Publications by authors named "Jose G Vilches-Moure"

26 Publications

  • Page 1 of 1

Management of Morbidity and Mortality in a New Zealand White Rabbit Model of SteroidInduced Osteonecrosis of the Femoral Head.

Comp Med 2021 Feb 26;71(1):86-98. Epub 2021 Jan 26.

Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a condition documented in humans and animals exposed to chronic steroid administration. The rabbit has become a preferred animal model for investigating the pathogenesis and treatment of SONFH due to its shared femoral vascular anatomy with human patients, relative size of the femoral head, and general fecundity. However, morbidity and mortality are frequent during the steroid induction period, prior to surgical manipulation. These problems are poorly reported and inadequately described in the literature. In this study, we report the clinical, gross, and histopathologic findings of New Zealand white (NZW) rabbits undergoing the steroid induction phase of the SONFH model. Severe weight loss (>30%), lipemia, hypercholesterolemia, hyperglycemia, and elevations in ALT and AST were consistent findings across all rabbits, although these changes did not differentiate asymptomatic rabbits from those that became clinically symptomatic or died. Euthanized and spontaneously deceased rabbits exhibited hepatomegaly, hepatic lipidosis/glycogenosis, and hepatocellular necrosis, in addition to a lipid-rich and proteinaceous thoracic effusion. A subset of rabbits developed opportunistic pulmonary infections with and and small intestine infections with superimposed on hepatic and thoracic disease. Together, these findings allowed us to establish a clinical decision-making flowchart that reduced morbidities and mortalities in a subsequent cohort of SONFH rabbits. Recognition of these model-associated morbidities is critical for providing optimal clinical care during the disease induction phase of SONFH.
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http://dx.doi.org/10.30802/AALAS-CM-20-000071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898173PMC
February 2021

CTLA-4 expression by B-1a B cells is essential for immune tolerance.

Nat Commun 2021 01 22;12(1):525. Epub 2021 Jan 22.

Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.

CTLA-4 is an important regulator of T-cell function. Here, we report that expression of this immune-regulator in mouse B-1a cells has a critical function in maintaining self-tolerance by regulating these early-developing B cells that express a repertoire enriched for auto-reactivity. Selective deletion of CTLA-4 from B cells results in mice that spontaneously develop autoantibodies, T follicular helper (Tfh) cells and germinal centers (GCs) in the spleen, and autoimmune pathology later in life. This impaired immune homeostasis results from B-1a cell dysfunction upon loss of CTLA-4. Therefore, CTLA-4-deficient B-1a cells up-regulate epigenetic and transcriptional activation programs and show increased self-replenishment. These activated cells further internalize surface IgM, differentiate into antigen-presenting cells and, when reconstituted in normal IgH-allotype congenic recipient mice, induce GCs and Tfh cells expressing a highly selected repertoire. These findings show that CTLA-4 regulation of B-1a cells is a crucial immune-regulatory mechanism.
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http://dx.doi.org/10.1038/s41467-020-20874-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822855PMC
January 2021

A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions.

Proc Natl Acad Sci U S A 2021 Jan;118(1)

Department of Radiology, Bio-X Program and Molecular Imaging Program, Stanford University School of Medicine, Stanford, CA 94305;

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.
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http://dx.doi.org/10.1073/pnas.2008072118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817203PMC
January 2021

Metabolic profiling during malaria reveals the role of the aryl hydrocarbon receptor in regulating kidney injury.

Elife 2020 10 6;9. Epub 2020 Oct 6.

Department of Microbiology and Immunology, Stanford University, Stanford, United States.

Systemic metabolic reprogramming induced by infection exerts profound, pathogen-specific effects on infection outcome. Here, we detail the host immune and metabolic response during sickness and recovery in a mouse model of malaria. We describe extensive alterations in metabolism during acute infection, and identify increases in host-derived metabolites that signal through the aryl hydrocarbon receptor (AHR), a transcription factor with immunomodulatory functions. We find that mice are more susceptible to malaria and develop high plasma heme and acute kidney injury. This phenotype is dependent on AHR in -expressing radioresistant cells. Our findings identify a role for AHR in limiting tissue damage during malaria. Furthermore, this work demonstrates the critical role of host metabolism in surviving infection.
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http://dx.doi.org/10.7554/eLife.60165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7538157PMC
October 2020

Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.

Invest Radiol 2020 11;55(11):711-721

From the Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, Palo Alto, CA.

Early detection of pancreatic ductal adenocarcinoma (PDAC) represents the most significant step toward the treatment of this aggressive lethal disease. Previously, we engineered a preclinical Thy1-targeted microbubble (MBThy1) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MBThy1-scFv and test for its efficacy in vivo in murine PDAC imaging, and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo.

Materials And Methods: We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre; KRas; Ink4a/Arf) of PDAC and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining.

Results: Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2-6 mm). Imaging signal from mice with L-arginine-induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy.

Conclusions: Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.
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http://dx.doi.org/10.1097/RLI.0000000000000697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541735PMC
November 2020

Molecular Imaging of Infective Endocarditis With 6''-[F]Fluoromaltotriose Positron Emission Tomography-Computed Tomography.

Circulation 2020 May 26;141(21):1729-1731. Epub 2020 May 26.

Department of Radiology, Molecular Imaging Program at Stanford (MIPS), Bio-X Program (M.W., G.G., E.C., M.N., T.H., M.T.G., J.C.W., S.S.G.), Stanford University School of Medicine, CA.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043924DOI Listing
May 2020

Salmonella-Driven Polarization of Granuloma Macrophages Antagonizes TNF-Mediated Pathogen Restriction during Persistent Infection.

Cell Host Microbe 2020 01 26;27(1):54-67.e5. Epub 2019 Dec 26.

Department of Microbiology and Immunology, Stanford University, Stanford, CA, USA. Electronic address:

Many intracellular bacteria can establish chronic infection and persist in tissues within granulomas composed of macrophages. Granuloma macrophages exhibit heterogeneous polarization states, or phenotypes, that may be functionally distinct. Here, we elucidate a host-pathogen interaction that controls granuloma macrophage polarization and long-term pathogen persistence during Salmonella Typhimurium (STm) infection. We show that STm persists within splenic granulomas that are densely populated by CD11bCD11cLy6C macrophages. STm preferentially persists in granuloma macrophages reprogrammed to an M2 state, in part through the activity of the effector SteE, which contributes to the establishment of persistent infection. We demonstrate that tumor necrosis factor (TNF) signaling limits M2 granuloma macrophage polarization, thereby restricting STm persistence. TNF neutralization shifts granuloma macrophages toward an M2 state and increases bacterial persistence, and these effects are partially dependent on SteE activity. Thus, manipulating granuloma macrophage polarization represents a strategy for intracellular bacteria to overcome host restriction during persistent infection.
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http://dx.doi.org/10.1016/j.chom.2019.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065835PMC
January 2020

Chronic Model of Inflammatory Bowel Disease in IL-10 Transgenic Mice: Evaluation with Ultrasound Molecular Imaging.

Theranostics 2019 14;9(21):6031-6046. Epub 2019 Aug 14.

Department of Radiology, Stanford University, School of Medicine, Stanford, California, USA.

: Acute mouse models of inflammatory bowel disease (IBD) fail to mirror the chronic nature of IBD in patients. We sought to develop a chronic mouse IBD model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI) by using dual P- and E-selectin targeted microbubbles (MB). : Interleukin 10 deficient (IL-10 on a C57BL/6 genetic background; n=55) and FVB (n=16) mice were used. In IL-10mice, various experimental regimens including piroxicam, 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sulfate sodium (DSS), respectively were used for promoting colitis; colitis was induced with DSS in FVB mice. Using clinical and small animal ultrasound scanners, evolution of inflammation in proximal, middle and distal colon, was monitored with USMI by using MB at multiple time points. Imaged colon segments were analyzed for inflammatory changes on H&E staining and for P-selectin expression on immunofluorescence staining. : Sustained colitis was not detected with USMI in IL-10 or FVB mice with various experimental regimens. USMI signals either gradually decreased after the colitis enhancing/inducing drug/agents were discontinued, or the mortality rate of mice was high. Inflammation was observed on H&E staining in IL-10 mice with piroxicam promotion, while stable overexpression of P-selectin was not found on immunofluorescence staining in the same mice. : Sustained colitis in IL-10 mice induced with piroxicam, TNBS or DSS, and in FVB mice induced with DSS, was not detected with USMI using MB, and this was verified by immunofluorescence staining for inflammation marker P-selectin. Thus, these models may not be appropriate for long-term monitoring of chronic colitis and subsequent treatment response with dual-selectin targeted USMI.
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http://dx.doi.org/10.7150/thno.37397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735517PMC
September 2020

Validation of a geropathology grading system for aging mouse studies.

Geroscience 2019 08 29;41(4):455-465. Epub 2019 Aug 29.

Department of Comparative Medicine, University of Washington, Seattle, WA, USA.

An understanding of early-onset mechanisms underlying age-related changes can be obtained by evaluating changes that precede frailty and end of life using histological characterization of age-related lesions. Histopathology-based information as a component of aging studies in mice can complement and add context to molecular, cellular, and physiologic data, but there is a lack of information regarding scoring criteria and lesion grading guidelines. This report describes the validation of a grading system, designated as the geropathology grading platform (GGP), which generated a composite lesion score (CLS) for comparison of histological lesion scores in tissues from aging mice. To assess reproducibility of the scoring system, multiple veterinary pathologists independently scored the same slides from the heart, lung, liver, and kidney from two different strains (C57BL/6 and CB6F1) of male mice at 8, 16, 24, and 32 months of age. There was moderate to high agreement between pathologists, particularly when agreement within a 1-point range was considered. CLS for all organs was significantly higher in older versus younger mice, suggesting that the GGP was reliable for detecting age-related pathology in mice. The overall results suggest that the GGP guidelines reliably distinguish between younger and older mice and may therefore be accurate in distinguishing between experimental groups of mice with more, or less, age-related pathology.
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http://dx.doi.org/10.1007/s11357-019-00088-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815299PMC
August 2019

Investigating circulating tumor cells and distant metastases in patient-derived orthotopic xenograft models of triple-negative breast cancer.

Breast Cancer Res 2019 08 28;21(1):98. Epub 2019 Aug 28.

Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA.

Background: Circulating tumor cells (CTCs) represent a temporal "snapshot" of a patient's cancer and changes that occur during disease evolution. There is an extensive literature studying CTCs in breast cancer patients, and particularly in those with metastatic disease. In parallel, there is an increasing use of patient-derived models in preclinical investigations of human cancers. Yet studies are still limited demonstrating CTC shedding and metastasis formation in patient-derived models of breast cancer.

Methods: We used seven patient-derived orthotopic xenograft (PDOX) models generated from triple-negative breast cancer (TNBC) patients to study CTCs and distant metastases. Tumor fragments from PDOX tissue from each of the seven models were implanted into 57 NOD scid gamma (NSG) mice, and tumor growth and volume were monitored. Human CTC capture from mouse blood was first optimized on the marker-agnostic Vortex CTC isolation platform, and whole blood was processed from 37 PDOX tumor-bearing mice.

Results: Staining and imaging revealed the presence of CTCs in 32/37 (86%). The total number of CTCs varied between different PDOX tumor models and between individual mice bearing the same PDOX tumors. CTCs were heterogeneous and showed cytokeratin (CK) positive, vimentin (VIM) positive, and mixed CK/VIM phenotypes. Metastases were detected in the lung (20/57, 35%), liver (7/57, 12%), and brain (1/57, less than 2%). The seven different PDOX tumor models displayed varying degrees of metastatic potential, including one TNBC PDOX tumor model that failed to generate any detectable metastases (0/8 mice) despite having CTCs present in the blood of 5/5 tested, suggesting that CTCs from this particular PDOX tumor model may typify metastatic inefficiency.

Conclusion: PDOX tumor models that shed CTCs and develop distant metastases represent an important tool for investigating TNBC.
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http://dx.doi.org/10.1186/s13058-019-1182-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714238PMC
August 2019

Ultrasound/microbubble-mediated targeted delivery of anticancer microRNA-loaded nanoparticles to deep tissues in pigs.

J Control Release 2019 09 18;309:1-10. Epub 2019 Jul 18.

Department of Radiology, School of Medicine, Stanford University, Stanford, California, United States of America.

In this study, we designed and validated a platform for ultrasound and microbubble-mediated delivery of FDA-approved pegylated poly lactic-co-glycolic acid (PLGA) nanoparticles loaded with anticancer microRNAs (miRNAs) to deep tissues in a pig model. Small RNAs have been shown to reprogram tumor cells and sensitize them to clinically used chemotherapy. To overcome their short intravascular circulation half-life and achieve controlled and sustained release into tumor cells, anticancer miRNAs need to be encapsulated into nanocarriers. Focused ultrasound combined with gas-filled microbubbles provides a noninvasive way to improve the permeability of tumor vasculature and increase the delivery efficiency of drug-loaded particles. A single handheld, curvilinear ultrasound array was used in this study for image-guided therapy with clinical-grade SonoVue contrast agent. First, we validated the platform on phantoms to optimize the microbubble cavitation dose based on acoustic parameters, including peak negative pressure, pulse length, and pulse repetition frequency. We then tested the system in vivo by delivering PLGA nanoparticles co-loaded with antisense-miRNA-21 and antisense-miRNA-10b to pig liver and kidney. Enhanced miRNA delivery was observed (1.9- to 3.7-fold increase) as a result of the ultrasound treatment compared to untreated control regions. Additionally, we used highly fluorescent semiconducting polymer nanoparticles to visually assess nanoparticle extravasation. Fluorescent microscopy suggested the presence of nanoparticles in the extravascular compartment. Hematoxylin and eosin staining of treated tissues did not reveal tissue damage. The results presented in this manuscript suggest that the proposed platform may be used to safely and noninvasively enhance the delivery of miRNA-loaded nanoparticles to target regions in deep organs in large animal models.
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http://dx.doi.org/10.1016/j.jconrel.2019.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815710PMC
September 2019

Embryonic Chicken () as a Model of Cardiac Biology and Development.

Comp Med 2019 05 10;69(3):184-203. Epub 2019 Jun 10.

Department of Comparative Medicine, Stanford University School of Medicine, Stanford, California, Email:

Cardiovascular disease remains one of the top contributors to morbidity and mortality in the United States. Increasing evidence suggests that many processes, pathways, and programs observed during development and organogenesis are recapitulated in adults in the face of disease. Therefore, a heightened understanding of cardiac development and organogenesis will help increase our understanding of developmental defects and cardiovascular diseases in adults. Chicks have long served as a model system in which to study developmental problems. Detailed descriptions of morphogenesis, low cost, accessibility, ease of manipulation, and the optimization of genetic engineering techniques have made chicks a robust model for studying development and make it a powerful platform for cardiovascular research. This review summarizes the cardiac developmental milestones of embryonic chickens, practical considerations when working with chicken embryos, and techniques available for use in chicks (including tissue chimeras, genetic manipulations, and live imaging). In addition, this article highlights examples that accentuate the utility of the embryonic chicken as model system in which to study cardiac development, particularly epicardial development, and that underscore the importance of how studying development informs our understanding of disease.
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http://dx.doi.org/10.30802/AALAS-CM-18-000061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591676PMC
May 2019

Receptor subtype discrimination using extensive shape complementary designed interfaces.

Nat Struct Mol Biol 2019 06 13;26(6):407-414. Epub 2019 May 13.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.
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http://dx.doi.org/10.1038/s41594-019-0224-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582999PMC
June 2019

Multimodality Hyperpolarized C-13 MRS/PET/Multiparametric MR Imaging for Detection and Image-Guided Biopsy of Prostate Cancer: First Experience in a Canine Prostate Cancer Model.

Mol Imaging Biol 2019 10;21(5):861-870

Department of Radiology, Stanford University, Stanford, CA, USA.

Purpose: To assess whether simultaneous hyperpolarized C-13 magnetic resonance spectroscopy (MRS)/positron emission tomography (PET)/multiparametric magnetic resonance (mpMR) imaging is feasible in an orthotopic canine prostate cancer (PCa) model using a clinical PET/MR system and whether the combined imaging datasets can be fused with transrectal ultrasound (TRUS) in real time for multimodal image fusion-guided targeted biopsy of PCa.

Procedures: Institutional Animal Care and Use Committee approval was obtained for this study. Canine prostate adenocarcinoma (Ace-1) cells were orthotopically injected into the prostate of four dogs. Once tumor engraftment was confirmed by TRUS, simultaneous hyperpolarized C-13 MRS of [1-C]pyruvate, PET (2-deoxy-2-[F]fluoro-D-glucose ([F]FDG), [Ga]NODAGA-SCH1), and mpMR (T2W, DWI) imaging was performed using a clinical PET/MR system. Multimodality imaging data sets were then fused with TRUS and image-guided targeted biopsy was performed. Imaging results were then correlated with histological findings.

Results: Successful tumor engraftment was histologically confirmed in three of the four dogs (dogs 2, 3, and 4) and simultaneous C-13 MRS/PET/mpMR was feasible in all three. In dog 2, C-13 MRS showed increased lactate signal in the tumor (lactate/totalC = 0.47) whereas mpMR did not show any signal changes. In dog 3, [F]FDG-PET (SUV = 1.90) and C-13 MRS (lactate/totalC = 0.59) showed elevated metabolic activity in the tumor. In dog 4, [F]FDG (SUV = 2.43), [Ga]NODAGA-SCH1 (SUV = 0.75), and C-13 MRS (Lac/totalC = 0.53) showed elevated uptake in tumor compared to control tissue and multimodal image fusion-guided biopsy of the tumor was successfully performed.

Conclusion: Simultaneous C-13 MRS/PET/mpMR imaging and multimodal image fusion-guided biopsy is feasible in a canine PCa model.
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http://dx.doi.org/10.1007/s11307-018-1235-6DOI Listing
October 2019

A Bird's-Eye View of Regulatory, Animal Care, and Training Considerations Regarding Avian Flight Research.

Comp Med 2019 05 14;69(3):169-178. Epub 2019 Feb 14.

Comparative Medicine.

A thorough understanding of how animals fly is a central goal of many scientific disciplines. Birds are a commonly used model organism for flight research. The success of this model requires studying healthy and naturally flying birds in a laboratory setting. This use of a nontraditional laboratory animal species presents unique challenges to animal care staff and researchers alike. Here we review regulatory, animal care, and training considerations associated with avian flight research.
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http://dx.doi.org/10.30802/AALAS-CM-18-000033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591680PMC
May 2019

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

Nature 2019 01 10;565(7739):372-376. Epub 2018 Dec 10.

Department of Biology, Stanford University, Stanford, CA, USA.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.
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http://dx.doi.org/10.1038/s41586-018-0821-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511263PMC
January 2019

Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle.

Mol Ther Methods Clin Dev 2018 Sep 24;10:144-155. Epub 2018 Jul 24.

Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants and in various primary and immortalized myogenic lines . Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.
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http://dx.doi.org/10.1016/j.omtm.2018.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6077147PMC
September 2018

A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection.

Cell Host Microbe 2018 08 26;24(2):296-307.e7. Epub 2018 Jul 26.

Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA. Electronic address:

The intestinal microbiota provides colonization resistance against pathogens, limiting pathogen expansion and transmission. These microbiota-mediated mechanisms were previously identified by observing loss of colonization resistance after antibiotic treatment or dietary changes, which severely disrupt microbiota communities. We identify a microbiota-mediated mechanism of colonization resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) by comparing high-complexity commensal communities with different levels of colonization resistance. Using inbred mouse strains with different infection dynamics and S. Typhimurium intestinal burdens, we demonstrate that Bacteroides species mediate colonization resistance against S. Typhimurium by producing the short-chain fatty acid propionate. Propionate directly inhibits pathogen growth in vitro by disrupting intracellular pH homeostasis, and chemically increasing intestinal propionate levels protects mice from S. Typhimurium. In addition, administering susceptible mice Bacteroides, but not a propionate-production mutant, confers resistance to S. Typhimurium. This work provides mechanistic understanding into the role of individualized microbial communities in host-to-host variability of pathogen transmission.
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http://dx.doi.org/10.1016/j.chom.2018.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223613PMC
August 2018

US Molecular Imaging of Acute Ileitis: Anti-Inflammatory Treatment Response Monitored with Targeted Microbubbles in a Preclinical Model.

Radiology 2018 10 24;289(1):90-100. Epub 2018 Jul 24.

From the Department of Radiology, Stanford University School of Medicine, 300 Pasteur Dr, Grant SO62B, Stanford, CA 94305-5105 (H.W., A.M.L., J.K.W.); Bracco Suisse SA, Geneva, Switzerland (J.M.H., S.C., T.B.); Departments of Comparative Medicine (S.A.F., J.G.V.) and Health, Research & Policy (L.T.), Stanford University, Stanford, Calif; and Ultrasound Business Unit, Siemens Healthcare, Mountain View, Calif (I.G.).

Purpose To evaluate whether dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in an acute terminal ileitis model in swine. Materials and Methods The Institutional Animal Care and Use Committee approved all animal studies. Fourteen swine with chemically induced acute terminal ileitis (day 0) were randomized into the following groups: (a) an anti-inflammatory treatment group (n = 8; meloxicam, 0.25 mg per kilogram of body weight; prednisone, 0.5 mg/kg) and (b) a control group (n = 6; saline). US molecular imaging was performed with a clinical US machine after intravenous injection of clinically translatable dual P- and E-selectin-targeted microbubbles (5 × 10/kg). Three inflamed bowel segments per swine were imaged at baseline, as well as on days 1, 3, and 6 after treatment initiation. At day 6, bowel segments were analyzed ex vivo for selectin expression levels by using quantitative immunofluorescence. Results After induction of inflammation, US molecular imaging signal increased at day 1 in both animal groups (P < .001). At day 3, signal in the treatment group decreased (P < .001 vs day 1), while signal in control animals did not significantly change (P = .18 vs day 1) and was higher (P = .001) compared with that in the treatment group. At day 6, signal in the treatment group further decreased and remained lower (P = .02) compared with that in the control group. Immunofluorescence confirmed significant (P ≤ .04) downregulation of both P- and E-selectin expression levels in treated versus control bowel segments. Conclusion Dual-selectin-targeted US molecular imaging allows longitudinal monitoring of anti-inflammatory treatment effects in a large-animal model of acute ileitis. This supports further clinical development of this quantitative and radiation-free technique for monitoring inflammatory bowel disease. © RSNA, 2018 Online supplemental material is available for this article.
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http://dx.doi.org/10.1148/radiol.2018172600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6190483PMC
October 2018

The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion.

JCI Insight 2017 12 7;2(23). Epub 2017 Dec 7.

Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, USA.

Primary and secondary hypertension are major risk factors for cardiovascular disease, the leading cause of death worldwide. Elevated secretion of aldosterone resulting from primary aldosteronism (PA) is a key driver of secondary hypertension. Here, we report an unexpected role for the ubiquitin ligase Siah1 in adrenal gland development and PA. Siah1a-/- mice exhibit altered adrenal gland morphology, as reflected by a diminished X-zone, enlarged medulla, and dysregulated zonation of the glomerulosa as well as increased aldosterone levels and aldosterone target gene expression and reduced plasma potassium levels. Genes involved in catecholamine biosynthesis and cAMP signaling are upregulated in the adrenal glands of Siah1a-/- mice, while genes related to retinoic acid signaling and cholesterol biosynthesis are downregulated. Loss of Siah1 leads to increased expression of the Siah1 substrate PIAS1, an E3 SUMO protein ligase implicated in the suppression of LXR, a key regulator of cholesterol levels in the adrenal gland. In addition, SIAH1 sequence variants were identified in patients with PA; such variants impaired SIAH1 ubiquitin ligase activity, resulting in elevated PIAS1 expression. These data identify a role for the Siah1-PIAS1 axis in adrenal gland organization and function and point to possible therapeutic targets for hyperaldosteronism.
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http://dx.doi.org/10.1172/jci.insight.97128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752272PMC
December 2017

Anatomical Road Mapping Using CT and MR Enterography for Ultrasound Molecular Imaging of Small Bowel Inflammation in Swine.

Eur Radiol 2018 May 23;28(5):2068-2076. Epub 2017 Nov 23.

Department of Radiology, Stanford University, School of Medicine, 300 Pasteur Drive, Room H1307, Stanford, CA, 94305-5621, USA.

Objectives: To evaluate the feasibility and time saving of fusing CT and MR enterography with ultrasound for ultrasound molecular imaging (USMI) of inflammation in an acute small bowel inflammation of swine.

Methods: Nine swine with ileitis were scanned with either CT (n = 3) or MR (n = 6) enterography. Imaging times to load CT/MR images onto a clinical ultrasound machine, fuse them to ultrasound with an anatomical landmark-based approach, and identify ileitis were compared to the imaging times without anatomical road mapping. Inflammation was then assessed by USMI using dual selectin-targeted (MB) and control (MB) contrast agents in diseased and healthy control bowel segments, followed by ex vivo histology.

Results: Cross-sectional image fusion with ultrasound was feasible with an alignment error of 13.9 ± 9.7 mm. Anatomical road mapping significantly reduced (P < 0.001) scanning times by 40%. Localising ileitis was achieved within 1.0 min. Subsequently performed USMI demonstrated significantly (P < 0.001) higher imaging signal using MB compared to MB and histology confirmed a significantly higher inflammation score (P = 0.006) and P- and E-selectin expression (P ≤ 0.02) in inflamed vs. healthy bowel.

Conclusions: Fusion of CT and MR enterography data sets with ultrasound in real time is feasible and allows rapid anatomical localisation of ileitis for subsequent quantification of inflammation using USMI.

Key Points: • Real-time fusion of CT/MRI with ultrasound to localise ileitis is feasible. • Anatomical road mapping using CT/MRI significantly decreases the scanning time for USMI. • USMI allows quantification of inflammation in swine, verified with ex vivo histology.
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http://dx.doi.org/10.1007/s00330-017-5148-6DOI Listing
May 2018

Restrictive orbital myofibroblastic sarcoma in a cat--cross-sectional imaging (MRI & CT) appearance, treatment, and outcome.

Vet Ophthalmol 2013 Jul 3;16 Suppl 1:123-9. Epub 2013 Jan 3.

Veterinary Medical Teaching Hospital School of Veterinary Medicine, University of California-Davis, CA 95616, USA.

A 16-year-old spayed female cat was evaluated for lagophthalmos and chronic exposure keratitis in both eyes. Ophthalmic examination revealed upper and lower eyelid entropion of the left eye (OS) and markedly decreased retropulsion, restricted eye movement, marked episcleral congestion, and severe keratitis of both eyes (OU). Magnetic resonance imaging of both orbits revealed extensive, irregular, contrast-enhancing tissue without evidence of osteolysis considered compatible with diffuse inflammatory tissue. Feline herpesvirus DNA was not detected in conjunctival samples. Partial temporary tarsorrhaphies were placed OU, and the cat was treated with topically administered erythromycin ointment OU, orally administered famciclovir and prednisolone, and sublingually administered buprenorphine. Little improvement was noted after 2 weeks. Six weeks after initial presentation, a left exenteration was performed and histopathology was consistent with idiopathic sclerosing orbital pseudotumor (ISOP). Ten weeks after initial presentation, the patient represented for weight loss and jaw pain. Computed tomography demonstrated disease progression in the right orbit and the patient was euthanized. Histopathology of the decalcified skull revealed an aggressive and highly infiltrative mass involving the right orbit with extension to the maxilla, hard palate, nasal cavity and gingiva most consistent with feline restrictive orbital myofibroblastic sarcoma (FROMS). Clinical data from this patient support the reclassification of ISOP as FROMS. MRI and CT may provide supportive evidence for FROMS, but histopathology is necessary for definitive diagnosis. Aggressive and early surgical treatment, including bilateral exenteration, with adjunctive radiotherapy and/or chemotherapy should be considered for patients with FROMS.
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http://dx.doi.org/10.1111/vop.12016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3620966PMC
July 2013

Myenteric ganglionitis as a cause of recurrent colic in an adult horse.

J Am Vet Med Assoc 2012 Jun;240(12):1494-500

William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA.

Case Description: A 10-year-old Lipizzaner stallion was evaluated over the course of 1.5 years because of intermittent, recurrent colic.

Clinical Findings: The horse was initially treated medically for gastric ulcers; dietary changes were made, and a deworming protocol was instituted, without resolution of colic episodes. Subsequently, the horse underwent exploratory celiotomy and a large colon volvulus was identified with diffuse colonic wall thickening. A pelvic flexure biopsy sample was submitted for histologic examination, which revealed lymphocytic (CD3-positive T cells) myenteric ganglionitis (MG). The horse developed a cecal impaction after surgery, which did not resolve, despite aggressive medical management; subsequently a complete cecal bypass was performed. Cecal and colonic wall biopsy samples were evaluated histologically and confirmed the diagnosis of MG. After surgery, the horse developed a large colon impaction, which initially responded to aggressive medical treatment, and the horse was discharged.

Treatment And Outcome: Despite rigorous feed restrictions and prokinetic and corticosteroid treatment, the horse continued to have signs of colic and was euthanized 3 weeks after discharge from the hospital because of a recurrent large colon impaction. Intestinal biopsy samples obtained at the time of death revealed chronic changes in intramural ganglia consistent with generalized MG.

Clinical Relevance: MG is a rare disease in horses, causing gastrointestinal motility dysfunction and signs of colic, which is challenging to diagnose and treat successfully. Further studies are needed to identify the etiology of this disease and to explore treatment options.
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http://dx.doi.org/10.2460/javma.240.12.1494DOI Listing
June 2012

Ingested Shiga toxin 2 (Stx2) causes histopathological changes in kidney, spleen, and thymus tissues and mortality in mice.

J Agric Food Chem 2010 Aug 27;58(16):9281-6. Epub 2010 Jul 27.

Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California 94710, USA.

The Shiga toxin (Stx)-producing bacterial strain, Escherichia coli O157:H7, colonizes the distal small intestine and the colon, initiating serious illness, including hemolytic-uremic syndrome (HUS), characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Although intravenous administration of purified Stx to primates has been able to reproduce the features of HUS, it has not been conclusively established as to whether ingestion of Stx alone without the bacterium poses a potential health risk. To help answer this question, in this study, we fed Shiga toxin 2 (Stx2) directly into the stomachs of mice via gavage. Our data show that ingestion of Stx2 at a concentration of 50 μg/mouse induces weight loss and kills the mice at 3-5 days post-gavage. Additional studies revealed that the toxin retains activity at low pH, that its activity is neutralized by treatment with toxin-specific antibody, and that about 1% of the fed toxin is absorbed into the blood circulation. Lethality by intraperitoneal (IP) injection of Stx2 occurred at much lower doses than by ingestion. Detailed histopathological evaluation of stained tissues by light microscopy revealed severe histopathological changes in kidneys, spleen, and thymus but not in the pancreas, lymph nodes, heart, lungs, trachea, esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon. The pathological changes in the kidney appeared similar to those seen in humans with HUS. The cited data suggest that (a) most but not all of the toxin is inactivated in the digestive tract, (b) part of the oral-ingested toxin is absorbed from the digestive tract into the circulation, (c) enough active toxin reaches susceptible organs to induce damage, and (d) Stx2 in the absence of toxin-producing bacteria can be harmful to mice. The results are clinically relevant for food safety because we also found that heat treatments (pasteurization) that destroy bacteria did not inactivate the heat-resistant toxin produced and secreted by the bacteria.
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http://dx.doi.org/10.1021/jf101744zDOI Listing
August 2010

Genes unlinked to the leptin receptor influence urinary albumin excretion in obese Zucker rats.

Physiol Genomics 2010 May 16;41(3):297-305. Epub 2010 Feb 16.

Rowe Program in Genetics.

We have previously shown that 90% of outbred obese Zucker Lepr(fa/fa) rats die prematurely of renal disease. Thus, renal disease in obese Zucker Lepr(fa/fa) rats may be caused by the LEPR mutation on chromosome 5, by the obesity, or it may be influenced by Zucker susceptibility alleles of genes on other chromosomes. We have searched for susceptibility genes on other chromosomes using urinary albumin excretion (UAE) as an early indicator of altered renal function in a backcross of (Brown Norway × inbred Zucker) F1 × inbred Zucker, which we name the BZZ cross. We killed 237 BZZ backcross animals at 15 wk of age. All included animals were homozygous for the fatty mutation of LEPR and were obese. Urinary creatinine measurements were used to calculate the albumin-to-creatinine ratio (ACR). We identified direct effect quantitative trait loci (QTLs) for UAE and ACR on chromosome 1 (LOD scores = 3.6 and 2.86, respectively) in males, and chromosome 4 (LOD score = 2.9) in females. Significant QTLs were identified for left kidney weight for females on chromosomes 3 and 12. We also demonstrated that kidneys from 15 wk old obese inbred Zucker rats already show evidence of kidney pathology: tubular dilation, proteinaceous fluid accumulation, evidence for inflammation, and mild mesangial and tubular membrane basement membrane thickening. Both lean Zucker rats and the Brown Norway rats showed no evidence for these changes. Thus, by removing the influence of the Lepr(fa/fa) mutation from analysis we have identified UAE QTLs unlinked to LEPR.
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http://dx.doi.org/10.1152/physiolgenomics.90367.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869106PMC
May 2010

Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues.

J Vet Diagn Invest 2005 Jul;17(4):346-50

Animal Disease Diagnostic Laboratory, and Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47906, USA.

Rabbit monoclonal (RM) antibodies appear to have higher affinity for antigens than mouse monoclonal (MM) antibodies. However, RM antibodies have not been used in veterinary diagnostic immunohistochemistry. The authors compared reactivities of RM and MM antibodies on formalin-fixed, paraffin-embedded canine tissues, targeting 11 different antigens: CD3, CD79a, calcitonin, calretinin, chromogranin A, COX-2, estrogen receptor, Ki67, progesterone receptor, synaptophysin, and vimentin. Paraffin-embedded tissue sections were processed by 1 of 2 antigen-retrieval methods: 1) proteinase K digestion or 2) steam heat in citrate buffer. An additional set of slides did not receive antigen retrieval. Immunostaining was performed using an automated stainer, and scores were assigned to the different dilutions and antigen-retrieval methods on the basis of staining intensity and number of positive cells. Steam heat was usually the best antigen-retrieval method. The optimal dilution for each antibody was that which resulted in the highest specific staining and the lowest nonspecific (background) staining. The RM or MM antibodies yielded a specific reaction for all antigens examined except calretinin. The RM and MM antibodies yielded a specific reaction for 4 antigens only: COX-2, Ki67, synaptophysin, and vimentin. Three antigens (CD3, chromogranin A, and progesterone receptor) were detected only with RM antibodies, whereas the other 3 (CD79a, calcitonin, estrogen receptor) were detected only with MM antibodies. The results of this study differed from those reported for human tissues by the manufacturers of the antibodies. These results emphasize that, regardless of manufacturers' recommendations, each antibody must be individually standardized and validated before routine use in canine tissues.
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http://dx.doi.org/10.1177/104063870501700407DOI Listing
July 2005