RESUMO
Florid cemento-osseous dysplasia (FCOD) is a rare, benign, multifocal fibro-osseous dysplastic process affecting tooth-bearing areas of the jaw, characterized by replacement of normal trabecular bone with osseous tissue and dense acellular cementum in a fibrous stroma. It is one clinicopathologic variant in a spectrum of related non-neoplastic fibro-osseous lesions known as cemento-osseous dysplasias (CODs), thought to arise from elements of the periodontal ligament. Diagnosis primarily relies upon radiographic and clinical findings; unnecessary biopsy should be avoided, as inoculation with oral pathogens may precipitate chronic infection in these hypovascular lesions. Appropriate management of uncomplicated FCOD consists of periodic radiographic follow-up. Accordingly, it is important that both radiologists and clinicians performing endodontic interventions possess familiarity with this entity in order to prevent misdiagnosis and inappropriate intervention, which may result in a protracted clinical course. Lesions are usually asymptomatic in the absence of infection, typically discovered on routine dental radiographs or imaging performed for unrelated indications. Radiographically, the condition typically manifests as widespread non-expansile intraosseous masses of varying internal lucency and sclerosis that surround the root apices of vital teeth or edentulous areas in the posterior jaw. While all CODs share similar microscopic features, FCOD is distinguished by its multifocal distribution, involving two or more quadrants of the maxilla and mandible, often in a bilateral symmetric fashion. The vast majority of cases are sporadic, though few exhibit an autosomal dominant familial inheritance pattern. In this pictorial review, we discuss the radiologic characteristics of this entity, pertinent clinical and histologic features, differential diagnoses, and management options.
Assuntos
Displasia Fibrosa Óssea/diagnóstico por imagem , Doenças Maxilomandibulares/diagnóstico por imagem , Osteomielite/diagnóstico por imagem , Diagnóstico Diferencial , Feminino , Humanos , Arcada Osseodentária , Radiografia/métodosRESUMO
Microbial contamination of cell culture is a major problem encountered both in academic labs and in the biotechnology/pharmaceutical industries. A broad spectrum of microbes including mycoplasma, bacteria, fungi, and viruses are the causative agents of cell culture contamination. Unfortunately, the existing disinfection techniques lack selectivity and/or lead to the development of drug-resistance, and more importantly there is no universal method to address all microbes. Here, we report a novel, chemical-free visible ultrashort pulsed laser method for cell culture disinfection. The ultrashort pulsed laser technology inactivates pathogens with mechanical means, a paradigm shift from the traditional pharmaceutical and chemical approaches. We demonstrate that ultrashort pulsed laser treatment can efficiently inactivate mycoplasma, bacteria, yeast, and viruses with good preservation of mammalian cell viability. Our results indicate that this ultrashort pulsed laser technology has the potential to serve as a universal method for the disinfection of cell culture.
RESUMO
BACKGROUND: Low-power ultrashort pulsed (USP) lasers operating at wavelengths of 425 nm and near infrared region have been shown to effectively inactivate viruses such as human immunodeficiency virus (HIV), M13 bacteriophage, and murine cytomegalovirus (MCMV). It was shown previously that non-enveloped, helical viruses such as M13 bacteriophage, were inactivated by a USP laser through an impulsive stimulated Raman scattering (ISRS) process. Recently, enveloped virus like MCMV has been shown to be inactivated by a USP laser via protein aggregation induced by an ISRS process. However, the inactivation mechanism for a clinically important class of viruses--non-enveloped, icosahedral viruses remains unknown. RESULTS AND DISCUSSIONS: We have ruled out the following four possible inactivation mechanisms for non-enveloped, icosahedral viruses, namely, (1) inactivation due to ultraviolet C (UVC) photons produced by non-linear optical process of the intense, fundamental laser beam at 425 nm; (2) inactivation caused by thermal heating generated by the direct laser absorption/heating of the virion; (3) inactivation resulting from a one-photon absorption process via chromophores such as porphyrin molecules, or indicator dyes, potentially producing reactive oxygen or other species; (4) inactivation by the USP lasers in which the extremely intense laser pulse produces shock wave-like vibrations upon impact with the viral particle. We present data which support that the inactivation mechanism for non-enveloped, icosahedral viruses is the impulsive stimulated Raman scattering process. Real-time PCR experiments show that, within the amplicon size of 273 bp tested, there is no damage on the genome of MNV-1 caused by the USP laser irradiation. CONCLUSION: We conclude that our model non-enveloped virus, MNV-1, is inactivated by the ISRS process. These studies provide fundamental knowledge on photon-virus interactions on femtosecond time scales. From the analysis of the transmission electron microscope (TEM) images of viral particles before and after USP laser irradiation, the locations of weak structural links on the capsid of MNV-1 were revealed. This important information will greatly aid our understanding of the structure of non-enveloped, icosahedral viruses. We envision that this non-invasive, efficient viral eradication method will find applications in the disinfection of pharmaceuticals, biologicals and blood products in the near future.
Assuntos
Lasers , Viabilidade Microbiana/efeitos da radiação , Norovirus/fisiologia , Norovirus/efeitos da radiação , Inativação de Vírus/efeitos da radiação , Análise Espectral RamanRESUMO
Intratracheal administration of low molecular mass (LMM) hyaluronan (200 kDa) results in greater neutrophil infiltration in the lungs of TLR4(-/-) mice compared with that in wild-type mice. In general, enhanced neutrophil infiltration in tissue is due to cell influx; however, neutrophil apoptosis also plays an important role. We have assessed the effects of TLR4 in the regulation of neutrophil apoptosis in response to administration of LMM hyaluronan. We found that apoptosis of inflammatory neutrophils is impaired in TLR4(-/-) mice, an effect that depends upon the IFN-ß-mediated TRAIL/TRAILR system. IFN-ß levels were decreased in LMM hyaluronan-treated TLR4-deficient neutrophils. The treatment of inflammatory neutrophils with IFN-ß enhanced the levels of TRAIL and TRAILR 2. LMM hyaluronan-induced inflammatory neutrophil apoptosis was substantially prevented by anti-TRAIL neutralizing mAb. We conclude that decreased IFN-ß levels decrease the activity of the TRAIL/TRAILR system in TLR4-deficient neutrophils, leading to impaired apoptosis of neutrophils and resulting in abnormal accumulation of neutrophils in the lungs of LMM hyaluronan-treated mice. Thus, TLR4 plays a novel homeostatic role in noninfectious lung inflammation by accelerating the elimination of inflammatory neutrophils.
Assuntos
Apoptose/imunologia , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/fisiologia , Interferon beta/fisiologia , Neutrófilos/imunologia , Receptor 4 Toll-Like/fisiologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Homeostase/imunologia , Ácido Hialurônico/química , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/química , Mediadores da Inflamação/fisiologia , Interferon beta/deficiência , Interferon beta/genética , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
The threat of emerging pathogens and microbial drug resistance has spurred tremendous efforts to develop new and more effective antimicrobial strategies. Recently, a novel ultrashort pulsed (USP) laser technology has been developed that enables efficient and chemical-free inactivation of a wide spectrum of viral and bacterial pathogens. Such a technology circumvents the need to introduce potentially toxic chemicals and could permit safe and environmentally friendly pathogen reduction, with a multitude of possible applications including the sterilization of pharmaceuticals and blood products, and the generation of attenuated or inactivated vaccines.
Assuntos
Bactérias/efeitos da radiação , Lasers , Vírus/efeitos da radiação , Bactérias/patogenicidade , Humanos , Esterilização/métodos , Vacinas de Produtos Inativados/efeitos da radiação , Vírus/patogenicidadeRESUMO
Low m.w. hyaluronan (LMW HA) has been shown to elicit the expression of proinflammatory cytokines and chemokines in various cells in vitro. However, the effects of this molecule in vivo are unknown. In this study, we report that intratracheal administration of LMW HA (200 kDa) causes inflammation in mouse lung. A lack of TLR4 is associated with even stronger inflammatory response in the lung as shown by increased neutrophil counts and elevated cytokine and chemokine concentrations. We also demonstrate that TLR4 anti-inflammatory signaling is dependent upon a MyD88-independent pathway. TLR4-mediated IL-1R antagonist production plays a negative regulatory role in LMW HA (200 kDa) induced lung inflammation. These data provide a molecular level explanation for the function of TLR4 in LMW HA (200 kDa)-induced lung inflammation, as inhibition of the beta form of pro-IL-1 promotes an anti-inflammatory response.
Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Regulação para Baixo/imunologia , Mediadores da Inflamação/fisiologia , Pulmão/imunologia , Pulmão/patologia , Receptor 4 Toll-Like/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Permeabilidade da Membrana Celular/imunologia , Células Cultivadas , Regulação para Baixo/genética , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/antagonistas & inibidores , Ácido Hialurônico/química , Mediadores da Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Proteína Antagonista do Receptor de Interleucina 1/fisiologia , Interleucina-1beta/antagonistas & inibidores , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peso Molecular , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.
Assuntos
Farmacorresistência Bacteriana Múltipla , Staphylococcus aureus Resistente à Meticilina , Animais , Vacinas Bacterianas , Escherichia coli , Lasers , Camundongos , Esporos BacterianosRESUMO
BACKGROUND: The development and optimization of therapies for rheumatoid arthritis (RA) is currently hindered by a lack of methods for early non-invasive monitoring of treatment response. Annexin A2, an inflammation-associated protein whose presence and phosphorylation levels are upregulated in RA, represents a potential molecular target for tracking RA treatment response. METHODS: LS301, a near-infrared dye-peptide conjugate that selectively targets tyrosine 23-phosphorylated annexin A2 (pANXA2), was evaluated for its utility in monitoring disease progression, remission, and early response to drug treatment in mouse models of RA by fluorescence imaging. The intraarticular distribution and localization of LS301 relative to pANXA2 was determined by histological and immunohistochemical methods. RESULTS: In mouse models of spontaneous and serum transfer-induced inflammatory arthritis, intravenously administered LS301 showed selective accumulation in regions of joint pathology including paws, ankles, and knees with positive correlation between fluorescent signal and disease severity by clinical scoring. Whole-body near-infrared imaging with LS301 allowed tracking of spontaneous disease remission and the therapeutic response after dexamethasone treatment. Histological analysis showed preferential accumulation of LS301 within the chondrocytes and articular cartilage in arthritic mice, and colocalization was observed between LS301 and pANXA2 in the joint tissue. CONCLUSIONS: We demonstrate that fluorescence imaging with LS301 can be used to monitor the progression, remission, and early response to drug treatment in mouse models of RA. Given the ease of detecting LS301 with portable optical imaging devices, the agent may become a useful early treatment response reporter for arthritis diagnosis and drug evaluation.
Assuntos
Anexina A2 , Artrite Experimental , Artrite Reumatoide , Animais , Artrite Experimental/diagnóstico por imagem , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/tratamento farmacológico , Condrócitos , Camundongos , Imagem Óptica , TirosinaRESUMO
This paper presents a new method to reduce the topographical artifacts in scanning near-field optical microscopy (SNOM) images. The method uses the harmonics caused intrinsically by the nonlinearity in the oscillation of the SNOM probe even when the probe is working in a normal condition without extra excitation. Using these intrinsic harmonics, the gradient of the received SNOM signal with respect to the probe motion is obtained. Then, taking advantage of a SNOM capable of simultaneously obtaining both the topographical and optical signals, topographical artifacts are calculated from the product of the gradient and the topographical signal, and then removed from the received SNOM signal. The effectiveness of the proposed method is demonstrated experimentally.
Assuntos
Artefatos , Microscopia de Varredura por Sonda/métodos , Microscopia/métodos , Óptica e Fotônica , Algoritmos , Calibragem , Desenho de Equipamento , Microscopia/instrumentação , Microscopia de Varredura por Sonda/instrumentação , Microscopia Ultravioleta/métodos , Oscilometria/métodos , Raios UltravioletaRESUMO
A near-field ellipsometry method is presented for nano-scale thin film characterization. The technique fuses the topographic and ellipso-metric optical measurements that are simultaneously obtained by a scanning near-field optical microscopy (SNOM). It is shown that the proposed near-field ellipsometry is able to attain nano-scale lateral resolution and correct artifacts in characterization. The effectiveness of the proposed method is verified by simulation and experimental studies.
Assuntos
Teste de Materiais/métodos , Membranas Artificiais , Microscopia de Varredura por Sonda/métodos , Propriedades de SuperfícieRESUMO
The heterogeneity and continuous genetic adaptation of tumours complicate their detection and treatment via the targeting of genetic mutations. However, hallmarks of cancer such as aberrant protein phosphorylation and calcium-mediated cell signalling provide broadly conserved molecular targets. Here, we show that, for a range of solid tumours, a cyclic octapeptide labelled with a near-infrared dye selectively binds to phosphorylated Annexin A2 (pANXA2), with high affinity at high levels of calcium. Because of cancer-cell-induced pANXA2 expression in tumour-associated stromal cells, the octapeptide preferentially binds to the invasive edges of tumours and then traffics within macrophages to the tumour's necrotic core. As proof-of-concept applications, we used the octapeptide to detect tumour xenografts and metastatic lesions, and to perform fluorescence-guided surgical tumour resection, in mice. Our findings suggest that high levels of pANXA2 in association with elevated calcium are present in the microenvironment of most solid cancers. The octapeptide might be broadly useful for selective tumour imaging and for delivering drugs to the edges and to the core of solid tumours.
Assuntos
Anexina A2/metabolismo , Cálcio/metabolismo , Diagnóstico por Imagem/métodos , Neoplasias/diagnóstico por imagem , Células A549 , Animais , Anexina A2/genética , Apoptose , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HEK293 , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias Pancreáticas/diagnóstico por imagem , Fosforilação , Proteômica , Células Estromais , Transplante HeterólogoRESUMO
BACKGROUND: Gene therapy with plasmid DNA is emerging as a promising strategy for the treatment of many diseases. One of the major obstacles to such therapy is the poor transfection efficiency of DNA in vivo. METHODS: In this report, we employed a very low power, near-infrared femtosecond laser technique to enhance the transfection efficiency of intradermally and intratumorally administered DNA plasmid. RESULTS: We found that femtosecond laser treatment can significantly enhance the delivery of DNA into the skin and into established tumors in mice. In addition, we found that both laser power density as well as duration of laser treatment are critical parameters for augmenting DNA transfection efficiency. The femtosecond laser technique employs a relatively unfocused laser beam that maximizes the transfected area, minimizes damage to tissue and simplifies its implementation. CONCLUSION: This femtosecond new laser technology represents a safe and innovative technology for enhancing DNA gene transfer in vivo.
Assuntos
DNA , Técnicas de Transferência de Genes , Terapia Genética , Lasers , Transfecção , Animais , DNA/administração & dosagem , DNA/genética , Feminino , Técnicas de Transferência de Genes/instrumentação , Terapia Genética/instrumentação , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Fatores de Tempo , Transfecção/instrumentação , Transfecção/métodosRESUMO
Thymic stromal lymphopoietin (TSLP) emerges as a central mediator of T helper cell (Th)2-dominant allergic diseases. However, the role of TSLP receptor (TSLPR) in allergen-induced Th2 priming, and the effects of TSLP signaling blocking on the development of asthma remain unclear. Here we showed that allergen challenge caused a rapid accumulation of TSLP in the airways of asthmatic mice, correlating well with eosinophils counts and interleukin (IL)-5 productions. When TSLP signaling was blocked by intratracheal administration of anti-TSLPR antibody before sensitization, eosinophilic airway inflammation, goblet cell hyperplasia and Th2 cytokines productions were significantly reduced. The alleviating effects of TSLPR blocking were achieved by inhibition of maturation and migration of airway dendritic cells (DCs), as well as their abilities of initiating CD4+T cell responses. Thus, local application of anti-TSLPR prevented Th2-mediated airway inflammation, at least partly, by regulating DCs function, which might be exploited to develop novel treatments for asthma.
Assuntos
Asma/prevenção & controle , Células Dendríticas/fisiologia , Pulmão/imunologia , Receptores de Citocinas/fisiologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/química , Movimento Celular , Polaridade Celular , Citocinas/análise , Feminino , Imunoglobulinas , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Muco/metabolismo , Receptores de Citocinas/antagonistas & inibidores , Linfócitos T/fisiologia , Células Th2/fisiologia , Linfopoietina do Estroma do TimoRESUMO
BACKGROUND: Resonant microwave absorption has been proposed in the literature to excite the vibrational states of microorganisms in an attempt to destroy them. But it is extremely difficult to transfer microwave excitation energy to the vibrational energy of microorganisms due to severe absorption of water in this spectral range. We demonstrate for the first time that, by using a visible femtosecond laser, it is effective to inactivate viruses such as bacteriophage M13 through impulsive stimulated Raman scattering. RESULTS AND DISCUSSION: By using a very low power (as low as 0.5 nj/pulse) visible femtosecond laser having a wavelength of 425 nm and a pulse width of 100 fs, we show that M13 phages were inactivated when the laser power density was greater than or equal to 50 MW/cm2. The inactivation of M13 phages was determined by plaque counts and had been found to depend on the pulse width as well as power density of the excitation laser. CONCLUSION: Our experimental findings lay down the foundation for an innovative new strategy of using a very low power visible femtosecond laser to selectively inactivate viruses and other microorganisms while leaving sensitive materials unharmed by manipulating and controlling with the femtosecond laser system.
Assuntos
Bacteriófago M13/efeitos da radiação , Lasers , Luz , Inativação de Vírus , Viabilidade Microbiana , Análise Espectral Raman , Ensaio de Placa ViralRESUMO
Raman spectroscopy is used to study low-wave-number (=20 cm(-1)) acoustic vibrations of the M13 phage. A well-defined Raman line is observed at around 8.5 cm(-1). The experimental results are compared with theoretical calculations based on an elastic continuum model and appropriate Raman selection rules derived from a bond polarizability model. The observed Raman mode is shown to belong to one of the Raman-active axial modes of the M13 phage protein coat. It is expected that the detection and characterization of this low-frequency vibrational mode can be used for applications in biomedical nanotechnology such as for monitoring the process of virus functionalization and self-assembly.
Assuntos
Bacteriófago M13/isolamento & purificação , Bacteriófago M13/fisiologia , Análise Espectral Raman/métodos , Montagem de Vírus/fisiologia , Água , Bacteriófago M13/classificação , Espalhamento de Radiação , VibraçãoRESUMO
The inactivation of viruses such as M13 bacteriophages subject to excitations by a very low power visible femtosecond laser has been studied. Our experimental results show that for a visible femtosecond laser having lambda = 425 nm and a pulse width of 100 fs, the M13 bacteriophages are inactivated when the laser power density is greater than or equal to 49 MW/cm(2). The medium lethal laser power density (LD(50)) is 51.94+/-0.14 MW/cm(2). The functionality of M13 bacteriophages has been shown to be critically dependent on the pulse width as well as power density of the excitation laser. Our work demonstrates that by using a very low power visible femtosecond laser, it is plausible to inactivate viruses such as the M13 bacteriophages through impulsive stimulated Raman scattering process. These experimental findings suggest a novel avenue of selectively inactivating microorganisms while leaving the sensitive materials unharmed by manipulating and controlling with femtosecond laser systems.
Assuntos
Bacteriófago M13/efeitos da radiação , Lasers , Análise Espectral Raman/métodos , Tomografia de Coerência Óptica/métodos , Tomografia de Coerência Óptica/estatística & dados numéricos , Ensaio de Placa ViralRESUMO
BACKGROUND: Recently, a technique which departs radically from conventional approaches has been proposed. This novel technique utilizes biological objects such as viruses as nano-templates for the fabrication of nanostructure elements. For example, rod-shaped viruses such as the M13 phage and tobacco mosaic virus have been successfully used as biological templates for the synthesis of semiconductor and metallic nanowires. RESULTS AND DISCUSSION: Low wave number (Assuntos
Bacteriófago M13/fisiologia
, Vibração
, Água/química
, Análise Espectral Raman
, Proteínas do Envelope Viral/fisiologia
, Montagem de Vírus/fisiologia
RESUMO
Energy transfer processes between beta carotene, lycopene, and singlet oxygen ((1)O(2)) have been studied by ultrafast Raman spectroscopy. Our experimental results demonstrate that during the neutralization of singlet oxygen by beta carotene the excitation energy of singlet oxygen is transferred directly to the first excited electronic state S(1) of beta carotene. In contrast, the excitation energy of singlet oxygen is transferred directly to the ground excited vibronic state S(0) of lycopene. Our data not only provide the first direct experimental elucidation of energy transfer processes in such important biological systems but also help explain why lycopene is a more potent antioxidant than beta carotene in the neutralization of singlet oxygen.
Assuntos
Carotenoides/química , Transferência de Energia , Oxigênio Singlete/química , Análise Espectral Raman/métodos , beta Caroteno/química , LicopenoRESUMO
There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.
Assuntos
Vacinas contra Influenza/química , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/citologia , Linhagem Celular , Cães , Feminino , Testes de Hemaglutinação , Humanos , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/prevenção & controle , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Testes de Neutralização , Vacinação , Vacinas de Produtos Inativados/químicaRESUMO
Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens.