Browsing by Subject "Mice, Inbred C57BL"
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Item Open Access A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.(The Journal of neuroscience : the official journal of the Society for Neuroscience, 2014-05) Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, NicolasMechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.Item Open Access A cord blood monocyte-derived cell therapy product accelerates brain remyelination.(JCI insight, 2016-08-18) Saha, Arjun; Buntz, Susan; Scotland, Paula; Xu, Li; Noeldner, Pamela; Patel, Sachit; Wollish, Amy; Gunaratne, Aruni; Gentry, Tracy; Troy, Jesse; Matsushima, Glenn K; Kurtzberg, Joanne; Balber, Andrew EMicroglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.Item Open Access A craniofacial-specific monosynaptic circuit enables heightened affective pain.(Nature neuroscience, 2017-12) Rodriguez, Erica; Sakurai, Katsuyasu; Xu, Jennie; Chen, Yong; Toda, Koji; Zhao, Shengli; Han, Bao-Xia; Ryu, David; Yin, Henry; Liedtke, Wolfgang; Wang, FanHumans often rank craniofacial pain as more severe than body pain. Evidence suggests that a stimulus of the same intensity induces stronger pain in the face than in the body. However, the underlying neural circuitry for the differential processing of facial versus bodily pain remains unknown. Interestingly, the lateral parabrachial nucleus (PBL), a critical node in the affective pain circuit, is activated more strongly by noxious stimulation of the face than of the hindpaw. Using a novel activity-dependent technology called CANE developed in our laboratory, we identified and selectively labeled noxious-stimulus-activated PBL neurons and performed comprehensive anatomical input-output mapping. Surprisingly, we uncovered a hitherto uncharacterized monosynaptic connection between cranial sensory neurons and the PBL-nociceptive neurons. Optogenetic activation of this monosynaptic craniofacial-to-PBL projection induced robust escape and avoidance behaviors and stress calls, whereas optogenetic silencing specifically reduced facial nociception. The monosynaptic circuit revealed here provides a neural substrate for heightened craniofacial affective pain.Item Open Access A Diffusion MRI Tractography Connectome of the Mouse Brain and Comparison with Neuronal Tracer Data.(Cereb Cortex, 2015-11) Calabrese, Evan; Badea, Alexandra; Cofer, Gary; Qi, Yi; Johnson, G AllanInterest in structural brain connectivity has grown with the understanding that abnormal neural connections may play a role in neurologic and psychiatric diseases. Small animal connectivity mapping techniques are particularly important for identifying aberrant connectivity in disease models. Diffusion magnetic resonance imaging tractography can provide nondestructive, 3D, brain-wide connectivity maps, but has historically been limited by low spatial resolution, low signal-to-noise ratio, and the difficulty in estimating multiple fiber orientations within a single image voxel. Small animal diffusion tractography can be substantially improved through the combination of ex vivo MRI with exogenous contrast agents, advanced diffusion acquisition and reconstruction techniques, and probabilistic fiber tracking. Here, we present a comprehensive, probabilistic tractography connectome of the mouse brain at microscopic resolution, and a comparison of these data with a neuronal tracer-based connectivity data from the Allen Brain Atlas. This work serves as a reference database for future tractography studies in the mouse brain, and demonstrates the fundamental differences between tractography and neuronal tracer data.Item Open Access A genetically engineered thermally responsive sustained release curcumin depot to treat neuroinflammation.(J Control Release, 2013-10-10) Sinclair, S Michael; Bhattacharyya, Jayanta; McDaniel, Jonathan R; Gooden, David M; Gopalaswamy, Ramesh; Chilkoti, Ashutosh; Setton, Lori ARadiculopathy, a painful neuroinflammation that can accompany intervertebral disc herniation, is associated with locally increased levels of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Systemic administration of TNF antagonists for radiculopathy in the clinic has shown mixed results, and there is growing interest in the local delivery of anti-inflammatory drugs to treat this pathology as well as similar inflammatory events of peripheral nerve injury. Curcumin, a known antagonist of TNFα in multiple cell types and tissues, was chemically modified and conjugated to a thermally responsive elastin-like polypeptide (ELP) to create an injectable depot for sustained, local delivery of curcumin to treat neuroinflammation. ELPs are biopolymers capable of thermally-triggered in situ depot formation that have been successfully employed as drug carriers and biomaterials in several applications. ELP-curcumin conjugates were shown to display high drug loading, rapidly release curcumin in vitro via degradable carbamate bonds, and retain in vitro bioactivity against TNFα-induced cytotoxicity and monocyte activation with IC50 only two-fold higher than curcumin. When injected proximal to the sciatic nerve in mice via intramuscular (i.m.) injection, ELP-curcumin conjugates underwent a thermally triggered soluble-insoluble phase transition, leading to in situ formation of a depot that released curcumin over 4days post-injection and decreased plasma AUC 7-fold.Item Open Access A high-throughput in vitro drug screen in a genetically engineered mouse model of diffuse intrinsic pontine glioma identifies BMS-754807 as a promising therapeutic agent.(PLoS One, 2015) Halvorson, Kyle G; Barton, Kelly L; Schroeder, Kristin; Misuraca, Katherine L; Hoeman, Christine; Chung, Alex; Crabtree, Donna M; Cordero, Francisco J; Singh, Raj; Spasojevic, Ivan; Berlow, Noah; Pal, Ranadip; Becher, Oren JDiffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.Item Open Access A neural network-based method for spectral distortion correction in photon counting x-ray CT.(Physics in medicine and biology, 2016-08) Touch, Mengheng; Clark, Darin P; Barber, William; Badea, Cristian TSpectral CT using a photon counting x-ray detector (PCXD) shows great potential for measuring material composition based on energy dependent x-ray attenuation. Spectral CT is especially suited for imaging with K-edge contrast agents to address the otherwise limited contrast in soft tissues. We have developed a micro-CT system based on a PCXD. This system enables both 4 energy bins acquisition, as well as full-spectrum mode in which the energy thresholds of the PCXD are swept to sample the full energy spectrum for each detector element and projection angle. Measurements provided by the PCXD, however, are distorted due to undesirable physical effects in the detector and can be very noisy due to photon starvation in narrow energy bins. To address spectral distortions, we propose and demonstrate a novel artificial neural network (ANN)-based spectral distortion correction mechanism, which learns to undo the distortion in spectral CT, resulting in improved material decomposition accuracy. To address noise, post-reconstruction denoising based on bilateral filtration, which jointly enforces intensity gradient sparsity between spectral samples, is used to further improve the robustness of ANN training and material decomposition accuracy. Our ANN-based distortion correction method is calibrated using 3D-printed phantoms and a model of our spectral CT system. To enable realistic simulations and validation of our method, we first modeled the spectral distortions using experimental data acquired from (109)Cd and (133)Ba radioactive sources measured with our PCXD. Next, we trained an ANN to learn the relationship between the distorted spectral CT projections and the ideal, distortion-free projections in a calibration step. This required knowledge of the ground truth, distortion-free spectral CT projections, which were obtained by simulating a spectral CT scan of the digital version of a 3D-printed phantom. Once the training was completed, the trained ANN was used to perform distortion correction on any subsequent scans of the same system with the same parameters. We used joint bilateral filtration to perform noise reduction by jointly enforcing intensity gradient sparsity between the reconstructed images for each energy bin. Following reconstruction and denoising, the CT data was spectrally decomposed using the photoelectric effect, Compton scattering, and a K-edge material (i.e. iodine). The ANN-based distortion correction approach was tested using both simulations and experimental data acquired in phantoms and a mouse with our PCXD-based micro-CT system for 4 bins and full-spectrum acquisition modes. The iodine detectability and decomposition accuracy were assessed using the contrast-to-noise ratio and relative error in iodine concentration estimation metrics in images with and without distortion correction. In simulation, the material decomposition accuracy in the reconstructed data was vastly improved following distortion correction and denoising, with 50% and 20% reductions in material concentration measurement error in full-spectrum and 4 energy bins cases, respectively. Overall, experimental data confirms that full-spectrum mode provides superior results to 4-energy mode when the distortion corrections are applied. The material decomposition accuracy in the reconstructed data was vastly improved following distortion correction and denoising, with as much as a 41% reduction in material concentration measurement error for full-spectrum mode, while also bringing the iodine detectability to 4-6 mg ml(-1). Distortion correction also improved the 4 bins mode data, but to a lesser extent. The results demonstrate the experimental feasibility and potential advantages of ANN-based distortion correction and joint bilateral filtration-based denoising for accurate K-edge imaging with a PCXD. Given the computational efficiency with which the ANN can be applied to projection data, the proposed scheme can be readily integrated into existing CT reconstruction pipelines.Item Open Access A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity.(Free radical biology & medicine, 2012-05) Rajic, Zrinka; Tovmasyan, Artak; Spasojevic, Ivan; Sheng, Huaxin; Lu, Miaomiao; Li, Alice M; Gralla, Edith B; Warner, David S; Benov, Ludmil; Batinic-Haberle, InesThe Mn porphyrins of k(cat)(O(2)(.-)) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP(5+) is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP(5+) has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH(2) group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP(5+). The high SOD-like activity is preserved and even enhanced: log k(cat)(O(2)(.-))=7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP(5+) and MnTnHep-2-PyP(5+), respectively. MnTnBuOE-2-PyP(5+) was tested in an O(2)(.-) -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5-30 μM. MnTnHep-2-PyP(5+) was already toxic at 5 μM, and MnTnHex-2-PyP(5+) became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP(5+) was several-fold less toxic than either MnTnHex-2-PyP(5+) or MnTnHep-2-PyP(5+).Item Open Access A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.(Neuron, 2015-11-04) Gu, Bin; Huang, Yang Zhong; He, Xiao-Ping; Joshi, Rasesh B; Jang, Wonjo; McNamara, James OThe BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.Item Open Access A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus.(Nucleic acids research, 2020-09) Zhao, Hao; Li, Zhaoqiang; Zhu, Yongchang; Bian, Shasha; Zhang, Yan; Qin, Litao; Naik, Abani Kanta; He, Jiangtu; Zhang, Zhenhai; Krangel, Michael S; Hao, BingtaoThe regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.Item Open Access Activation of the ATF6 (Activating Transcription Factor 6) Signaling Pathway in Neurons Improves Outcome After Cardiac Arrest in Mice.(Journal of the American Heart Association, 2021-06-11) Shen, Yuntian; Li, Ran; Yu, Shu; Zhao, Qiang; Wang, Zhuoran; Sheng, Huaxin; Yang, WeiBackground Ischemia/reperfusion injury impairs proteostasis, and triggers adaptive cellular responses, such as the unfolded protein response (UPR), which functions to restore endoplasmic reticulum homeostasis. After cardiac arrest (CA) and resuscitation, the UPR is activated in various organs including the brain. However, the role of the UPR in CA has remained largely unknown. Here we aimed to investigate effects of activation of the ATF6 (activating transcription factor 6) UPR branch in CA. Methods and Results Conditional and inducible sATF6-KI (short-form ATF6 knock-in) mice and a selective ATF6 pathway activator 147 were used. CA was induced in mice by KCl injection, followed by cardiopulmonary resuscitation. We first found that neurologic function was significantly improved, and neuronal damage was mitigated after the ATF6 pathway was activated in neurons of sATF6-KI mice subjected to CA/cardiopulmonary resuscitation. Further RNA sequencing analysis indicated that such beneficial effects were likely attributable to increased expression of pro-proteostatic genes regulated by ATF6. Especially, key components of the endoplasmic reticulum-associated degradation process, which clears potentially toxic unfolded/misfolded proteins in the endoplasmic reticulum, were upregulated in the sATF6-KI brain. Accordingly, the CA-induced increase in K48-linked polyubiquitin in the brain was higher in sATF6-KI mice relative to control mice. Finally, CA outcome, including the survival rate, was significantly improved in mice treated with compound 147. Conclusions This is the first experimental study to determine the role of the ATF6 UPR branch in CA outcome. Our data indicate that the ATF6 UPR branch is a prosurvival pathway and may be considered as a therapeutic target for CA.Item Open Access Adenovirus F protein as a delivery vehicle for botulinum B.(BMC Immunol, 2010-07-07) Clapp, Beata; Golden, Sarah; Maddaloni, Massimo; Staats, Herman F; Pascual, David WBACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.Item Open Access Allo-Specific Humoral Responses: New Methods for Screening Donor-Specific Antibody and Characterization of HLA-Specific Memory B Cells.(Frontiers in immunology, 2021-01) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, GarnettAntibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.Item Open Access An aberrant SREBP-dependent lipogenic program promotes metastatic prostate cancer.(Nature genetics, 2018-02) Chen, Ming; Zhang, Jiangwen; Sampieri, Katia; Clohessy, John G; Mendez, Lourdes; Gonzalez-Billalabeitia, Enrique; Liu, Xue-Song; Lee, Yu-Ru; Fung, Jacqueline; Katon, Jesse M; Menon, Archita Venugopal; Webster, Kaitlyn A; Ng, Christopher; Palumbieri, Maria Dilia; Diolombi, Moussa S; Breitkopf, Susanne B; Teruya-Feldstein, Julie; Signoretti, Sabina; Bronson, Roderick T; Asara, John M; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Pandolfi, Pier PaoloLipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.Item Open Access An entirely cell-based system to generate single-chain antibodies against cell surface receptors.(2008) Chen, Yu-Hsun JasonThe generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen (Ag). Traditionally, the generation of single chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single chain Abs that does not require the use of purified protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high throughputs creening of arrayed phage clones, and characterization of recombinant single chain variable regions(scFvs). This strategy was used to generate a panel of single chain Abs specific for the innate immunity receptor Toll‐like receptor2 (TLR2). Once generated, individual scFvs were subcloned into an expression vector allowing the production of recombinant antibodies in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell‐based system efficiently generates Abs that bind native molecules displayed on cell surfaces, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination. However, an inconvenience of this strategy is that it requires construction of a new library for each target TLR. This problem might be solved by using non‐immune antibody libraries to obtain antibodies against multiple TLRs. Non‐immune libraries contain a wide variety of antibodies but these are often low affinity, while immune libraries, derived from immunized animals, containa high frequency of high affinity antibodies, but are typically limited to a single antigen. In addition, it can be difficult to produce non‐immune libraries with sufficient complexity to select Abs against multiple Ags. Because the re‐assortment of VH and VL regions that occurs during antibody library construction greatly increases library complexity, we hypothesized that an immune antibody library produced against one member of a protein family would contain antibodies specific for other members of the same protein family. Here, we tested this hypothesis by mining an existing anti‐hTLR2 antibody library for antibodies specific for other members of the TLR family. This procedure, which we refer to as homolog mining, proved to be effective. Using a cell‐based system to pan and screen our anti‐hTLR2 library, we identified single chain antibodies specific for three of the four hTLR2 homologs we targeted. The antibodies identified, anti‐murine TLR2, anti‐hTLR5, and anti‐hTLR6, bind specifically to their target, with no cross‐reactivity to hTLR2 or other TLRs tested. These results demonstrate that combinatorial re‐assortment of VH and VL fragments during Ab library construction increases Ab repertoire complexity, allowing antibody libraries produced by immunization with one antigen to be used to obtain antibodies specific to related antigens. The principle of homolog mining may be extended to other protein families and will facilitate and accelerate antibody production processes. In conclusion, we developed an entirely cell‐based method to generate antibodies that bind to native molecules on the cell surface, while eliminating the requirement of recombinant proteins and the risk of microbial component contamination. With homolog mining, this system is capable of generating antibodies not only against the original immunized Ag, but also against homologous Ags. In combination, this system proved to be an effective and efficient means for generating multiple antibodies that bind to multiple related Ags as they are displayed on cell surfaces.Item Open Access An Evolutionary Insertion in the Mxra8 Receptor-Binding Site Confers Resistance to Alphavirus Infection and Pathogenesis.(Cell host & microbe, 2020-03) Kim, Arthur S; Zimmerman, Ofer; Fox, Julie M; Nelson, Christopher A; Basore, Katherine; Zhang, Rong; Durnell, Lorellin; Desai, Chandni; Bullock, Christopher; Deem, Sharon L; Oppenheimer, Jonas; Shapiro, Beth; Wang, Ting; Cherry, Sara; Coyne, Carolyn B; Handley, Scott A; Landis, Michael J; Fremont, Daved H; Diamond, Michael SAlphaviruses are emerging, mosquito-transmitted RNA viruses with poorly understood cellular tropism and species selectivity. Mxra8 is a receptor for multiple alphaviruses including chikungunya virus (CHIKV). We discovered that while expression of mouse, rat, chimpanzee, dog, horse, goat, sheep, and human Mxra8 enables alphavirus infection in cell culture, cattle Mxra8 does not. Cattle Mxra8 encodes a 15-amino acid insertion in its ectodomain that prevents Mxra8 binding to CHIKV. Identical insertions are present in zebu, yak, and the extinct auroch. As other Bovinae lineages contain related Mxra8 sequences, this insertion likely occurred at least 5 million years ago. Removing the Mxra8 insertion in Bovinae enhances alphavirus binding and infection, while introducing the insertion into mouse Mxra8 blocks CHIKV binding, prevents infection by multiple alphaviruses in cells, and mitigates CHIKV-induced pathogenesis in mice. Our studies on how this insertion provides resistance to CHIKV infection could facilitate countermeasures that disrupt Mxra8 interactions with alphaviruses.Item Open Access An operant-based detection method for inferring tinnitus in mice.(Journal of neuroscience methods, 2017-11) Zuo, Hongyan; Lei, Debin; Sivaramakrishnan, Shobhana; Howie, Benjamin; Mulvany, Jessica; Bao, JianxinBackground
Subjective tinnitus is a hearing disorder in which a person perceives sound when no external sound is present. It can be acute or chronic. Because our current understanding of its pathology is incomplete, no effective cures have yet been established. Mouse models are useful for studying the pathophysiology of tinnitus as well as for developing therapeutic treatments.New method
We have developed a new method for determining acute and chronic tinnitus in mice, called sound-based avoidance detection (SBAD). The SBAD method utilizes one paradigm to detect tinnitus and another paradigm to monitor possible confounding factors, such as motor impairment, loss of motivation, and deficits in learning and memory.Results
The SBAD method has succeeded in monitoring both acute and chronic tinnitus in mice. Its detection ability is further validated by functional studies demonstrating an abnormal increase in neuronal activity in the inferior colliculus of mice that had previously been identified as having tinnitus by the SBAD method.Comparison with existing methods
The SBAD method provides a new means by which investigators can detect tinnitus in a single mouse accurately and with more control over potential confounding factors than existing methods.Conclusion
This work establishes a new behavioral method for detecting tinnitus in mice. The detection outcome is consistent with functional validation. One key advantage of mouse models is they provide researchers the opportunity to utilize an extensive array of genetic tools. This new method could lead to a deeper understanding of the molecular pathways underlying tinnitus pathology.Item Open Access Anti-fibrotic effects of different sources of MSC in bleomycin-induced lung fibrosis in C57BL6 male mice.(Respirology (Carlton, Vic.), 2021-02) Periera-Simon, Simone; Xia, Xiaomei; Catanuto, Paola; Coronado, Ramon; Kurtzberg, Joanne; Bellio, Michael; Lee, Yee-Shuan; Khan, Aisha; Smith, Robin; Elliot, Sharon J; Glassberg, Marilyn KBackground and objective
IPF is a fatal and debilitating lung disorder increasing in incidence worldwide. To date, two approved treatments only slow disease progression, have multiple side effects and do not provide a cure. MSC have promising therapeutic potential as a cell-based therapy for many lung disorders based on the anti-fibrotic properties of the MSC.Methods
Critical questions remain surrounding the optimal source, timing and efficacy of cell-based therapies. The present study examines the most effective sources of MSC. Human MSC were derived from adipose, WJ, chorionic membrane (CSC) and chorionic villi (CVC). MSC were injected into the ageing mouse model of BLM-induced lung fibrosis.Results
All sources decreased Aschroft and hydroxyproline levels when injected into BLM-treated mice at day 10 with the exception of CSC cells that did not change hydroxyproline levels. There were also decreases in mRNA expression of αv -integrin and TNFα in all sources except CSC. Only ASC- and WJ-derived cells reduced AKT and MMP-2 activation, while Cav-1 was increased by ASC treatment as previously reported. BLM-induced miR dysregulation of miR-29 and miR-199 was restored only by ASC treatment.Conclusion
Our data suggest that sources of MSC may differ in the pathway(s) involved in repair.Item Open Access Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.(Nat Commun, 2015-01-22) Kanayama, M; Inoue, M; Danzaki, K; Hammer, G; He, Y; Shinohara, MLImmune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.Item Open Access Axonal regrowth after spinal cord injury via chondroitinase and the tissue plasminogen activator (tPA)/plasmin system.(The Journal of neuroscience : the official journal of the Society for Neuroscience, 2011-10) Bukhari, Noreen; Torres, Luisa; Robinson, John K; Tsirka, Stella ESpinal cord injury (SCI) causes permanent debilitation due to the inability of axons to grow through established scars. Both the sugar chains and core proteins of chondroitin sulfate proteoglycans (CSPGs) are inhibitory for neurite regrowth. Chondroitinase ABC (ChABC) degrades the sugar chains and allows for synaptic plasticity, suggesting that after the sugar chain cleavage additional steps occur promoting a permissive microenvironment in the glial scar region. We report that the clearance of the core protein by the tissue plasminogen activator (tPA)/plasmin proteolytic system partially contributes to ChABC-promoted plasticity. tPA and plasmin are upregulated after SCI and degrade the deglycosylated CSPG proteins. Mice lacking tPA (tPA(-/-)) exhibit attenuated neurite outgrowth and blunted sensory and motor recovery despite ChABC treatment. Coadministration of ChABC and plasmin enhanced the tPA(-/-) phenotype and supported recovery in WT SCI mice. Collectively, these findings show that the tPA/plasmin cascade may act downstream of ChABC to allow for synergistic sensory and motor improvement compared with each treatment alone and suggest a potential new approach to enhance functional recovery after SCI.