José Vilches-Moure, DVM, PhD

All Publications

• GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism. Development (Cambridge, England) Yuki, K., Vallon, M., Ding, J., Rada, C. C., Tang, A. T., Vilches-Moure, J. G., McCormick, A. K., Echeverri, M. F., Alwahabi, S., Braunger, B. M., Ergün, S., Kahn, M. L., Kuo, C. J. 2024

  Abstract

  The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion 7-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7, which is transferred to Frizzled (FZD) to initiate canonical b-catenin signaling. GPR124 remains enigmatic; while its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124/FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function by mouse deletion (Gpr124ΔC). Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, versus Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Further, Gpr124ΔC and recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function is exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion 7-pass transmembrane receptors.

  View details for DOI 10.1242/dev.202794

  View details for PubMedID 38682276

• Design of a mucin-selective protease for targeted degradation of cancer-associated mucins. Nature biotechnology Pedram, K., Shon, D. J., Tender, G. S., Mantuano, N. R., Northey, J. J., Metcalf, K. J., Wisnovsky, S. P., Riley, N. M., Forcina, G. C., Malaker, S. A., Kuo, A., George, B. M., Miller, C. L., Casey, K. M., Vilches-Moure, J. G., Ferracane, M. J., Weaver, V. M., Läubli, H., Bertozzi, C. R. 2023

  Abstract

  Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

  View details for DOI 10.1038/s41587-023-01840-6

  View details for PubMedID 37537499

  View details for PubMedCentralID 6031612

• Gut regulatory T cells mediate immunological tolerance in <i>Salmonella</i>-infected superspreader hosts. Di Luccia, B., Massis, L., Ruddle, S., Narasimhan, R., Pham, T., Vilches-Moure, J., Amieva, M. R., Monack, D. M. AMER ASSOC IMMUNOLOGISTS. 2023

  View details for Web of Science ID 001106506500499

• Lysine-Derived Charge-Altering Releasable Transporters: Targeted Delivery of mRNA and siRNA to the Lungs. Bioconjugate chemistry Blake, T. R., Haabeth, O. A., Sallets, A., McClellan, R. L., Del Castillo, T. J., Vilches-Moure, J. G., Ho, W. C., Wender, P. A., Levy, R., Waymouth, R. M. 2023

  Abstract

  Targeted delivery of nucleic acid therapeutics to the lungs could transform treatment options for pulmonary disease. We have previously developed oligomeric charge-altering releasable transporters (CARTs) for in vivo mRNA transfection and demonstrated their efficacy for use in mRNA-based cancer vaccination and local immunomodulatory therapies against murine tumors. While our previously reported glycine-based CART-mRNA complexes (G-CARTs/mRNA) show selective protein expression in the spleen (mouse, >99%), here, we report a new lysine-derived CART-mRNA complex (K-CART/mRNA) that, without additives or targeting ligands, shows selective protein expression in the lungs (mouse, >90%) following systemic IV administration. We further show that by delivering siRNA using the K-CART, we can significantly decrease expression of a lung-localized reporter protein. Blood chemistry and organ pathology studies demonstrate that K-CARTs are safe and well-tolerated. We report on the new step economical, organocatalytic synthesis (two steps) of functionalized polyesters and oligo-carbonate-co-α-aminoester K-CARTs from simple amino acid and lipid-based monomers. The ability to direct protein expression selectively in the spleen or lungs by simple, modular changes to the CART structure opens fundamentally new opportunities in research and gene therapy.

  View details for DOI 10.1021/acs.bioconjchem.3c00019

  View details for PubMedID 36996808

• Focused ultrasound-induced inhibition of peripheral nerve fibers in an animal model of acute pain. Regional anesthesia and pain medicine Anderson, T. A., Pacharinsak, C., Vilches-Moure, J., Kantarci, H., Zuchero, J. B., Butts-Pauly, K., Yeomans, D. 2023

  Abstract

  Moderate-to-severe acute pain is prevalent in many healthcare settings and associated with adverse outcomes. Peripheral nerve blockade using traditional needle-based and local anesthetic-based techniques improves pain outcomes for some patient populations but has shortcomings limiting use. These limitations include its invasiveness, potential for local anesthetic systemic toxicity, risk of infection with an indwelling catheter, and relatively short duration of blockade compared with the period of pain after major injuries. Focused ultrasound is capable of inhibiting the peripheral nervous system and has potential as a pain management tool. However, investigations of its effect on peripheral nerve nociceptive fibers in animal models of acute pain are lacking. In an in vivo acute pain model, we investigated focused ultrasound's effects on behavior and peripheral nerve structure.Focused ultrasound was applied directly to the sciatic nerve of rats just prior to a hindpaw incision; three control groups (focused ultrasound sham only, hindpaw incision only, focused ultrasound sham+hindpaw incision) were also included. For all four groups (intervention and controls), behavioral testing (thermal and mechanical hyperalgesia, hindpaw extension and flexion) took place for 4 weeks. Structural changes to peripheral nerves of non-focused ultrasound controls and after focused ultrasound application were assessed on days 0 and 14 using light microscopy and transmission electron microscopy.Compared with controls, after focused ultrasound application, animals had (1) increased mechanical nociceptive thresholds for 2 weeks; (2) sustained increase in thermal nociceptive thresholds for ≥4 weeks; (3) a decrease in hindpaw motor response for 0.5 weeks; and (4) a decrease in hindpaw plantar sensation for 2 weeks. At 14 days after focused ultrasound application, alterations to myelin sheaths and nerve fiber ultrastructure were observed both by light and electron microscopy.Focused ultrasound, using a distinct parameter set, reversibly inhibits A-delta peripheral nerve nociceptive, motor, and non-nociceptive sensory fiber-mediated behaviors, has a prolonged effect on C nociceptive fiber-mediated behavior, and alters nerve structure. Focused ultrasound may have potential as a peripheral nerve blockade technique for acute pain management. However, further investigation is required to determine C fiber inhibition duration and the significance of nerve structural changes.

  View details for DOI 10.1136/rapm-2022-104060

  View details for PubMedID 36822815

• Ablation of Adar1 in myeloid cells imprints a global antiviral state in the lung and heightens early immunity against SARS-CoV-2. Cell reports Adamska, J. Z., Verma, R., Gupta, S., Hagan, T., Wimmers, F., Floyd, K., Li, Q., Valore, E. V., Wang, Y., Trisal, M., Vilches-Moure, J. G., Subramaniam, S., Walkley, C. R., Suthar, M. S., Li, J. B., Pulendran, B. 2023; 42 (1): 112038

  Abstract

  Under normal homeostatic conditions, self-double-stranded RNA (self-dsRNA) is modified by adenosine deaminase acting on RNA 1 (ADAR1) to prevent the induction of a type I interferon-mediated inflammatory cascade. Antigen-presenting cells (APCs) sense pathogen-associated molecular patterns, such as dsRNA, to activate the immune response. The impact of ADAR1 on the function of APCs and the consequences to immunity are poorly understood. Here, we show that ADAR1 deletion in CD11c+ APCs leads to (1) a skewed myeloid cell compartment enriched in inflammatory cDC2-like cells, (2) enhanced numbers of activated tissue resident memory Tcells in the lung, and (3) the imprinting of a broad antiviral transcriptional signature across both immune and non-immune cells. The resulting changes can be partially reversed by blocking IFNAR1 signaling and promote early resistance against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our study provides insight into the consequences of self-dsRNA sensing in APCs on the immune system.

  View details for DOI 10.1016/j.celrep.2023.112038

  View details for PubMedID 36732946

• Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos. Cell reports Roodgar, M., Suchy, F. P., Nguyen, L. H., Bajpai, V. K., Sinha, R., Vilches-Moure, J. G., Van Bortle, K., Bhadury, J., Metwally, A., Jiang, L., Jian, R., Chiang, R., Oikonomopoulos, A., Wu, J. C., Weissman, I. L., Mankowski, J. L., Holmes, S., Loh, K. M., Nakauchi, H., VandeVoort, C. A., Snyder, M. P. 2022; 40 (9): 111264

  Abstract

  As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

  View details for DOI 10.1016/j.celrep.2022.111264

  View details for PubMedID 36044843

• Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell Labanieh, L., Majzner, R. G., Klysz, D., Sotillo, E., Fisher, C. J., Vilches-Moure, J. G., Pacheco, K. Z., Malipatlolla, M., Xu, P., Hui, J. H., Murty, T., Theruvath, J., Mehta, N., Yamada-Hunter, S. A., Weber, E. W., Heitzeneder, S., Parker, K. R., Satpathy, A. T., Chang, H. Y., Lin, M. Z., Cochran, J. R., Mackall, C. L. 2022

  Abstract

  Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

  View details for DOI 10.1016/j.cell.2022.03.041

  View details for PubMedID 35483375

• Dose-dependent effects of high intensity focused ultrasound on compound action potentials in an ex vivo rodent peripheral nerve model: comparison to local anesthetics. Regional anesthesia and pain medicine Anderson, T. A., Delgado, J., Sun, S., Behzadian, N., Vilches-Moure, J., Szlavik, R. B., Butts-Pauly, K., Yeomans, D. 1800

  Abstract

  BACKGROUND: In animal models, focused ultrasound can reversibly or permanently inhibit nerve conduction, suggesting a potential role in managing pain. We hypothesized focused ultrasound's effects on action potential parameters may be similar to those of local anesthetics.METHODS: In an ex vivo rat sciatic nerve model, action potential amplitude, area under the curve, latency to 10% peak, latency to 100% peak, rate of rise, and half peak width changes were assessed after separately applying increasing focused ultrasound pressures or concentrations of bupivacaine and ropivacaine. Focused ultrasound's effects on nerve structure were examined histologically.RESULTS: Increasing focused ultrasound pressures decreased action potential amplitude, area under the curve, and rate of rise, increased latency to 10% peak, and did not change latency to 100% peak or half peak width. Increasing local anesthetic concentrations decreased action potential amplitude, area under the curve, and rate of rise and increased latency to 10% peak, latency to 100% peak, and half peak width. At the highest focused ultrasound pressures, nerve architecture was altered compared with controls.DISCUSSION: While some action potential parameters were altered comparably by focused ultrasound and local anesthetics, there were small but notable differences. It is not evident if these differences may lead to differences in clinical pain effects when focused ultrasound is applied in vivo or if focused ultrasound pressures that result in clinically relevant changes damage nerve structures. Given the potential advantages of a non-invasive technique for managing pain conditions, further investigation may be warranted in an in vivo pain model.

  View details for DOI 10.1136/rapm-2021-103115

  View details for PubMedID 35115412

• Local Sound Speed Estimation for Pulse-Echo Ultrasound in Layered Media IEEE TRANSACTIONS ON ULTRASONICS FERROELECTRICS AND FREQUENCY CONTROL Ali, R., Telichko, A., Wang, H., Sukumar, U. K., Vilches-Moure, J. G., Paulmurugan, R., Dahl, J. J. 2022; 69 (2): 500-511

  Abstract

  Our previous methodology in local sound speed estimation utilized time delays measured by the cross correlation of delayed full-synthetic aperture channel data to estimate the average speed of sound. However, focal distortions in this methodology lead to biased estimates of the average speed of sound, which, in turn, leads to biased estimates of the local speed of sound. Here, we demonstrate the bias in the previous methodology and introduce a coherence-based average sound speed estimator that eliminates this bias and is computationally much cheaper in practice. Because this coherence-based approach estimates the average sound speed in the medium over an equally spaced grid in depth rather than time, we derive a refined model that relates the local and average speeds of sound as a function of depth in layered media. A fast, closed-form inversion of this model yields highly accurate local sound speed estimates. The root-mean-square (rms) error of local sound speed reconstruction in simulations of two-layer media is 4.6 and 2.5 m/s at 4 and 8 MHz, respectively. This work examines the impact of frequency, f -number, aberration, and reverberation on sound speed estimation. Phantom and in vivo experiments in rats further validate the coherence-based sound speed estimator.

  View details for DOI 10.1109/TUFFC.2021.3124479

  View details for Web of Science ID 000748372800009

  View details for PubMedID 34723801

• Contrast Enhanced Ultrasound Molecular Imaging of Spontaneous Chronic Inflammatory Bowel Disease in an Interleukin-2 Receptor alpha-/- Transgenic Mouse Model Using Targeted Microbubbles. Nanomaterials (Basel, Switzerland) Wang, H., Vilches-Moure, J. G., Bettinger, T., Cherkaoui, S., Lutz, A., Paulmurugan, R. 1800; 12 (2)

  Abstract

  Inflammatory bowel disease (IBD) is a lifelong inflammatory disorder with relapsing-remission cycles, which is currently diagnosed by clinical symptoms and signs, along with laboratory and imaging findings. However, such clinical findings are not parallel to the disease activity of IBD and are difficult to use in treatment monitoring. Therefore, non-invasive quantitative imaging tools are required for the multiple follow-up exams of IBD patients in order to monitor the disease activity and determine treatment regimens. In this study, we evaluated a dual P- and E-selectin-targeted microbubble (MBSelectin) in an interleukin-2 receptor alpha deficient (IL-2Ralpha-/-) spontaneous chronic IBD mouse model for assessing long-term anti-inflammatory effects with ultrasound molecular imaging (USMI). We used IL-2Ralpha-/- (male and female on a C57BL/6 genetic background; n = 39) and C57BL/6 wild-type (negative control; n = 6) mice for the study. USMI of the proximal, middle, and distal colon was performed with MBSelectin using a small animal scanner (Vevo 2100) up to six times in each IL-2Ralpha-/- mouse between 6-30 weeks of age. USMI signals were compared between IL-2Ralpha-/- vs. wild-type mice, and sexes in three colonic locations. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E-stained sections and for selectin expression by immunofluorescence staining. We successfully detected spontaneous chronic colitis in IL-2Ralpha-/- mice between 6-30 weeks (onset at 6-14 weeks) compared to wild-type mice. Both male and female IL-2Ralpha-/- mice were equally (p = 0.996) affected with the disease, and there was no significant (p > 0.05) difference in USMI signals of colitis between the proximal, middle, and distal colon. We observed the fluctuating USMI signals in IL-2Ralpha-/- mice between 6-30 weeks, which might suggest a resemblance of the remission-flare pattern of human IBD. The ex vivo H&E and immunostaining further confirmed the inflammatory changes, and the high expression of P- and E-selectin in the colon. The results of this study highlight the IL-2Ralpha-/- mice as a chronic colitis model and are suitable for the long-term assessment of treatment response using a dual P- and E-selectin-targeted USMI.

  View details for DOI 10.3390/nano12020280

  View details for PubMedID 35055297

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity CANCER CELL Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 2022; 40 (1): 53-+

  View details for DOI 10.1016/j.ccell.2021.12-005

  View details for Web of Science ID 000744691500012

• Mitochondrial Gene Diversity and Host Specificity of Isospora in Passerine Birds. Frontiers in veterinary science Kubiski, S. V., Witte, C., Burchell, J. A., Conradson, D., Zmuda, A., Barbon, A. R., Vilches-Moure, J. G., Felt, S. A., Rideout, B. A. 2022; 9: 847030

  Abstract

  Isospora infections are common in both wild and captive passerine species. Many bird species have been shown to have co-evolved with a particular species of Isospora. Disease can range from subclinical to severe and fatal, making infection and transmission of this parasite a concern for birds under managed care, particularly in institutions housing endangered species for breeding and reintroduction purposes. Whether birds in mixed-species enclosures represent a risk factor for severe isosporiasis due to infection with non-host-adapted strains is of concern for institutions managing these populations. To begin answering this question, we sought to characterize the host-specificity of Isospora spp. in a large number of passerine birds via retrospective sequencing of mitochondrial gene cytochrome c oxidase subunit I (COI). Despite outliers, Isospora sequences largely grouped by host species and/or host family. Additional research is warranted into the degree of interspecies transmission and host-switching of Isospora parasites, and risk factors for the development of severe disease in passerine birds.

  View details for DOI 10.3389/fvets.2022.847030

  View details for PubMedID 35847651

• GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity. Cancer cell Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 1800

  Abstract

  Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (5,000 molecules/cell, range 1-6* 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.

  View details for DOI 10.1016/j.ccell.2021.12.005

  View details for PubMedID 34971569

• Noninvasive estimation of local speed of sound by pulse-echo ultrasound in a rat model of nonalcoholic fatty liver. Physics in medicine and biology Telichko, A. V., Ali, R., Brevett, T., Wang, H., Vilches-Moure, J., Kumar, S. U., Paulmurugan, R., Dahl, J. J. 1800

  Abstract

  Objective:Speed of sound has previously been demonstrated to correlate with fat concentration in the liver. However, estimating speed of sound in the liver noninvasively can be biased by the speed of sound of the tissue layers overlying the liver. Here, we demonstrate a noninvasive local speed of sound estimator, which is based on a layered media assumption, that can accurately capture the speed of sound in the liver. We validate the estimator using an obese Zucker rat model of non-alcoholic fatty liver disease and correlate the local speed of sound with liver steatosis.Approach:We estimated the local and global average speed of sound noninvasively in 4 lean Zucker rats fed a normal diet and 16 obese Zucker rats fed a high fat diet for up to 8 weeks. The ground truth speed of sound and fat concentration were measured from the excised liver using established techniques.Main Results:The noninvasive, local speed of sound estimates of the livers were similar in value to their corresponding "ground truth'' measurements, having a slope ± standard error of the regression of 0.82 ± 0.15 (R2= 0.74 and p < 0.001). Measurement of the noninvasive global average speed of sound did not reliably capture the ``ground truth'' speed of sound in the liver, having a slope of 0.35 ± 0.07 (R2= 0.74 and p < 0.001). Decreasing local speed of sound was observed with increasing hepatic fat accumulation (approximately -1.7 m/s per 1% increase in hepatic fat) and histopathology steatosis grading (approximately -10 to -13 m/s per unit increase in steatosis grade). Local speed of sound estimates were highly correlated with steatosis grade, having Pearson and Spearman correlation coefficients both ranging from -0.87 to -0.78. In addition, a lobe-dependent speed of sound in the liver was observed by theex vivomeasurements, with speed of sound differences of up to 25 m/s (p < 0.003) observed between lobes in the liver of the same animal.Significance:The findings of this study suggest that local speed of sound estimation has the potential to be used to predict or assist in the measurement of hepatic fat concentration and that the global average speed of sound should be avoided in hepatic fat estimation due to significant bias in the speed of sound estimate.

  View details for DOI 10.1088/1361-6560/ac4562

  View details for PubMedID 34933288

• Potent activity of CAR T cells targeting the oncofetal protein GPC2 engineered to recognize low antigen density in neuroblastoma. Heitzeneder, S., Bosse, K. R., Zhu, Z., Jelev, D., Dhingra, S., Majzner, R., Sotillo-Pineiro, E., Buongervino, S., Xu, P., Huang, J., Delaidelli, A., Hasselblatt, M., Parker, K., Anbunathan, H., Alag, A., Hwang, J., Huang, M., Klysz, D. D., Theruvath, J. L., Vilches-Moure, J. G., Satpathy, A. T., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000680263502187

• Management of Morbidity and Mortality in a New Zealand White Rabbit Model of Steroid-Induced Osteonecrosis of the Femoral Head COMPARATIVE MEDICINE Casey, K. M., Gore, F., Vilches-Moure, J. G., Maruyama, M., Goodman, S. B., Yang, Y., Baker, S. W. 2021; 71 (1): 86–98

  Abstract

  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 Bordetella bronchiseptica and Escherichia coli and small intestine infections with Lawsonia intracellularis 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.

  View details for DOI 10.30802/AALAS-CM-20-000071

  View details for Web of Science ID 000620257900005

  View details for PubMedID 33500020

  View details for PubMedCentralID PMC7898173

• A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions. Proceedings of the National Academy of Sciences of the United States of America Yim, J. J., Harmsen, S., Flisikowski, K., Flisikowska, T., Namkoong, H., Garland, M., van den Berg, N. S., Vilches-Moure, J. G., Schnieke, A., Saur, D., Glasl, S., Gorpas, D., Habtezion, A., Ntziachristos, V., Contag, C. H., Gambhir, S. S., Bogyo, M., Rogalla, S. 2021; 118 (1)

  Abstract

  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 m, 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.

  View details for DOI 10.1073/pnas.2008072118

  View details for PubMedID 33443161

• CTLA-4 expression by B-1a B cells is essential for immune tolerance. Nature communications Yang, Y. n., Li, X. n., Ma, Z. n., Wang, C. n., Yang, Q. n., Byrne-Steele, M. n., Hong, R. n., Min, Q. n., Zhou, G. n., Cheng, Y. n., Qin, G. n., Youngyunpipatkul, J. V., Wing, J. B., Sakaguchi, S. n., Toonstra, C. n., Wang, L. X., Vilches-Moure, J. G., Wang, D. n., Snyder, M. P., Wang, J. Y., Han, J. n., Herzenberg, L. A. 2021; 12 (1): 525

  Abstract

  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.

  View details for DOI 10.1038/s41467-020-20874-x

  View details for PubMedID 33483505

• Metabolic profiling during malaria reveals the role of the aryl hydrocarbon receptor in regulating kidney injury. eLife Lissner, M. M., Cumnock, K., Davis, N. M., Vilches-Moure, J. G., Basak, P., Navarrete, D. J., Allen, J. A., Schneider, D. 2020; 9

  Abstract

  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 Ahr-/- mice are more susceptible to malaria and develop high plasma heme and acute kidney injury. This phenotype is dependent on AHR in Tek-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.

  View details for DOI 10.7554/eLife.60165

  View details for PubMedID 33021470

• Molecular Imaging of Infective Endocarditis With 6''-[18F]Fluoromaltotriose Positron Emission Tomography-Computed Tomography. Circulation Wardak, M., Gowrishankar, G., Zhao, X., Liu, Y., Chang, E., Namavari, M., Haywood, T., Gabr, M. T., Neofytou, E., Chour, T., Qin, X., Vilches-Moure, J. G., Hardy, J., Contag, C. H., McConnell, M. V., Wu, J. C., Gambhir, S. S. 2020; 141 (21): 1729–31

  View details for DOI 10.1161/CIRCULATIONAHA.119.043924

  View details for PubMedID 32453662

• Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma. Investigative radiology Bam, R. n., Daryaei, I. n., Abou-Elkacem, L. n., Vilches-Moure, J. G., Meuillet, E. J., Lutz, A. n., Marinelli, E. R., Unger, E. C., Gambhir, S. S., Paulmurugan, R. n. 2020

  Abstract

  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.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.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.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.

  View details for DOI 10.1097/RLI.0000000000000697

  View details for PubMedID 32569010

• Sound Speed Estimation in Layered Media Using the Angular Coherence of Plane Waves Ali, R., Maredia, S., Telichko, A., Wang, H., Paulmurugan, R., Vilches-Moure, J., Dahl, J. J., Byram, B. C., Ruiter, N. V. SPIE-INT SOC OPTICAL ENGINEERING. 2020

  View details for DOI 10.1117/12.2548878

  View details for Web of Science ID 000558363700011

• Salmonella-Driven Polarization of Granuloma Macrophages Antagonizes TNF-Mediated Pathogen Restriction during Persistent Infection. Cell host & microbe Pham, T. H., Brewer, S. M., Thurston, T., Massis, L. M., Honeycutt, J., Lugo, K., Jacobson, A. R., Vilches-Moure, J. G., Hamblin, M., Helaine, S., Monack, D. M. 2019

  Abstract

  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 CD11b+CD11c+Ly6C+ 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.

  View details for DOI 10.1016/j.chom.2019.11.011

  View details for PubMedID 31883922

• TOTAL ABDOMINAL ULTRA-RAPID FLASH IRRADIATION DEMONSTRATES DECREASED GASTROINTESTINAL TOXICITY COMPARED TO CONVENTIONAL TOTAL ABDOMINAL IRRADIATION IN MICE Levy, K., Eggold, J., Rafat, M., Schuler, E., Shehade, H., Fregoso, D., Vilches-Moure, J., Koong, A., Maxim, P., Loo, B. W., Rankin, E. AMER ASSOC CANCER RESEARCH. 2019: 182

  View details for Web of Science ID 000497337700109

• 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 MOLECULAR IMAGING AND BIOLOGY Bachawal, S. V., Park, J., Valluru, K. S., Loft, M., Felt, S. A., Vilches-Moure, J. G., Saenz, Y. F., Daniel, B., Iagaru, A., Sonn, G., Cheng, Z., Spielman, D. M., Willmann, J. K. 2019; 21 (5): 861–70

  View details for DOI 10.1007/s11307-018-1235-6

  View details for Web of Science ID 000483788100009

• Investigating circulating tumor cells and distant metastases in patient-derived orthotopic xenograft models of triple-negative breast cancer. Breast cancer research : BCR Ramani, V. C., Lemaire, C. A., Triboulet, M., Casey, K. M., Heirich, K., Renier, C., Vilches-Moure, J. G., Gupta, R., Razmara, A. M., Zhang, H., Sledge, G. W., Sollier, E., Jeffrey, S. S. 2019; 21 (1): 98

  Abstract

  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.

  View details for DOI 10.1186/s13058-019-1182-4

  View details for PubMedID 31462307

• Chronic Model of Inflammatory Bowel Disease in IL-10-/- Transgenic Mice: Evaluation with Ultrasound Molecular Imaging. Theranostics Wang, H., Vilches-Moure, J. G., Cherkaoui, S., Tardy, I., Alleaume, C., Bettinger, T., Lutz, A., Paulmurugan, R. 2019; 9 (21): 6031-6046

  Abstract

  Objective: 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 (MBSelectin). Materials and Methods: Interleukin 10 deficient (IL-10-/- on a C57BL/6 genetic background; n=55) and FVB (n=16) mice were used. In IL-10-/-mice, 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 MBSelectin at multiple time points. Imaged colon segments were analyzed ex vivo for inflammatory changes on H&E staining and for P-selectin expression on immunofluorescence staining. Results: 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. Conclusion: 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 MBSelectin, 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.

  View details for DOI 10.7150/thno.37397

  View details for PubMedID 31534535

  View details for PubMedCentralID PMC6735517

• Ultrasound/microbubble-mediated targeted delivery of anticancer microRNA-loaded nanoparticles to deep tissues in pigs. Journal of controlled release : official journal of the Controlled Release Society Di Ianni, T., Bose, R. J., Sukumar, U. K., Bachawal, S., Wang, H., Telichko, A., Herickhoff, C., Robinson, E., Baker, S., Vilches-Moure, J. G., Felt, S. A., Gambhir, S. S., Paulmurugan, R., Dahl, J. D. 2019

  Abstract

  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.

  View details for DOI 10.1016/j.jconrel.2019.07.024

  View details for PubMedID 31326463

• Receptor subtype discrimination using extensive shape complementary designed interfaces NATURE STRUCTURAL & MOLECULAR BIOLOGY Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K., Baker, D. 2019; 26 (6): 407-+

  View details for DOI 10.1038/s41594-019-0224-z

  View details for Web of Science ID 000470110200005

• Receptor subtype discrimination using extensive shape complementary designed interfaces. Nature structural & molecular biology Dang, L. T., Miao, Y., Ha, A., Yuki, K., Park, K., Janda, C. Y., Jude, K. M., Mohan, K., Ha, N., Vallon, M., Yuan, J., Vilches-Moure, J. G., Kuo, C. J., Garcia, K. C., Baker, D. 2019

  Abstract

  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.

  View details for PubMedID 31086346

• Embryonic Chicken (Gallus gallus domesticus) as a Model of Cardiac Biology and Development. Comparative medicine Vilches-Moure, J. G. 2019; 69 (3): 184-203

  Abstract

  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.

  View details for DOI 10.30802/AALAS-CM-18-000061

  View details for PubMedID 31182184

  View details for PubMedCentralID PMC6591676

• 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. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging Bachawal, S. V., Park, J. M., Valluru, K. S., Loft, M. D., Felt, S. A., Vilches-Moure, J. G., Saenz, Y. F., Daniel, B., Iagaru, A., Sonn, G., Cheng, Z., Spielman, D. M., Willmann, J. K. 2019

  Abstract

  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 [1-13C]pyruvate, PET (2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), [68Ga]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, [18F]FDG-PET (SUVmean=1.90) and C-13 MRS (lactate/totalC=0.59) showed elevated metabolic activity in the tumor. In dog 4, [18F]FDG (SUVmean=2.43), [68Ga]NODAGA-SCH1 (SUVmean=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.

  View details for PubMedID 30793241

• SETD3 is an actin histidine methyltransferase that prevents primary dystocia NATURE Wilkinson, A. W., Diep, J., Dai, S., Liu, S., Ooi, Y., Song, D., Li, T., Horton, J. R., Zhang, X., Liu, C., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J., Cowan, T., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X., Carette, J. E., Gozani, O. 2019; 565 (7739): 372-+

  View details for DOI 10.1038/s41586-018-0821-8

  View details for Web of Science ID 000455781600044

• Validation of a geropathology grading system for aging mouse studies. GeroScience Snyder, J. M., Snider, T. A., Ciol, M. A., Wilkinson, J. E., Imai, D. M., Casey, K. M., Vilches-Moure, J. G., Pettan-Brewer, C. n., Pillai, S. P., Carrasco, S. E., Salimi, S. n., Ladiges, W. n. 2019

  Abstract

  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.

  View details for DOI 10.1007/s11357-019-00088-w

  View details for PubMedID 31468322

• Chronic Model of Inflammatory Bowel Disease in IL-10(-/-) Transgenic Mice: Evaluation with Ultrasound Molecular Imaging THERANOSTICS Wang, H., Vilches-Moure, J. G., Cherkaoui, S., Tardy, I., Alleaume, C., Bettinger, T., Lutz, A., Paulmurugan, R. 2019; 9 (21): 6031–46

  View details for DOI 10.7150/thno.37397

  View details for Web of Science ID 000481603700001

• A Bird's-Eye View of Regulatory, Animal Care, and Training Considerations Regarding Avian Flight Research. Comparative medicine Baker, S. W., Tucci, E. R., Felt, S. A., Zehnder, A. n., Lentink, D. n., Vilches-Moure, J. G. 2019

  Abstract

  A thorough understanding of how animals fly is a central goal of many scientific disciplines. Birds are a commonly usedmodel 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.

  View details for DOI 10.30802/AALAS-CM-18-000033

  View details for PubMedID 30764892

• US Molecular Imaging of Acute Ileitis: Anti-Inflammatory Treatment Response Monitored with Targeted Microbubbles in a Preclinical Model RADIOLOGY Wang, H., Hyvelin, J., Felt, S. A., Guracar, I., Vilches-Moure, J. G., Cherkaoui, S., Bettinger, T., Tian, L., Lutz, A. M., Willmann, J. K. 2018; 289 (1): 90–100

  View details for DOI 10.1148/radiol.2018172600

  View details for Web of Science ID 000444990900015

• An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo. Nature biomedical engineering Vermesh, O., Aalipour, A., Ge, T. J., Saenz, Y., Guo, Y., Alam, I. S., Park, S. M., Adelson, C. N., Mitsutake, Y., Vilches-Moure, J., Godoy, E., Bachmann, M. H., Ooi, C. C., Lyons, J. K., Mueller, K., Arami, H., Green, A., Solomon, E. I., Wang, S. X., Gambhir, S. S. 2018; 2 (9): 696-705

  Abstract

  The detection and analysis of rare blood biomarkers is necessary for early diagnosis of cancer and to facilitate the development of tailored therapies. However, current methods for the isolation of circulating tumour cells (CTCs) or nucleic acids present in a standard clinical sample of only 5-10 ml of blood provide inadequate yields for early cancer detection and comprehensive molecular profiling. Here, we report the development of a flexible magnetic wire that can retrieve rare biomarkers from the subject's blood in vivo at a much higher yield. The wire is inserted and removed through a standard intravenous catheter and captures biomarkers that have been previously labelled with injected magnetic particles. In a proof-of-concept experiment in a live porcine model, we demonstrate the in vivo labelling and single-pass capture of viable model CTCs in less than 10 s. The wire achieves capture efficiencies that correspond to enrichments of 10-80 times the amount of CTCs in a 5-ml blood draw, and 500-5,000 times the enrichments achieved using the commercially available Gilupi CellCollector.

  View details for DOI 10.1038/s41551-018-0257-3

  View details for PubMedID 30505627

  View details for PubMedCentralID PMC6261517

• Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Paulk, N. K., Pekrun, K., Charville, G. W., Maguire-Nguyen, K., Wosczyna, M. N., Xu, J., Zhang, Y., Lisowski, L., Yoo, B., Vilches-Moure, J. G., Lee, G. K., Shrager, J. B., Rando, T. A., Kay, M. A. 2018; 10: 144–55

  View details for DOI 10.1016/j.omtm.2018.06.001

  View details for Web of Science ID 000451115100014

• An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo NATURE BIOMEDICAL ENGINEERING Vermesh, O., Aalipour, A., Ge, T., Saenz, Y., Guo, Y., Alam, I. S., Park, S., Adelson, C. N., Mitsutake, Y., Vilches-Moure, J., Godoy, E., Bachmann, M. H., Ooi, C., Lyons, J. K., Mueller, K., Arami, H., Green, A., Solomon, E., Wang, S. X., Gambhir, S. S. 2018; 2 (9): 696–705

  View details for DOI 10.1038/s41551-018-0257-3

  View details for Web of Science ID 000444282800014

• A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection CELL HOST & MICROBE Jacobson, A., Lam, L., Rajendram, M., Tamburini, F., Honeycutt, J., Trung Pham, Van Treuren, W., Pruss, K., Stabler, S., Lugo, K., Bouley, D. M., Vilches-Moure, J. G., Smith, M., Sonnenburg, J. L., Bhatt, A. S., Huang, K., Monack, D. 2018; 24 (2): 296-+

  View details for DOI 10.1016/j.chom.2018.07.002

  View details for Web of Science ID 000441083900016

• US Molecular Imaging of Acute Ileitis: Anti-Inflammatory Treatment Response Monitored with Targeted Microbubbles in a Preclinical Model. Radiology Wang, H., Hyvelin, J., Felt, S. A., Guracar, I., Vilches-Moure, J. G., Cherkaoui, S., Bettinger, T., Tian, L., Lutz, A. M., Willmann, J. K. 2018: 172600

  Abstract

  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 * 108/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.

  View details for PubMedID 30040040

• A Gut Commensal-Produced Metabolite Mediates Colonization Resistance to Salmonella Infection. Cell host & microbe Jacobson, A., Lam, L., Rajendram, M., Tamburini, F., Honeycutt, J., Pham, T., Van Treuren, W., Pruss, K., Stabler, S. R., Lugo, K., Bouley, D. M., Vilches-Moure, J. G., Smith, M., Sonnenburg, J. L., Bhatt, A. S., Huang, K. C., Monack, D. 2018

  Abstract

  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 invitro 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.

  View details for PubMedID 30057174

• SETD3 is an actin histidine methyltransferase that prevents primary dystocia. Nature Wilkinson, A. W., Diep, J. n., Dai, S. n., Liu, S. n., Ooi, Y. S., Song, D. n., Li, T. M., Horton, J. R., Zhang, X. n., Liu, C. n., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J. n., Cowan, T. n., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X. n., Carette, J. E., Gozani, O. n. 2018

  Abstract

  For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. 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.

  View details for PubMedID 30626964

• Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle. Molecular therapy. Methods & clinical development Paulk, N. K., Pekrun, K. n., Charville, G. W., Maguire-Nguyen, K. n., Wosczyna, M. N., Xu, J. n., Zhang, Y. n., Lisowski, L. n., Yoo, B. n., Vilches-Moure, J. G., Lee, G. K., Shrager, J. B., Rando, T. A., Kay, M. A. 2018; 10: 144–55

  Abstract

  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 ex vivo and in various primary and immortalized myogenic lines in vitro. 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.

  View details for PubMedID 30101152

• Anatomical Road Mapping Using CT and MR Enterography for Ultrasound Molecular Imaging of Small Bowel Inflammation in Swine. European radiology Wang, H. n., Felt, S. A., Guracar, I. n., Taviani, V. n., Zhou, J. n., Sigrist, R. M., Zhang, H. n., Liau, J. n., Vilches-Moure, J. G., Tian, L. n., Saenz, Y. n., Bettinger, T. n., Hargreaves, B. A., Lutz, A. M., Willmann, J. K. 2018; 28 (5): 2068–76

  Abstract

  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.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 (MBSelectin) and control (MBControl) contrast agents in diseased and healthy control bowel segments, followed by ex vivo histology.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 MBSelectin compared to MBControl 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.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.• 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.

  View details for PubMedID 29170798

• The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion. JCI insight Scortegagna, M., Berthon, A., Settas, N., Giannakou, A., Garcia, G., Li, J. L., James, B., Liddington, R. C., Vilches-Moure, J. G., Stratakis, C. A., Ronai, Z. A. 2017; 2 (23)

  Abstract

  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.

  View details for DOI 10.1172/jci.insight.97128

  View details for PubMedID 29212953

  View details for PubMedCentralID PMC5752272

• The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion JCI INSIGHT Scortegagna, M., Berthon, A., Settas, N., Giannakou, A., Garcia, G., Li, J., James, B., Liddington, R. C., Vilches-Moure, J. G., Stratakis, C. A., Ronai, Z. A. 2017; 2 (23)

  View details for DOI 10.1172/jci.insight.97128

  View details for Web of Science ID 000417307600010

• AAV Capsid Evolution for Enhanced Antibody Delivery to Human Skeletal Muscle for Use in Next-Generation HIV Vaccines and Muscle Gene Therapies Paulk, N. K., Charville, G., Maguire, K., Pekrun, K., Zhang, Y., Tiffany, M., Vilches-Moure, J., Lee, G., Shrager, J., Rando, T., Kay, M. A. NATURE PUBLISHING GROUP. 2016: S284–S285

  View details for DOI 10.1016/S1525-0016(16)33530-4

  View details for Web of Science ID 000375264200710

• Restrictive orbital myofibroblastic sarcoma in a cat--cross-sectional imaging (MRI & CT) appearance, treatment, and outcome. Veterinary ophthalmology Thomasy, S. M., Cissell, D. D., Arzi, B., Vilches-Moure, J. G., Lo, W. Y., Wisner, E. R., Dubielzig, R. R., Maggs, D. J. 2013; 16 Suppl 1 (0 0): 123-9

  Abstract

  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.

  View details for DOI 10.1111/vop.12016

  View details for PubMedID 23281709

  View details for PubMedCentralID PMC3620966

• Hepatosplenic and hepatocytotropic T-cell lymphoma: two distinct types of T-cell lymphoma in dogs. Veterinary pathology Keller, S. M., Vernau, W., Hodges, J., Kass, P. H., Vilches-Moure, J. G., McElliot, V., Moore, P. F. 2013; 50 (2): 281-90

  Abstract

  The clinical, clinicopathologic, and pathological findings of 9 dogs with T-cell lymphoma that involved the liver in the absence of peripheral lymphadenopathy were assessed. Seven dogs had hepatosplenic T-cell lymphoma (HS-TCL). Dogs with HS-TCL presented with hepato- and/or splenomegaly, regenerative anemia, thrombocytopenia, and hypoproteinemia. The clinical course was rapidly progressive with all dogs but 1 dead within 24 days of initial presentation. Neoplastic lymphocytes were centered on hepatic and splenic sinusoids and had a CD3+ (5/7), TCRαβ- (5/5), TCRγδ+ (3/5), CD11d+ (6/7), granzyme B+ (5/7) immunophenotype. Bone marrow and lungs were consistently but variably involved. These findings closely resemble the human disease and support the classification of HS-TCL as a distinct World Health Organization entity in dogs. The remaining 2 dogs markedly differed in the pattern of hepatic involvement by neoplastic lymphocytes, which were not confined to hepatic sinusoids but invaded hepatic cords. In addition, neoplastic cells had a CD11d- immunophenotype, and clinicopathologic data indicated marked cholestasis and mild to absent anemia. Based on the distinct tropism of neoplastic lymphocytes for hepatocytes, the name hepatocytotropic T-cell lymphoma (HC-TCL) is proposed. Given the histomorphologic, clinicopathologic, and immunophenotypic differences, HC-TCL likely represents a separate biological entity rather than a histomorphologic variant of HS-TCL.

  View details for DOI 10.1177/0300985812451625

  View details for PubMedID 22711745

• Myenteric ganglionitis as a cause of recurrent colic in an adult horse. Journal of the American Veterinary Medical Association Blake, K. R., Affolter, V. K., Lowenstine, L. J., Vilches-Moure, J. G., le Jeune, S. S. 2012; 240 (12): 1494-500

  Abstract

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

  View details for DOI 10.2460/javma.240.12.1494

  View details for PubMedID 22657934

• Ingested Shiga toxin 2 (Stx2) causes histopathological changes in kidney, spleen, and thymus tissues and mortality in mice. Journal of agricultural and food chemistry Rasooly, R., Do, P. M., Griffey, S. M., Vilches-Moure, J. G., Friedman, M. 2010; 58 (16): 9281-6

  Abstract

  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.

  View details for DOI 10.1021/jf101744z

  View details for PubMedID 20681531

• Genes unlinked to the leptin receptor influence urinary albumin excretion in obese Zucker rats. Physiological genomics Kim, K., Warden, C. H., Griffey, S. M., Vilches-Moure, J. G., Hansen, S., Cuppen, E., Nijman, I. J., Chiu, S., Stern, J. S. 2010; 41 (3): 297-305

  Abstract

  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.

  View details for DOI 10.1152/physiolgenomics.90367.2008

  View details for PubMedID 20159938

  View details for PubMedCentralID PMC2869106

• Comparison of rabbit monoclonal and mouse monoclonal antibodies in immunohistochemistry in canine tissues. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc Vilches-Moure, J. G., Ramos-Vara, J. A. 2005; 17 (4): 346-50

  Abstract

  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.

  View details for DOI 10.1177/104063870501700407

  View details for PubMedID 16130992