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1.
Front Neurol ; 14: 1281790, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38107632

RESUMO

Introduction: Posture extraction from videos is fundamental to many real-world applications, including health screenings. In this study, we extend the utility and specificity of a well-established protocol, the balance beam, for examining balance and active motor coordination in adult mice of both sexes. Objectives: The primary objective of this study is to design a workflow for analyzing the postures of mice walking on a balance beam. Methods: We developed new tools and scripts based on the FluoRender architecture, which can interact with DeepLabCut (DLC) through Python code. Notably, twenty input videos were divided into four feature point groups (head, body, tail, and feet), based on camera positions relative to the balance beam (left and right), and viewing angles (90° and 45° from the beam). We determined key feature points on the mouse to track posture in a still video frame. We extracted a standard walk cycle (SWC) by focusing on foot movements, which were computed by a weighted average of the extracted walk cycles. The correlation of each walk cycle to the SWC was used as the weight. Results: We learned that positions of the camera angles significantly improved the performance of 2D pose estimation (90°) and 3D (45°). Comparing the SWCs from age-matched mice, we found a consistent pattern of supporting feet on the beam. Two feet were consistently on the beam followed by three feet and another three feet in a 2-3-3 pattern. However, this pattern can be mirrored among individual subjects. A subtle phase shift of foot movement was also observed from the SWCs. Furthermore, we compared the SWCs with speed values to reveal anomalies in mouse walk postures. Some anomalies can be explained as the start or finish of the traversal, while others may be correlated to the distractions of the test environment, which will need further investigation. Conclusion: Our posture analysis workflow improves the classical behavioral testing and analysis, allowing the detection of subtle, but significant differences in vestibular function and motor coordination.

2.
Comput Sci Eng ; 24(6): 60-65, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-38094600

RESUMO

FluoRender is a software program used for the visualization and analysis of 3-D biological image data, particularly from fluorescence microscopy. We examine FluoRender's script system to demonstrate its translation process. In this article, we borrow the concept of lingua franca from linguistics. We designed a connecting language between the source and target domains for translation, thereby augmenting understanding and acceptance. In FluoRender's script system, the lingua franca consists of the mapping between the control of the media player and the computational and interactive subroutines of an analysis workflow. Workflows supporting automatic, semiautomatic, and manual operations were made available and easily accessible to end users. The formalization of the lingua franca as a technique for translational computer science provides guidance for future development.

3.
Comput Graph ; 98: 138-149, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34602661

RESUMO

The main objective for understanding fluorescence microscopy data is to investigate and evaluate the fluorescent signal intensity distributions as well as their spatial relationships across multiple channels. The quantitative analysis of 3D fluorescence microscopy data needs interactive tools for researchers to select and focus on relevant biological structures. We developed an interactive tool based on volume visualization techniques and GPU computing for streamlining rapid data analysis. Our main contribution is the implementation of common data quantification functions on streamed volumes, providing interactive analyses on large data without lengthy preprocessing. Data segmentation and quantification are coupled with brushing and executed at an interactive speed. A large volume is partitioned into data bricks, and only user-selected structures are analyzed to constrain the computational load. We designed a framework to assemble a sequence of GPU programs to handle brick borders and stitch analysis results. Our tool was developed in collaboration with domain experts and has been used to identify cell types. We demonstrate a workflow to analyze cells in vestibular epithelia of transgenic mice.

4.
Front Neurosci ; 15: 710076, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34566562

RESUMO

Hair cells in the mammalian inner ear sensory epithelia are surrounded by supporting cells which are essential for function of cochlear and vestibular systems. In mice, support cells exhibit spontaneous intracellular Ca2+ transients in both auditory and vestibular organs during the first postnatal week before the onset of hearing. We recorded long lasting (>200 ms) Ca2+ transients in cochlear and vestibular support cells in neonatal mice using the genetic calcium indicator GCaMP5. Both cochlear and vestibular support cells exhibited spontaneous intracellular Ca2+ transients (GCaMP5 ΔF/F), in some cases propagating as waves from the apical (endolymph facing) to the basolateral surface with a speed of ∼25 µm per second, consistent with inositol trisphosphate dependent calcium induced calcium release (CICR). Acetylcholine evoked Ca2+ transients were observed in both inner border cells in the cochlea and vestibular support cells, with a larger change in GCaMP5 fluorescence in the vestibular support cells. Adenosine triphosphate evoked robust Ca2+ transients predominantly in the cochlear support cells that included Hensen's cells, Deiters' cells, inner hair cells, inner phalangeal cells and inner border cells. A Ca2+ event initiated in one inner border cells propagated in some instances longitudinally to neighboring inner border cells with an intercellular speed of ∼2 µm per second, and decayed after propagating along ∼3 cells. Similar intercellular propagation was not observed in the radial direction from inner border cell to inner sulcus cells, and was not observed between adjacent vestibular support cells.

5.
iScience ; 23(8): 101407, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32771977

RESUMO

Sense of motion, spatial orientation, and balance in vertebrates relies on sensory hair cells in the inner ear vestibular system. Vestibular supporting cells can regenerate hair cells that are lost from aging, ototoxicity, and trauma, although not all factors or specific cell types are known. Here we report a population of GAD2-positive cells in the mouse crista ampullaris and trace GAD2 progenitor-like cells that express pluripotent transcription factors SOX2, PROX1, and CTBP2. GAD2 progenitor-like cells organize into rosettes around a central branched structure in the eminentia cruciatum (EC) herein named the EC plexus. GCaMP5G calcium indicator shows spontaneous and acetylcholine-evoked whole-cell calcium waves in neonatal and adult mice. We present a hypothetical model that outlines the lineage and potential regenerative capacity of GAD2 cells in the mammalian vestibular neuroepithelium.

6.
Front Cell Neurosci ; 13: 186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31133810

RESUMO

Spontaneous calcium transients are present during early postnatal development in the mouse retina and cochlea, and play an important role in maturation of the sensory organs and neural circuits in the central nervous system (CNS). It is not known whether similar calcium transients occur during postnatal development in the vestibular sensory organs. Here we demonstrate spontaneous intracellular calcium transients in sensory hair cells (HCs) and supporting cells (SCs) in the murine utricular macula during the first two postnatal weeks. Calcium transients were monitored using a genetically encoded calcium indicator, GCaMP5G (G5), at 100 ms-frame-1 in excised utricle sensory epithelia, including HCs, SCs, and neurons. The reporter line expressed G5 and tdTomato (tdT) in a Gad2-Cre dependent manner within a subset of utricular HCs, SCs and neurons. Kinetics of the G5 reporter limited temporal resolution to calcium events lasting longer than 200 ms. Spontaneous calcium transients lasting 1-2 s were observed in the expressing population of HCs at birth and slower spontaneous transients lasting 10-30 s appeared in SCs by P3. Beginning at P5, calcium transients could be modulated by application of the efferent neurotransmitter acetylcholine (ACh). In mature mice, calcium transients in the utricular macula occurred spontaneously, had a duration 1-2 s, and could be modulated by the exogenous application of acetylcholine (ACh) or muscarine. Long-lasting calcium transients evoked by ACh in mature mice were blocked by atropine, consistent with previous reports describing the role of muscarinic receptors expressed in calyx bearing afferents in efferent control of vestibular sensation. Large spontaneous and ACh evoked transients were reversibly blocked by the inositol trisphosphate receptor (IP3R) antagonist aminoethoxydiphenyl borate (2-APB). Results demonstrate long-lasting calcium transients are present in the utricular macula during the first postnatal week, and that responses to ACh mature over this same time period.

7.
BMC Bioinformatics ; 18(1): 280, 2017 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-28549411

RESUMO

BACKGROUND: Image segmentation and registration techniques have enabled biologists to place large amounts of volume data from fluorescence microscopy, morphed three-dimensionally, onto a common spatial frame. Existing tools built on volume visualization pipelines for single channel or red-green-blue (RGB) channels have become inadequate for the new challenges of fluorescence microscopy. For a three-dimensional atlas of the insect nervous system, hundreds of volume channels are rendered simultaneously, whereas fluorescence intensity values from each channel need to be preserved for versatile adjustment and analysis. Although several existing tools have incorporated support of multichannel data using various strategies, the lack of a flexible design has made true many-channel visualization and analysis unavailable. The most common practice for many-channel volume data presentation is still converting and rendering pseudosurfaces, which are inaccurate for both qualitative and quantitative evaluations. RESULTS: Here, we present an alternative design strategy that accommodates the visualization and analysis of about 100 volume channels, each of which can be interactively adjusted, selected, and segmented using freehand tools. Our multichannel visualization includes a multilevel streaming pipeline plus a triple-buffer compositing technique. Our method also preserves original fluorescence intensity values on graphics hardware, a crucial feature that allows graphics-processing-unit (GPU)-based processing for interactive data analysis, such as freehand segmentation. We have implemented the design strategies as a thorough restructuring of our original tool, FluoRender. CONCLUSION: The redesign of FluoRender not only maintains the existing multichannel capabilities for a greatly extended number of volume channels, but also enables new analysis functions for many-channel data from emerging biomedical-imaging techniques.


Assuntos
Software , Algoritmos , Animais , Batracoidiformes/metabolismo , Extremidades/anatomia & histologia , Olho/anatomia & histologia , Olho/patologia , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Camundongos , Microscopia de Fluorescência , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia
8.
J Assoc Res Otolaryngol ; 17(6): 525-540, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27619213

RESUMO

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4 h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.


Assuntos
Compostos de Boro/química , Orelha Interna/química , Glicosaminoglicanos/análise , Imagem Óptica/métodos , Xilose/análogos & derivados , Animais , Batracoidiformes , Microscopia Confocal , Microscopia de Fluorescência , Modelos Teóricos , Xilose/química
9.
Biophys J ; 106(8): 1570-7, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24739156

RESUMO

Brief heat shocks delivered to cells by pulsed laser light can evoke action potentials in neurons and contraction in cardiomyocytes, but the primary biophysical mechanism has been elusive. In this report we show in the neuromuscular junction of Caenorhabditis elegans that application of a 500°C/s heat shock for 500 µs evoked ~35 pA of excitatory current and injected ~23 fC(femtocoulomb) of charge into the cell while raising the temperature only 0.25°C. The key variable driving the current was the rate of change of temperature (dT/dt heat shock), not temperature itself. The photothermal heat shock current was voltage-dependent and was from thermally driven displacement of ions near the plasma membrane. The charge movement was rapid during the heat shock and slow during thermal relaxation, thus leading to an asymmetrical capacitive current that briefly depolarized the cell. A simple quantitative model is introduced to describe modulation of the membrane potential and facilitate practical application of optical heat shock stimuli.


Assuntos
Caenorhabditis elegans/fisiologia , Membrana Celular/fisiologia , Resposta ao Choque Térmico , Animais , Caenorhabditis elegans/metabolismo , Raios Infravermelhos , Mutação/genética , Técnicas de Patch-Clamp , Temperatura
10.
J Biol Chem ; 286(1): 354-62, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21036903

RESUMO

Mitochondria undergo continuous cycles of homotypic fusion and fission, which play an important role in controlling organelle morphology, copy number, and mitochondrial DNA maintenance. Because mitochondria cannot be generated de novo, the motility and distribution of these organelles are essential for their inheritance by daughter cells during division. Mitochondrial Rho (Miro) GTPases are outer mitochondrial membrane proteins with two GTPase domains and two EF-hand motifs, which act as receptors to regulate mitochondrial motility and inheritance. Here we report that although all of these domains are biochemically active, only the GTPase domains are required for the mitochondrial inheritance function of Gem1p (the yeast Miro ortholog). Mutations in either of the Gem1p GTPase domains completely abrogated mitochondrial inheritance, although the mutant proteins retained half the GTPase activity of the wild-type protein. Although mitochondrial inheritance was not dependent upon Ca(2+) binding by the two EF-hands of Gem1p, a functional N-terminal EF-hand I motif was critical for stable expression of Gem1p in vivo. Our results suggest that basic features of Miro protein function are conserved from yeast to humans, despite differences in the cellular machinery mediating mitochondrial distribution in these organisms.


Assuntos
Genes Mitocondriais , Mitocôndrias/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Hidrólise , Mitocôndrias/metabolismo , Mutação , Nucleotídeos/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Proteínas rho de Ligação ao GTP/genética
11.
J Cell Biol ; 180(6): 1245-60, 2008 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-18362183

RESUMO

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.


Assuntos
Carcinoma/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Citocromo-B(5) Redutase/metabolismo , Neoplasias/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/ultraestrutura , Citocromo-B(5) Redutase/genética , Regulação para Baixo/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Forminas , Invasividade Neoplásica/fisiopatologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Ratos , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína rhoA de Ligação ao GTP/metabolismo
12.
J Neurovirol ; 14(1): 28-40, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18300073

RESUMO

The herpes simplex virus type 1 (HSV-1) diploid gene gamma(1)34.5 encodes a neurovirulent factor, infected cell protein 34.5 (ICP34.5). The promoter to gamma(1)34.5 is located within the HSV-1 genome where there are repeated sequences. This region of the genome also contains important overlapping transcripts involved with the virus's ability to establish lytic and latent infections and reactivation. These transcripts include the latency-associated transcripts and regulator proteins ICP0 and ICP4. This study aimed to separate ICP34.5 from these overlapping transcripts and test if its expression from a single gene could restore wild-type HSV-1 strain 17+ virulence. To address these aims, different recombinant viruses were constructed using the Delta gamma(1)34.5 mutant 1716. Immunoblots probed with different ICP34.5 antisera demonstrated that one of the newly generated recombinant viruses, 1622, overexpresses ICP34.5 relative to a panel of wild-type viruses. Interestingly, the overexpression of ICP34.5 does not yield a more virulent virus. The onset of ICP34.5 expression from 1622-infected cells in vitro matched that of 17+, and its expression restored the function of maintaining protein synthesis in human neuroblastoma cells. Replication of 1622, however, was only partially restored to 17+ levels in vivo. Additionally, plaque morphology from 1622-infected cells indicates there is an additional defect. The authors report that the mutant virus 1622 can express ICP34.5 from a single gamma(1)34.5 gene and restore most (but not all) wild-type function. These findings are discussed with respect to the use of the gamma(1)34.5 deleted mutant, 1716, in oncolytic viral vector therapies and future studies for ICP34.5.


Assuntos
Herpesvirus Humano 1/genética , Proteínas Virais/genética , Animais , Linhagem Celular/virologia , Cricetinae , Encefalite por Herpes Simples/virologia , Regulação Viral da Expressão Gênica , Homologia de Genes , Genes Sintéticos , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/patogenicidade , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fenótipo , Regiões Promotoras Genéticas/genética , Vírus Reordenados/genética , Sequências Repetitivas de Aminoácidos , Deleção de Sequência , Proteínas do Envelope Viral/genética , Ensaio de Placa Viral , Proteínas Virais/biossíntese , Proteínas Virais/fisiologia , Virulência/genética , Replicação Viral
13.
Curr Biol ; 17(7): 579-91, 2007 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-17398099

RESUMO

BACKGROUND: Mammalian Diaphanous (mDia)-related formins and the N-WASP-activated Arp2/3 complex initiate the assembly of filamentous actin. Dia-interacting protein (DIP) binds via its amino-terminal SH3 domain to the proline-rich formin homology 1 (FH1) domain of mDia1 and mDia2 and to the N-WASp proline-rich region. RESULTS: Here, we investigated an interaction between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain that nucleates, processively elongates, and bundles actin filaments. DIP binding to mDia2 was regulated by the same Rho-GTPase-controlled autoinhibitory mechanism modulating formin-mediated actin assembly. DIP was previously shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3. Despite direct binding to both mDia1 and mDia2 FH2 domains, DIP LRR inhibited only mDia2-dependent filament assembly and bundling in vitro. DIP expression interfered with filopodia formation, consistent with a role for mDia2 in assembly of these structures. After filopodia retraction into the cell body, DIP expression induced excessive nonapoptotic membrane blebbing, a physiological process involved in both cytokinesis and amoeboid cell movement. DIP-induced blebbing was dependent on mDia2 but did not require the activities of either mDia1 or Arp2/3. CONCLUSIONS: These observations point to a pivotal role for DIP in the control of nonbranched and branched actin-filament assembly that is mediated by Diaphanous-related formins and activators of Arp2/3, respectively. The ability of DIP to trigger blebbing also suggests a role for mDia2 in the assembly of cortical actin necessary for maintaining plasma-membrane integrity.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Musculares/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Forminas , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Mutação , Estrutura Terciária de Proteína , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteína cdc42 de Ligação ao GTP/metabolismo
14.
J Biol Chem ; 281(7): 4300-7, 2006 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-16361707

RESUMO

Mammalian diaphanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal GTPase-binding domains (GBDs) causes the adjacent Dia-inhibitory domain (DID) to release the carboxyl-terminal Dia-autoregulatory (DAD) domain that flanks the formin homology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.


Assuntos
Proteínas de Transporte/química , NADPH Desidrogenase/química , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/antagonistas & inibidores , Forminas , Homeostase , Camundongos , Proteínas Associadas aos Microtúbulos , Dados de Sequência Molecular , NADPH Desidrogenase/antagonistas & inibidores , Células NIH 3T3 , Estrutura Terciária de Proteína
15.
Virol J ; 1: 5, 2004 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-15507126

RESUMO

Inflammatory cytokines and infiltrating T cells are readily detected in herpes simplex virus (HSV) infected mouse cornea and trigeminal ganglia (TG) during the acute phase of infection, and certain cytokines continue to be expressed at lower levels in infected TG during the subsequent latent phase. Recent results have shown that HSV infection activates Toll-like receptor signaling. Thus, we hypothesized that chemokines may be broadly expressed at both primary sites and latent sites of HSV infection for prolonged periods of time. Real-time reverse transcriptase-polymrease chain reaction (RT-PCR) to quantify expression levels of transcripts encoding chemokines and their receptors in cornea and TG following corneal infection. RNAs encoding the inflammatory-type chemokine receptors CCR1, CCR2, CCR5, and CXCR3, which are highly expressed on activated T cells, macrophages and most immature dendritic cells (DC), and the more broadly expressed CCR7, were highly expressed and strongly induced in infected cornea and TG at 3 and 10 days postinfection (dpi). Elevated levels of these RNAs persisted in both cornea and TG during the latent phase at 30 dpi. RNAs for the broadly expressed CXCR4 receptor was induced at 30 dpi but less so at 3 and 10 dpi in both cornea and TG. Transcripts for CCR3 and CCR6, receptors that are not highly expressed on activated T cells or macrophages, also appeared to be induced during acute and latent phases; however, their very low expression levels were near the limit of our detection. RNAs encoding the CCR1 and CCR5 chemokine ligands MIP-1alpha, MIP-1beta and RANTES, and the CCR2 ligand MCP-1 were also strongly induced and persisted in cornea and TG during the latent phase. These and other recent results argue that HSV antigens or DNA can stimulate expression of chemokines, perhaps through activation of Toll-like receptors, for long periods of time at both primary and latent sites of HSV infection. These chemokines recruit activated T cells and other immune cells, including DC, that express chemokine receptors to primary and secondary sites of infection. Prolonged activation of chemokine expression could provide mechanistic explanations for certain aspects of HSV biology and pathogenesis.


Assuntos
Quimiocinas/genética , Regulação da Expressão Gênica , Herpes Simples/genética , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Receptores de Quimiocinas/genética , Latência Viral , Animais , Córnea/metabolismo , Gânglios/metabolismo , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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