including PET with [(18)F]fluorodeoxyglucose and [(18)F]sodium fluoride. Our results offer a preclinical proof of concept that rationalizes clinical evaluation in patients with advanced prostate cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234089PMC
October 2014