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The bacterial pathogen Shigella flexneri causes 270 million cases of bacillary dysentery (blood in stool) worldwide every year, resulting in more than 200,000 deaths. A major challenge in combating bacillary dysentery is the lack of a small-animal model that recapitulates the symptoms observed in infected individuals, including bloody diarrhea. Here, we show that similar to humans, infant rabbits infected with S. flexneri experience severe inflammation, massive ulceration of the colonic mucosa, and bloody diarrhea. T3SS-dependent invasion of epithelial cells is necessary and sufficient for mediating immune cell infiltration and vascular lesions. However, massive ulceration of the colonic mucosa, bloody diarrhea, and dramatic weight loss are strictly contingent on the ability of the bacteria to spread from cell to cell. The infant rabbit model features bacterial dissemination as a critical determinant of S. flexneri pathogenesis and provides a unique small-animal model for research and development of therapeutic interventions.
Purpose - To use our intra-arterial chemotherapy (IAC) rabbit model to assess the impact of IAC procedure, drug, dose, and choice of technique on ocular structure and function, to study the nature and etiology of IAC toxicity, and to compare to observations in patients.
Methods - Rabbits received IAC melphalan (0.4-0.8 mg/kg), carboplatin (25-50 mg), or saline, either by direct ophthalmic artery cannulation, or with a technique emulating nonocclusion. Ocular structure/function were assessed with examination, electroretinography (ERG), fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT angiography, prior to and 5 to 6 weeks after IAC. Blood counts were obtained weekly. We reviewed our last 50 IAC treatments in patients for evidence of ocular or systemic complications.
Results - No toxicity was seen in the saline control group. With standard (0.4 mg/kg) melphalan, no vascular/microvascular abnormalities were seen with either technique. However, severe microvascular pruning and arteriolar occlusions were seen occasionally at 0.8 mg/kg doses. ERG reductions were dose-dependent. Histology showed melphalan dose-dependent degeneration in all retinal layers, restricted geographically to areas of greatest vascular density. Carboplatin caused massive edema of ocular/periocular structures. IAC patients experienced occasional periocular swelling/rash, and only rarely experienced retinopathy or vascular events/hemorrhage in eyes treated multiple times with triple (melphalan/carboplatin/topotecan) therapy. Transient neutropenia occurred after 46% of IAC procedures, generally after triple therapy.
Conclusions - IAC toxicity appears to be related to the specific drug being used and is dose-dependent, rather than related to the IAC procedure itself or the specific technique selected. These rabbit findings are corroborated by our clinical findings in patients.
Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with HO and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.
BACKGROUND - Diffusion tensor tractography (DTT) has recently been shown to accurately detect nerve injury and regeneration. This study assesses whether 7-tesla (7T) DTT imaging is a viable modality to observe axonal outgrowth in a 4 cm rabbit sciatic nerve injury model fixed by a reverse autograft (RA) surgical technique.
METHODS - Transection injury of unilateral sciatic nerve (4 cm long) was performed in 25 rabbits and repaired using a RA surgical technique. Analysis of the nerve autograft was performed at 3, 6, and 11 weeks postoperatively and compared to normal contralateral sciatic nerve, used as control group. High-resolution DTT from ex vivo sciatic nerves were obtained using 3D diffusion-weighted spin-echo acquisitions at 7-T. Total axons and motor and sensory axons were counted at defined lengths along the graft.
RESULTS - At 11 weeks, histologically, the total axon count of the RA group was equivalent to the contralateral uninjured nerve control group. Similarly, by qualitative DTT visualization, the 11-week RA group showed increased fiber tracts compared to the 3 and 6 weeks counterparts. Upon immunohistochemical evaluation, 11-week motor axon counts did not significantly differ between RA and control; but significantly decreased sensory axon counts remained. Nerves explanted at 3 weeks and 6 weeks showed decreased motor and sensory axon counts.
DISCUSSION - 7-T DTT is an effective imaging modality that may be used qualitatively to visualize axonal outgrowth and regeneration. This has implications for the development of technology that non-invasively monitors peripheral nerve regeneration in a variety of clinical settings.
IMPACT STATEMENT - The development of autograft extenders is a significant clinical need in bone tissue engineering. We report new settable poly(thioketal urethane)-based autograft extenders that have bone-like mechanical properties and handling properties comparable to calcium phosphate bone cements. These settable autograft extenders remodeled to form new bone in a biologically stringent intertransverse process model of bone formation that does not heal when treated with calcium phosphate bone void fillers or cements alone. This is the first study to report settable autograft extenders with bone-like strength and handling properties comparable to ceramic bone cements, which have the potential to improve treatment of bone fractures and other orthopedic conditions.
OBJECTIVE - The objective of this study was to test the hypothesis that a compression-resistant bone graft augmented with recombinant human morphogenetic protein-2 (rhBMP-2) will promote lateral ridge augmentation without the use of protective mesh in a canine model.
MATERIALS & METHODS - Compression-resistant (CR) bone grafts were evaluated in a canine model of lateral ridge augmentation. Bilateral, right trapezoidal prism-shaped defects (13-14 mm long × 8-9 mm wide × 3-4 mm deep at the base) in 13 hounds (two defects per hound) were treated with one of four groups: (i) absorbable collagen sponge + 400 μg rhBMP-2/ml (ACS, clinical control) protected by titanium mesh, (ii) CR without rhBMP-2 (CR, negative control), (iii) CR + 200 μg rhBMP-2 (CR-L), or (iv) CR + 400 μg rhBMP-2 (CR-H). All animals were euthanized after 16 weeks. Ridge height and width and new bone formation were assessed by μCT, histology, and histomorphometry. The release kinetics of rhBMP-2 from CR bone grafts in vitro and in vivo in a femoral condyle defect model in rabbits was also evaluated.
RESULTS - All four bone grafts promoted new bone formation (11-31.6 volume%) in the lateral ridge defects. For CR grafts, ridge height and width increased in a dose-responsive manner with increasing rhBMP-2 concentration. Ridge height and width measured for CR-H without the use of protective mesh was comparable to that measured for ACS with a protective mesh.
CONCLUSIONS - At the same dose of rhBMP-2, an injectable, compression-resistant bone graft resulted in a comparable volume of new bone formation with the clinical control (ACS). These findings highlight the potential of compression-resistant bone grafts without the use of protective mesh for lateral ridge augmentation.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Purpose - Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy.
Methods - Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity.
Results - OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 μM and area under the curve (AUC0→∞) was 5.26 μM·h. Treated eye vitreous Cmax was 2.24 μM and AUC0→∞ was 4.19 μM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 μM and AUC0→∞ was 2.07 μM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan.
Conclusions - This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.
The events required for the induction of broad neutralizing antibodies (bnAbs) following HIV-1 envelope (Env) vaccination are unknown, and their induction in animal models as proof of concept would be critical. Here, we describe the induction of plasma antibodies capable of neutralizing heterologous primary (tier 2) HIV-1 strains in one macaque and two rabbits. Env immunogens were designed to induce CD4 binding site (CD4bs) bnAbs, but surprisingly, the macaque developed V1V2-glycan bnAbs. Env immunization of CD4bs bnAb heavy chain rearrangement (VDJ) knockin mice similarly induced V1V2-glycan neutralizing antibodies (nAbs), wherein the human CD4bs V chains were replaced with mouse rearrangements bearing diversity region (D)-D fusions, creating antibodies with long, tyrosine-rich HCDR3s. Our results show that Env vaccination can elicit broad neutralization of tier 2 HIV-1, demonstrate that V1V2-glycan bnAbs are more readily induced than CD4bs bnAbs, and define V replacement and diversity region fusion as potential mechanisms for generating V1V2-glycan bnAb site antibodies.
Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Studies of regulatory activity and gene expression have revealed an intriguing dichotomy: There is substantial turnover in the regulatory activity of orthologous sequences between species; however, the expression level of orthologous genes is largely conserved. Understanding how distal regulatory elements, for example, enhancers, evolve and function is critical, as alterations in gene expression levels can drive the development of both complex disease and functional divergence between species. In this study, we investigated determinants of the conservation of regulatory enhancer activity for orthologous sequences across mammalian evolution. Using liver enhancers identified from genome-wide histone modification profiles in ten diverse mammalian species, we compared orthologous sequences that exhibited regulatory activity in all species (conserved-activity enhancers) to shared sequences active only in a single species (species-specific-activity enhancers). Conserved-activity enhancers have greater regulatory potential than species-specific-activity enhancers, as quantified by both the density and diversity of transcription factor binding motifs. Consistent with their greater regulatory potential, conserved-activity enhancers have greater regulatory activity in humans than species-specific-activity enhancers: They are active across more cellular contexts, and they regulate more genes than species-specific-activity enhancers. Furthermore, the genes regulated by conserved-activity enhancers are expressed in more tissues and are less tolerant of loss-of-function mutations than those targeted by species-specific-activity enhancers. These consistent results across various stages of gene regulation demonstrate that conserved-activity enhancers are more pleiotropic than their species-specific-activity counterparts. This suggests that pleiotropy is associated with the conservation of regulatory across mammalian evolution.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
BACKGROUND - The challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model.
METHODS - LV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% β-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts' capability to encourage spinal fusion.
RESULTS - These materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 μg rhBMP-2/ml. Images of μCT and histological sections revealed evidence of bone fusion near the transverse processes.
CONCLUSION - This study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion.