Al and Mary Agnes McQuinn, the Richard M. Schulze Foundation, and an NIH Relief Grant from the Mayo Clinic funded representative work described here.

The authors have nothing to disclose.

A concepção, a composição e a localização dos elementos de resposta microARN dentro do genoma viral ditará segmentação eficácia e especificidade. Otimizar estes irão exigir tentativa e erro. No entanto, design racional baseada na análise estrutural de RNA e estudos anteriores de replicação viral e microRNA assinaturas auxilia na implementação dessa técnica com otimização mínima 10, 11, 12, <sup cl...

O desenvolvimento de um método amplamente aplicável, fácil e eficaz para a engenharia de um vector com tropismo restrito proporciona uma grande oportunidade para melhorar a segurança, a compreensão biológica e utilidade terapêutica de vírus. Existem vários mecanismos para atingir tropismo viral incluindo transdução, da transcrição, e as técnicas baseadas em translação. No entanto, estes métodos não são geralmente aplicáveis ​​a todos os sistemas de vectores, pode exigir vias de sinalização defeituosos em células alvo ou exigir a inserção de sequências codificadoras grandes no genoma viral. Além disso, esses métodos podem resultar na atenuação do vírus, o que dificulta significativamente a sua actividade terapêutica e limitando uma visão sobre o sistema não modificado. MicroRNAs são pequenos (22-25 nucleótidos), RNAs não-codificantes que medeiam o silenciamento de genes em células eucarióticas. função MicroRNAs ligando sequências alvo complementares (elementos de resposta) em RNAs mensageiro (mRNA) resulti<html> ng na desestabilização da transcrição, a degradação ou a repressão de translação. MicroRNAs normalmente ligam-se elementos de resposta com complementaridade parcial e produzir pequenas modificações na expressão do gene 1, 2, 3, 4, 5. Alterações mais significativas na expressão do gene pode ser conseguido aumentando a complementaridade do elemento de resposta a seis. Milhares de microARNs maduros têm sido identificados numa variedade de espécies e muitas exibem padrões de expressão diferenciais em uma variedade de tipos de células e tecidos 7, 8, 9. Estas assinaturas microRNA pode ser explorada para a restrição específica da célula de amplificação do vírus, incorporando elementos de resposta perfeitamente complementares ao genoma viral 10,= &quot;xref&quot;&gt; 11, 12, 13. O objetivo geral desta técnica de metas de microRNA é controlar o tropismo de um genoma vector sem atenuação adicional. A utilidade deste método para regular o tropismo viral foi inicialmente demonstrada em vectores lentivirais para restringir a expressão do transgene em tecidos específicos, 14, 15, 16. Esta técnica foi subsequentemente aplicada a uma vasta gama de replicar e não replicantes vectores virais para terapia génica aumentada, bem como para melhorar os perfis de segurança de muitos vírus oncolí ticos, eliminando toxicidades indesejadas em tecidos normais 10, 11, 12, 13, 17 . Também tem sido utilizado para gerar e e seguravacinas vivas atenuadas FICAZ, bem como para melhorar o vírus e fabrico de vacinas de processos 18, 19, 20, 21. MicroRNA-alvo de um vector pode permitir a atenuação em hospedeiros vacinados ou sistemas segmentados, mantendo níveis de crescimento do tipo selvagem em sistemas de produtores. MicroRNA-direccionamento podem também ser utilizadas para melhorar a segurança biológica do vírus para fins de investigação, restringindo a transmissão em uma espécie (por exemplo, seres humanos), mantendo a transmissão em outros hospedeiros 22. Finalmente, microRNA-alvo pode permitir análises em profundidade de ciclo de vida viral e papéis específicos de tipos de células em patogênese e imunidade por segregar o crescimento viral 23, 24, 25, 26. Esta técnica oferece uma alternative método que pode ser facilmente implementada e aplicáveis ​​segmentação para todos os sistemas de vírus. Além disso, a recolha em constante expansão de microARNs maduras, com padrões de expressão diferenciais em tipos específicos de células torna esta técnica altamente versátil. -MicroRNA segmentação baseada provou eficaz para uma variedade de sistemas vírus sem comprometer a função do sistema. As principais limitações desta técnica incluem tentativa e otimização de erro, o potencial para mutações de escape, e potenciais efeitos fora do alvo em transcrições endógenos. No entanto, estas limitações geralmente podem ser superados com design otimizado e racional elemento de resposta. vírus de ARN de sentido positivo tende a ser particularmente sensível a microARN-direccionamento devido à orientação de sentido positivo do seu genoma e a disponibilidade dos transcritos à maquinaria microARN durante o ciclo de replicação completamente citoplasmática. Aqui nós descrevemos um protocolo para gerar um picornavírus alvejado-microRNA eo experimmétodos ental para verificar a eficiência e especificidade de que a segmentação in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="780">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">RE encoding Oligonucleotides</td>
			<td style="width:119px;">IDT</td>
			<td style="width:167px;">PAGE-Purified Ultramer</td>
			<td style="width:247px;">Sequence Designed by Investigator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Oligonucleotides encoding unique restriction site</td>
			<td style="width:119px;">IDT</td>
			<td style="width:167px;">25nM</td>
			<td style="width:247px;">Sequence Designed by Investigator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Expand High Fidelity PCR Kit</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:167px;">11732641001</td>
			<td style="width:247px;">Many other High Fidelity Polymerase PCR kits available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">T4 DNA Ligase System</td>
			<td style="width:119px;">NEB</td>
			<td style="width:167px;">M0202S</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">MEGAscript Kit</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM1333</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">MEGAclear Kit</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM1908</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">0.5 M EDTA</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">AM9260G</td>
			<td style="width:247px;">RNase-free</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">5 M NH4 Acetate</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">N/A</td>
			<td style="width:247px;">Comes in MEGAclear Kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Ethanol</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">BP2818100</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Nuclease-free Water</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:167px;">AM9938</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">TransIT-2020 Transfection Reagent</td>
			<td style="width:119px;">Mirus</td>
			<td style="width:167px;">MIR 5404</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">TransIT-mRNA Transfection Reagent</td>
			<td style="width:119px;">Mirus</td>
			<td style="width:167px;">MIR 2225</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">0.2 &#956;m syringe filter</td>
			<td style="width:119px;">Millipore</td>
			<td style="width:167px;">SLGP033RS</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">2mL Screw-Cap Tubes</td>
			<td style="width:119px;">Sarstedt</td>
			<td style="width:167px;">72.694.005</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Cell Scrapers</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:167px;">08-100-241</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">MicroRNA Mimics</td>
			<td style="width:119px;">Dharmacon</td>
			<td style="width:167px;">Varied</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">MTT Cell Proliferation Assay</td>
			<td style="width:119px;">ATCC</td>
			<td style="width:167px;">30-1010K</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td style="width:119px;">ThermoFisher Scientific</td>
			<td style="width:167px;">18265017</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:247px;">pBlueScript II Vectors</td>
			<td style="width:119px;">Agilent Technologies</td>
			<td style="width:167px;">Variable (e.g. 212205)</td>
			<td style="width:247px;">There are different plasmids with T7 or T3 promoters and variable cloning sites to enable cloning and RNA transcription.</td>
		</tr>
	</tbody>
</table>

microrna-based regulation of picornavirus tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

A Tabela 1 representa os resultados típicos de um ensaio de titulação para um picornavírus e descreve a forma de calcular a dose infecciosa de cultura de tecido de 50%. Uma representação esquemática do conceito global de regulação baseada em microARN de tropismo viral descrito neste manuscrito é mostrado na Figura 1. A orientação do elemento de resposta para microARN durante interacções intracelulares, concepção adequada de elemento de re...

Watch this Scientific Journal Video about MicroRNA-based Regulation of Picornavirus Tropism at JoVE.com

MicroRNA-based Regulation of Picornavirus Tropism

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Regulamento baseada MicroRNA de Picornavirus tropismo

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

microrna-based-regulation-of-picornavirus-tropism

Research

JoVE Journal

Immunology and Infection

7.4K visualizações.  Mayo Clinic College of Medicine. We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle. 

Vídeo: MicroRNA-based Regulation of Picornavirus Tropism

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferência genotípica do HIV-1 Tropismo Usando de base populacional de Seqüenciamento V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Imunologia e Infecção

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

Previsão do HIV-1 de uso co-receptor (tropismo) por análise de seqüência usando uma abordagem genotípica

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Atraso Tipo Ensaio Hipersensibilidade para o Antigen Regulamento Específico

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Quantificação simultânea de celulares e extracelulares Componentes de biofilmes

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Grande Escala da Purificação de suínos ou bovinos fotorreceptores Outer Segmentos para a fagocitose Ensaios sobre Retinal Pigment células epiteliais

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

Visualização de Twitching motilidade e Caracterização do papel da<em&gt; PilG</em&gt; in<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...