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INTRODUCTION: Esophageal anastomotic stricture is a well-known complication after transhiatal esophagectomy (THE), but there is limited data regarding the initial management and subsequent outcomes after stricture dilation. There is concern that dilating to larger diameters upon the initial encounter, specifically with high-grade strictures, will lead to increased risk for complications. We therefore reviewed one surgeon's experience with esophageal dilations after THE and provided data and treatment recommendations based upon these findings. METHODS: A retrospective review of patients who underwent esophageal dilations ≥ 18 mm up to 20 mm after THE between 2006 and 2019 at our institution was performed. Patient demographics were n = 97, age = 70, 81 males. RESULTS: For all cases, the mean location, length, diameter of the stricture, and number of days from surgery and initial dilation were 20 cm, 1.9 cm, 6.7 mm, and 106 days, respectively. Most dilations (79%) occurred within 2 weeks to 3 months from surgery. 29.9% were dilated up to 18 mm, 10.3% were dilated up to 19 mm, and 59.8% were dilated up to 20 mm upon initial dilation. Even 1-mm-diameter lesions could be safely dilated upon 18-20 mm. In this study group there were no complications after endoscopic dilation that required hospitalization or further surgical or endoscopic interventions. CONCLUSION: These results suggest that early aggressive endoscopic management of esophageal anastomotic strictures after THE can be safely performed.
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Neoplasias Esofágicas , Estenose Esofágica , Anastomose Cirúrgica/efeitos adversos , Constrição Patológica , Dilatação , Neoplasias Esofágicas/cirurgia , Estenose Esofágica/etiologia , Estenose Esofágica/cirurgia , Esofagectomia/efeitos adversos , Humanos , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
We developed a new method to manufacture dense, aligned, and porous collagen scaffolds using biaxial plastic compression of type I collagen gels. Using a novel compression apparatus that constricts like an iris diaphragm, low density collagen gels were compressed to yield a permanently densified, highly aligned collagen material. Micro-porosity scaffolds were created using hydrophilic elastomer porogens that can be selectively removed following biaxial compression, with porosity modulated by using different porogen concentrations. The resulting scaffolds exhibit collagen densities that are similar to native connective tissues (â¼10% collagen by weight), pronounced collagen alignment across multiple length scales, and an interconnected network of pores, making them highly relevant for use in tissue culture, the study of physiologically relevant cell-matrix interactions, and tissue engineering applications. The scaffolds exhibited highly anisotropic material behavior, with the modulus of the scaffolds in the fiber direction over 100 times greater than the modulus in the transverse direction. Adipose-derived mesenchymal stem cells were seeded onto the biaxially compressed scaffolds with minimal cell death over seven days of culture, along with cell proliferation and migration into the pore spaces. This fabrication method provides new capabilities to manufacture structurally and mechanically relevant cytocompatible scaffolds that will enable more physiologically relevant cell culture studies. Further improvement of manufacturing techniques has the potential to produce engineered scaffolds for direct replacement of dense connective tissues such as meniscus and annulus fibrosus. STATEMENT OF SIGNIFICANCE: In vitro studies of cell-matrix interactions and the engineering of replacement materials for collagenous connective tissues require biocompatible scaffolds that replicate the high collagen density (15-25%/wt), aligned fibrillar organization, and anisotropic mechanical properties of native tissues. However, methods for creating scaffolds with these characteristics are currently lacking. We developed a new apparatus and method to create high density, aligned, and porous collagen scaffolds using a biaxial compression with porogens technique. These scaffolds have a highly directional structure and mechanical properties, with the tensile strength and modulus up to 100 times greater in the direction of alignment. We also demonstrated that the scaffolds are a suitable material for cell culture, promoting cell adhesion, viability, and an aligned cell morphology comparable to the cell morphology observed in native aligned tissues.
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Materiais Biocompatíveis , Colágeno Tipo I , Tecido Conjuntivo , Engenharia Tecidual/métodos , Alicerces Teciduais , Tecido Adiposo/citologia , Anisotropia , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Géis , Humanos , Células-Tronco Mesenquimais/citologia , Microscopia Eletrônica de Varredura , Porosidade , Resistência à TraçãoRESUMO
Back pain is the leading cause of disability globally and the second most common cause of doctors' visits. Despite extensive research efforts, the underlying mechanism of back pain has not been fully elucidated. The intervertebral disc (IVD) is a viscoelastic tissue that provides flexibility to the spinal column and acts as a shock absorber in the spine. When viscoelastic materials like the IVD are cyclically loaded, they dissipate energy as heat. Thus, diurnal, regular movements of the vertebral column that deform the IVD could increase disc temperature through viscoelastic heating. This temperature rise has the potential to influence cell function, drive cell death and induce nociception in innervating nociceptive neurons within the IVD. The present study was conducted to investigate the capacity of IVD to increase in temperature due to viscoelastic heating. Insulated caudal bovine IVD were subjected to physiological cyclic uniaxial compression over a range of frequencies (0.1-15 Hz) and loading durations (1-10 min) ex vivo, and the temperature rise in the tissue was recorded. According to our findings, the IVD can experience a temperature rise of up to 2.5°C under cyclic loading. Furthermore, under similar conditions, the inner nucleus pulposus exhibits more viscoelastic heating than the outer annulus fibrosis, likely due to its more viscous composition. The measured temperature rise of the disc has physiological relevance as degenerative IVD tissue has been shown to produce a sensitization of nociceptive neurons that spontaneously fire at 37°C, with a T50 response at 37.3°C and a maximum response at 38°C. Our results suggest that viscoelastic heating of IVD could interact with sensitized nociceptive neurons in the degenerative IVD to play a role in back pain.
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The CRISPR/Cas9 system has emerged as a powerful tool for mammalian genome engineering. In basic and translational intervertebral disc (IVD) research, this technique has remarkable potential to answer fundamental questions on pathway interactions, to simulate IVD pathologies, and to promote drug development. Furthermore, the precisely targeted CRISPR/Cas9 gene therapy holds promise for the effective and targeted treatment of degenerative disc disease and low back pain. In this perspective, we provide an overview of recent CRISPR/Cas9 advances stemming from/with transferability to IVD research, outline possible treatment approaches for degenerative disc disease, and discuss current limitations that may hinder clinical translation.
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The intervertebral disc contributes to motion, weight bearing, and flexibility of the spine, but is susceptible to damage and morphological changes that contribute to pathology with age and injury. Engineering strategies that rely upon synthetic materials or composite implants that do not interface with the biological components of the disc have not met with widespread use or desirable outcomes in the treatment of intervertebral disc pathology. Here we review bioengineering advances to treat disc disorders, using cell-supplemented materials, or acellular, biologically based materials, that provide opportunity for cell-material interactions and remodeling in the treatment of intervertebral disc disorders. While a field still in early development, bioengineering-based strategies employing novel biomaterials are emerging as promising alternatives for clinical treatment of intervertebral disc disorders.
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Materiais Biocompatíveis/farmacologia , Disco Intervertebral/fisiologia , Regeneração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Humanos , Disco Intervertebral/efeitos dos fármacos , Disco Intervertebral/patologia , Engenharia TecidualRESUMO
The major regulatory protein ICP22 is unique among the immediate early proteins of herpes simplex virus. Viruses deleted for ICP22 replicate well in actively dividing cells, but not in quiescent cells or certain rodent lines. Accordingly, ICP22 represents an understudied herpes simplex virus (HSV) neurovirulence marker which is absolutely essential for viral neurogrowth. We utilized the bacterial artificial chromosome methodology to create a novel ICP22 truncation mutant, termed HSV-1(BACX). The integrity of HSV-1(BACX) was confirmed by detailed polymerase chain reaction analyses and immunoblotting using anti-ICP22 antibody. HSV-1(BACX) showed a reduced replication capacity in rabbit skin cells, consistent with previous studies using ICP22-null viruses. Importantly, HSV-1(BACX) localized to nuclei of infected primate Vero cells in a manner similar to wild-type ICP22. Thus, HSV-1(BACX) will serve as a useful tool to decipher the unusual biological properties and functions of the ICP22 protein.
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Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/genética , Animais , Núcleo Celular/genética , Núcleo Celular/virologia , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos , Herpesvirus Humano 1/patogenicidade , Humanos , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Mutação , Coelhos , Transcrição Gênica , Células Vero , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response. Thus, DCs link the innate and adaptive immune systems. The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs. Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs). Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs. Electroporation has been used with mixed results, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility. In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNß. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor, at both the mRNA and protein levels.
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Células Dendríticas/fisiologia , Técnicas de Silenciamento de Genes/métodos , Transfecção/métodos , Separação Celular/métodos , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Células Dendríticas/imunologia , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Plasmídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores ImunológicosRESUMO
BACKGROUND AND AIMS: Matrix metalloproteinase-2 (MMP-2), a type IV collagenase secreted by activated hepatic stellate cells (HSCs), is upregulated in chronic liver disease and is considered a profibrotic mediator due to its proliferative effect on cultured HSCs and ability to degrade normal liver matrix. Although associative studies and cell culture findings suggest that MMP-2 promotes hepatic fibrogenesis, no in vivo model has definitively established a pathologic role for MMP-2 in the development and progression of liver fibrosis. We therefore examined the impact of MMP-2 deficiency on liver fibrosis development during chronic CCl(4) liver injury and explored the effect of MMP-2 deficiency and overexpression on collagen I expression. METHODS: Following chronic CCl(4) administration, liver fibrosis was analyzed using Sirius Red staining with quantitative morphometry and real-time polymerase chain reaction (PCR) in MMP-2-/- mice and age-matched MMP-2+/+ controls. These studies were complemented by analyses of cultured human stellate cells. RESULTS: MMP-2-/- mice demonstrated an almost twofold increase in fibrosis which was not secondary to significant differences in hepatocellular injury, HSC activation or type I collagenase activity; however, type I collagen messenger RNA (mRNA) expression was increased threefold in the MMP-2-/- group by real-time PCR. Furthermore, targeted reduction of MMP-2 in cultured HSCs using RNA interference significantly increased collagen I mRNA and protein, while overexpression of MMP-2 resulted in decreased collagen I mRNA. CONCLUSIONS: These findings suggest that increased MMP-2 during the progression of liver fibrosis may be an important mechanism for inhibiting type I collagen synthesis by activated HSCs, thereby providing a protective rather than pathologic role.
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Colágeno Tipo I/metabolismo , Cirrose Hepática/induzido quimicamente , Metaloproteinase 2 da Matriz/metabolismo , Regulação para Cima/fisiologia , Animais , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Linhagem Celular , Colágeno Tipo I/genética , Relação Dose-Resposta a Droga , Células Estreladas do Fígado , Humanos , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Knockout , RNA/genética , RNA/metabolismo , Regulação para Cima/genéticaRESUMO
Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNß mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.
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Células Dendríticas , Técnicas de Silenciamento de Genes/métodos , Monócitos , Plasmídeos , RNA Interferente Pequeno , Transfecção/métodos , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Proteína DEAD-box 58 , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Citometria de Fluxo/métodos , Expressão Gênica , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Monócitos/citologia , Monócitos/metabolismo , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/metabolismo , Interferência de RNA , Receptores ImunológicosRESUMO
Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are ubiquitous human pathogens that infect their hosts for life and reactivate to cause disease at or near the initial site of infection. As the incidence of genital HSV-1 infections increase, there is an increased demand for valid viral typing diagnostics. In this report, we reconsidered and developed a triple-phase immune-typing procedure that compares differences in electrophoretic mobilities of viral ICP4, ICP27, and VP22 proteins between HSV-1 and HSV-2 strains. We isolated and immunotyped 5 primary HSV-1 strains derived from orofacial, ocular, and genital areas along with 2 primary HSV-2 strains from the genital area. Advantages of this methodology include its general technical simplicity, sensitivity, and ability to definitively type HSV. It is anticipated that this methodology will be useful in distinguishing viruses obtained in clinical cultures.
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Herpes Genital , Herpes Simples , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Immunoblotting/métodos , Proteínas Virais/análise , Animais , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Herpes Genital/diagnóstico , Herpes Genital/virologia , Herpes Simples/diagnóstico , Herpes Simples/virologia , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/isolamento & purificação , Herpesvirus Humano 2/classificação , Herpesvirus Humano 2/isolamento & purificação , Humanos , Proteínas Imediatamente Precoces/análise , Células Vero , Proteínas Estruturais Virais/análiseRESUMO
The present study evaluated the status of mood congruent memory bias in implicit memory tasks for threat related information. A literature review complemented by three experiments on high and low trait anxiety participants found no implicit memory bias for threat-related information in anxious individuals on either word fragment completion or tachistoscopic word identification tasks. The theoretical implications of these results are discussed.