RESUMEN

Peribunyaviridae is a large family of RNA viruses with several members that cause mild to severe diseases in humans and livestock. Despite their importance in public heath very little is known about the host cell factors hijacked by these viruses to support assembly and cell egress. Here we show that assembly of Oropouche virus, a member of the genus Orthobunyavirus that causes a frequent arboviral infection in South America countries, involves budding of virus particles toward the lumen of Golgi cisternae. As viral replication progresses, these Golgi subcompartments become enlarged and physically separated from Golgi stacks, forming Oropouche viral factory (Vfs) units. At the ultrastructural level, these virally modified Golgi cisternae acquire an MVB appearance, and while they lack typical early and late endosome markers, they become enriched in endosomal complex required for transport (ESCRT) proteins that are involved in MVB biogenesis. Further microscopy and viral replication analysis showed that functional ESCRT machinery is required for efficient Vf morphogenesis and production of infectious OROV particles. Taken together, our results indicate that OROV attracts ESCRT machinery components to Golgi cisternae to mediate membrane remodeling events required for viral assembly and budding at these compartments. This represents an unprecedented mechanism of how viruses hijack host cell components for coordinated morphogenesis.

Asunto(s)

Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Orthobunyavirus/metabolismo , Orthobunyavirus/fisiología , Técnicas de Cultivo de Célula , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células HeLa , Humanos , Orthobunyavirus/crecimiento & desarrollo , Orthobunyavirus/patogenicidad , Virión/metabolismo , Ensamble de Virus/fisiología , Liberación del Virus/fisiología , Replicación Viral/fisiología

RESUMEN

Quantitative alterations in mast cell numbers in pancreatic lymph nodes (PLNs) have been reported to be associated with type 1 diabetes (T1D) progression, but their potential role during T1D remains unclear. In this study, we evaluated the role of mast cells in T1D induced by multiple low-dose streptozotocin (MLD-STZ) treatments, using two strains of mast cell-deficient mice (W/W(v) or Wsh/Wsh) and the adoptive transfer of mast cells. Mast cell deficient mice developed severe insulitis and accelerated hyperglycemia, with 100% of mice becoming diabetic compared to their littermates. In parallel, these diabetic mice had decreased numbers of T regulatory (Treg) cells in the PLNs. Additionally, mast cell deficiency caused a significant reduction in IL-10, TGF-ß, and IL-6 expression in the pancreatic tissue. Interestingly, IL-6-deficient mice are more susceptible to T1D associated with reduced Treg-cell numbers in the PLNs, but mast cell transfer from wild-type mice induced protection to T1D in these mice. Finally, mast cell adoptive transfer prior to MLD-STZ administration conferred resistance to T1D, promoted increased Treg cells, and decreased IL-17-producing T cells in the PLNs. Taken together, our results indicate that mast cells are implicated in resistance to STZ-induced T1D via an immunological tolerance mechanism mediated by Treg cells.

Asunto(s)

Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Regulación de la Expresión Génica/inmunología , Mastocitos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Citocinas/genética , Citocinas/inmunología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Mastocitos/patología , Ratones , Ratones Noqueados , Linfocitos T Reguladores/patología , Células Th17/inmunología , Células Th17/patología

RESUMEN

Snake venom metalloproteinases have been described as responsible for several inflammatory effects. In this study, we investigated the edema and hyperalgesia induced in rats by Batroxase, a P-I metalloproteinase from Bothrops atrox venom, along with possible inflammatory mediators involved in these responses. Batroxase or sterile saline was injected into rat paws and the edema and hyperalgesic effects were evaluated for 6h by using a plethysmometer and a Von Frey system, respectively. Batroxase induced significant edematogenic and hyperalgesic peak responses in the first hours after administration. The inflammatory mediators involved in these responses were assayed by pretreatment of animals with synthesis inhibitors or receptor antagonists. Peak responses were significantly reduced by administration of the glucocorticoid dexamethasone, the H1 receptor antagonist diphenhydramine and the FLAP inhibitor MK-886. Rat paws injected with compound 48/80, a mast cell degranulating agent, followed by Batroxase injection resulted in significant reduction of the edema and hyperalgesia. However, Batroxase itself induced minor degranulation of RBL-2H3 mast cells in vitro. Additionally, the inflammatory responses did not seem to be related to prostaglandins, bradykinin or nitric oxide. Our results indicate a major involvement of histamine and leukotrienes in the edema and hyperalgesia induced by Batroxase, which could be related, at least in part, to mast cell degranulation.

Asunto(s)

Venenos de Crotálidos/enzimología , Edema/patología , Pie/patología , Hiperalgesia/patología , Mediadores de Inflamación/metabolismo , Metaloproteasas , Animales , Bothrops , Degranulación de la Célula/efectos de los fármacos , Edema/inducido químicamente , Hiperalgesia/inducido químicamente , Masculino , Mastocitos/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Wistar

RESUMEN

Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4- T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Exosomas/inmunología , Productos del Gen nef/inmunología , Infecciones por VIH/inmunología , Línea Celular , Regulación hacia Abajo , Células HEK293 , VIH-1 , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Microscopía Fluorescente

RESUMEN

Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.

Asunto(s)

Huesos/fisiología , Colágeno Tipo III/química , Colágeno Tipo I/química , Periodoncio/fisiología , Regeneración , Animales , Materiales Biocompatibles/química , Regeneración Ósea , Colorantes/química , Tejido Conectivo/patología , Perros , Femenino , Masculino , Microscopía , Diente Molar/fisiología , Ligamento Periodontal/patología , Ligamento Periodontal/fisiología , Politetrafluoroetileno/química , Factores Sexuales

RESUMEN

Eukaryotic translation initiation factor 5A (eIF5A) has a unique character: the presence of an unusual amino acid, hypusine, which is formed by post-translational modifications. Even before the identification of hypusination in eIF5A, the correlation between hypusine formation and protein synthesis, shifting cell proliferation rates, had already been observed. Embryogenesis is a complex process in which cellular proliferation and differentiation are intense. In spite of the fact that many studies have described possible functions for eIF5A, its precise role is under investigation, and to date nothing has been reported about its participation in embryonic development. In this study we show that eIF5A is expressed at all mouse embryonic post-implantation stages with increase in eIF5A mRNA and protein expression levels between embryonic days E10.5 and E13.5. Immunohistochemistry revealed the ubiquitous presence of eIF5A in embryonic tissues and organs at E13.5 day. Interestingly, stronger immunoreactivity to eIF5A was observed in the stomodeum, liver, ectoderm, heart, and eye, and the central nervous system; regions which are known to undergo active differentiation at this stage, suggesting a role of eIF5A in differentiation events. Expression analyses of MyoD, a myogenic transcription factor, revealed a significantly higher expression from day E12.5 on, both at the mRNA and the protein levels suggesting a possible correlation to eIF5A. Accordingly, we next evidenced that inhibiting eIF5A hypusination in mouse myoblast C2C12 cells impairs their differentiation into myotubes and decreases MyoD transcript levels. Those results point to a new functional role for eIF5A, relating it to embryogenesis, development, and cell differentiation.

Asunto(s)

Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Línea Celular , Ratones , Fibras Musculares Esqueléticas/citología , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Factores de Iniciación de Péptidos/antagonistas & inhibidores , Factores de Iniciación de Péptidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/genética , Factor 5A Eucariótico de Iniciación de Traducción

RESUMEN

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection.

Asunto(s)

Mastocitos/inmunología , Mycobacterium tuberculosis , Receptor Toll-Like 2/metabolismo , Tuberculosis Pulmonar/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/inmunología , Movimiento Celular , Células Cultivadas , Citocinas/metabolismo , Granuloma/inmunología , Granuloma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor Toll-Like 2/genética , Tuberculosis Pulmonar/prevención & control

RESUMEN

Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.

Asunto(s)

Infecciones por Actinomycetales/inmunología , Citocinas/inmunología , Galectina 3/metabolismo , Inmunidad Innata , Interleucina-1beta/inmunología , Macrófagos/microbiología , Infecciones por Actinomycetales/microbiología , Animales , Citocinas/biosíntesis , Citocinas/genética , Galectina 3/deficiencia , Galectina 3/inmunología , Técnicas de Silenciamiento del Gen , Interleucina-12/biosíntesis , Interleucina-1beta/biosíntesis , Hígado/citología , Hígado/inmunología , Hígado/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rhodococcus equi/inmunología , Bazo/citología , Bazo/inmunología , Bazo/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo

RESUMEN

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

Asunto(s)

Endocitosis , Células Endoteliales/efectos de los fármacos , Fibrinolíticos/farmacología , Heparina/análogos & derivados , Heparina/farmacología , Lisosomas/efectos de los fármacos , Proteoglicanos/metabolismo , Animales , Sitios de Unión , Línea Celular , Cloroquina/farmacología , Células Endoteliales/enzimología , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Fibrinolíticos/metabolismo , Heparina/metabolismo , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Lisosomas/ultraestructura , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microscopía por Video , Unión Proteica , Conejos , Factores de Tiempo

RESUMEN

Exposure of endothelial cells to heparin and other antithrombotic drugs specifically stimulates the synthesis of an antithrombotic heparan sulfate (HS). In the present work, biotinylated heparin (BiotHep) was used to characterize the binding site(s) of heparin responsible for the stimulus in HS synthesis on endothelial cells. No differences were observed between biotinylated and non-biotinylated heparin in their ability to increase the synthesis of HS. In kinetic studies the BiotHep showed fast, saturable and specific binding with an apparent K(D) of 83 nM to adherent cells and 44 nM to the extracellular matrix (ECM) in the absence of cells. By confocal and electron microscopy, BiotHep bound only to the ECM, co-localizing with fibronectin. The same pattern of binding to the ECM was observed using heparin conjugated with FITC or Alexa Fluor 488 in the presence or absence of fetal calf serum. However, after degradation of HS, heparin binds to the cell surface, indicating that endogenous HS possibly occupied the heparin binding sites. Analyses by flow cytometry and confocal microscopy of cells with non-associated ECM, showed labeling of the cell surface using syndecan-4 monoclonal antibody as well as wheat germ agglutinin, but no binding of heparin. Furthermore, the stimulation in HS synthesis is not elicited by heparin in the absence of ECM. These results indicate that the stimulus for the synthesis of HS does not require binding of the heparin to the cell surface, and the signaling may be mediated through the ECM.

Asunto(s)

Células Endoteliales/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Fibrinolíticos/farmacología , Heparina/análogos & derivados , Heparina/farmacología , Proteoglicanos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Sitios de Unión , Biotinilación , Células COS , Línea Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Fibrinolíticos/metabolismo , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Heparina/metabolismo , Humanos , Hidrazinas , Cinética , Ligandos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Unión Proteica , Conejos , Regulación hacia Arriba

RESUMEN

OBJECTIVE: The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS: We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS: Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION: Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.

Asunto(s)

Antígeno CD146/genética , Fibroblastos/citología , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Pericitos/citología , Cordón Umbilical/citología , Antígeno CD146/fisiología , Diferenciación Celular/fisiología , Separación Celular/métodos , Células Cultivadas , Análisis por Conglomerados , Fibroblastos/fisiología , Citometría de Flujo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/fisiología , Pericitos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genética , Cordón Umbilical/fisiología

RESUMEN

BACKGROUND: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. METHODS: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. RESULTS: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. CONCLUSION: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

RESUMEN

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co-glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.

RESUMEN

Dentre os vários tipos de laser usados em Medicina, o laser de CO2 é o mais usado na Otorrinolaringologia e Cirurgia de Cabeça e Pescoço. As vantagens de seu uso são a diminuição do sangramento, a diminuição do edema no pós-operatório e a facilidade de acesso ao campo operatório, entre outras. Desde os trabalhos de Jako e Strong em 1972(1,2), quando o laser de CO2 passou a ser usado no tratamento de papilomatose laríngea e de lesões malignas glóticas iniciais, suas indicações têm aumetado, principalmente em lesões benignas, a partir da alta tecnologia desenvolvida dos últimos anos como, por exemplo, a diminuição do microspot e o uso do superpulso, reduzindo conseqüentemente seu efeito térmico sobre os tecidos. MÉTODOS: Neste trabalho foram realizadas incisões com instrumental a frio e com laser de CO2 1 watt de modo contínuo e superpulso, em pregas vocais caninas e observado, através de cortes histológicos corados pelo método de Sirius Red, a quantidade de colágeno depositada sobre as mesmas. RESULTADOS: A quantidade de colágeno das pregas vocais foi maior do que no grupo controle, e estatisticamente maior no grupo de animais submetidos a procedimentos com instrumental a frio do que com laser de CO2. Não houve diferença estatística entre o grupo controle e o grupo submetido a incisões com instrumentos a frio. CONCLUSÃO: A microcirurgia de laringe com o laser de CO2, quando este é usado em baixa potência, com pequeno "microspot" e com superpulso, é um método seguro em relação à deposição de colágeno, quando comparado com instrumentos com lâmina a frio, obedecendo os princípios da fonomicrocirurgia.