RESUMO
Isoniazid-filled Fe2 O3 hollow nanospheres (INH@Fe2 O3 , diameter <30 nm, 48 wt % INH-load) are prepared for the first time and suggested for tuberculosis therapy. After dextran-functionalization, the INH@Fe2 O3 @DEX nanocontainers show strong activity against Mycobacterium tuberculosis (M.tb.) and M.tb.-infected macrophages. The nanocontainers can be considered as "Trojan horses" and show efficient, active uptake into both M.tb.-infected macrophages and even into mycobacterial cells.
Assuntos
Antituberculosos/administração & dosagem , Antituberculosos/farmacologia , Compostos Férricos/química , Isoniazida/administração & dosagem , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Nanosferas/química , Animais , Células Cultivadas , Humanos , Macrófagos/microbiologia , Camundongos , Nanosferas/ultraestrutura , Tuberculose/tratamento farmacológicoRESUMO
Mutations in human DNA mismatch repair (MMR) genes are commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). MLH1 protein heterodimerizes with PMS2, PMS1, and MLH3 to form MutLα, MutLß, and MutLγ, respectively. We reported recently stable expression of GFP-linked MLH3 in human cell lines. Monitoring these cell lines during the cell cycle using live cell imaging combined with confocal microscopy, we detected accumulation of MLH3 at the centrosomes. Fluorescence recovery after photobleaching (FRAP) revealed high mobility and fast exchange rates at the centrosomes as it has been reported for other DNA repair proteins. MLH3 may have a role in combination with other repair proteins in the control of centrosome numbers.
Assuntos
Proteínas de Transporte/metabolismo , Centrossomo/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Células HEK293 , Humanos , Microscopia Confocal , Proteínas MutLRESUMO
The human DNA mismatch repair (MMR) gene family comprises four MutL paralogues capable of forming heterodimeric MutLα (MLH1-PMS2), MutLß (MLH1-PMS1), and MutLγ (MLH1-MLH3) protein complexes. Human MutL subunits PMS2 and MLH3 contain an evolutionarily conserved amino acid motif DQHA(X)2E(X)4E identified as an endonucleolytic domain capable of incising a defective DNA strand. PMS2 of MutLα is generally accepted to be the sole executor of endonucleolytic activity, but since MLH3 was shown to be able to perform DNA repair at low levels in vitro, our aim was to investigate whether or not MLH3 is activated as a backup under MutLα-deficient conditions. Here, we report stable expression of GFP-tagged MLH3 in the isogenic cell lines 293 and 293T which are functional or defective for MLH1 expression, respectively. As expected, MLH3 formed dimeric complexes with endogenous and recombinant MLH1. MutLγ dimers were recruited to sites of DNA damage induced by UVA micro-irradiation as shown for MutLα. Surprisingly, splicing variant MLH3Δ7 lacking the endonucleolytic motif displayed congruent foci formation, implying that recruitment is not necessarily representing active DNA repair. As an alternative test for repair enzyme activity, we combined alkylation-directed DNA damage with comet formation assays. While recombinant MutLα led to full recovery of DNA damage response in MMR deficient cells, expression of MutLγ or single MLH3 failed to do so. These experiments show recruitment and persistence of MutLγ-heterodimers at UVA-induced DNA lesions. However, we demonstrate that in a MutLα-deficient background no DNA repair-specific function carried out by MutLγ can be detected in living cells.
Assuntos
Reparo de Erro de Pareamento de DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , DNA/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Ensaio Cometa , DNA/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo de Erro de Pareamento de DNA/genética , Enzimas Reparadoras do DNA/genética , Humanos , ImunoprecipitaçãoRESUMO
Salmonella Typhimurium causes diarrhea by infecting the epithelium and lamina propria of the intestinal mucosa and by secreting various effector proteins through type III secretion systems (TTSSs). However, the mechanisms by which Salmonella transverses the epithelium and is subsequently released into the lamina propria are poorly understood. Using a murine Salmonella-diarrhea model and in vivo microscopy, we show that epithelial traversal requires TTSS-1-mediated invasion and TTSS-2-dependent trafficking to the basolateral side. After being released into the lamina propria, the bacterium is transiently extracellular before being taken up by phagocytes, including CD11c(+)CX(3)CR1(high) monocytic phagocytes (MPs), which were found to constitutively sample cellular material shed from the basolateral side of the epithelium. Thus, Salmonella infects the cecal mucsa through a step-wise process wherein the bacterium transverses the epithelium through TTSS-2-dependent trafficking and then likely exploits lamina propria MPs, which are sampling the epithelium, to enter and replicate within the host.
Assuntos
Epitélio/microbiologia , Trato Gastrointestinal/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Mucosa/microbiologia , Fagócitos/microbiologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Microscopia , Mucosa/citologia , Salmonelose AnimalRESUMO
Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination.
RESUMO
BACKGROUND: Dendritic cells (DCs) are applied worldwide in several clinical studies of immune therapy of malignancies, autoimmune diseases, and transplantations. Most legislative bodies are demanding high standards for cultivation and transduction of cells. Closed-cell cultivating systems like cell culture bags would simplify and greatly improve the ability to reach these cultivation standards. We investigated if a new polyolefin cell culture bag enables maturation and adenoviral modification of human DCs in a closed system and compare the results with standard polystyrene flasks. STUDY DESIGN AND METHODS: Mononuclear cells were isolated from HLA-A*0201-positive blood donors by leukapheresis. A commercially available separation system (CliniMACS, Miltenyi Biotec) was used to isolate monocytes by positive selection using CD14-specific immunomagnetic beads. The essentially homogenous starting cell population was cultivated in the presence of granulocyte-macrophage-colony-stimulating factor and interleukin-4 in a closed-bag system in parallel to the standard flask cultivation system. Genetic modification was performed on Day 4. After induction of maturation on Day 5, mature DCs could be harvested and cryopreserved on Day 7. During the cultivation period comparative quality control was performed using flow cytometry, gene expression profiling, and functional assays. RESULTS: Both flasks and bags generated mature genetically modified DCs in similar yields. Surface membrane markers, expression profiles, and functional testing results were comparable. The use of a closed-bag system facilitated clinical applicability of genetically modified DCs. CONCLUSIONS: The polyolefin bag-based culture system yields DCs qualitatively and quantitatively comparable to the standard flask preparation. All steps including cryopreservation can be performed in a closed system facilitating standardized, safe, and reproducible preparation of therapeutic cells.
Assuntos
Técnicas de Cultura de Células/métodos , Células Dendríticas/fisiologia , Adulto , Antígenos CD/análise , Antígenos CD/genética , Antígenos de Superfície/análise , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Divisão Celular/fisiologia , Senescência Celular/fisiologia , Criança , Células Dendríticas/citologia , Células Dendríticas/ultraestrutura , Feminino , Técnicas de Transferência de Genes , Humanos , Leucaférese/métodos , Leucócitos/citologia , Leucócitos/fisiologia , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/genética , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: Dendritic cells (DCs) play a central role in the initiation and regulation of immune responses. DCs for clinical applications can be generated with high yield from leukapheresis products. Using adenoviral transduction we genetically modified human DCs to produce and present melanoma-associated antigens. Coexpression of green fluorescent protein and epitope tags were used to monitor genetic modification. Generation, genetic modification, and cryoconservation of gene modified human DCs on a clinical scale in a closed system is feasible. STUDY DESIGN AND METHODS: CD14-positive monomuclear cells were isolated from leukapheresis products of HLA-A* 0201 positive voluntary blood donors using immunomagnetic beads. Selected cells were cultivated for 7 days. Adenovirus transduction was optimal on Day 4. Maturation was induced on Day 5. Mature DC were aliquoted and cryoconserved on Day 7. Quality control was performed using flow cytometry, expression profiling, and functional assays (ELISPOT, CBA). RESULTS: We were able to generate sufficient genetically modified mature DCs in serum-free cultures that could be stored by cryopreservation. The use of a closed system facilitated development of methods for standardized production of clinically applicable genetically modified DCs. The adenoviral transduction system allowed simultaneous and flexible expression of tumor-associated antigens for prolonged presentation of multiple epitopes. CONCLUSION: The feasibility of a closed-bag system for the cultivation of genetically modified human DCs is shown. The immature DCs were genetically modified by recombinant replication-deficient adenoviruses to express multiple epitopes of tumor-associated proteins and then differentiated to mature antigen-presenting DCs.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/fisiologia , Antígenos HLA-A/imunologia , Proteínas de Neoplasias/análise , Organismos Geneticamente Modificados/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD19/imunologia , Sobrevivência Celular/imunologia , Células Dendríticas/imunologia , Antígenos HLA-A/genética , Antígeno HLA-A2 , Teste de Histocompatibilidade/métodos , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Ativação Linfocitária/imunologia , Reação em Cadeia da Polimerase , Linfócitos T/imunologia , Vacinação/métodosRESUMO
Serology, defined as antibody-based diagnostics, has been regarded as the diagnostic gold standard in transfusion medicine. Nowadays however the impact of molecular diagnostics in transfusion medicine is rapidly growing. Molecular diagnostics can improve tissue typing (HLA typing), increase safety of blood products (NAT testing of infectious diseases), and enable blood group typing in difficult situations (after transfusion of blood products or prenatal non-invasive RhD typing). Most of the molecular testing involves the determination of the presence of single nucleotide polymorphisms (SNPs). Antigens (e.g. blood group antigens) mostly result from single nucleotide differences in critical positions. However, most blood group systems cannot be determined by looking at a single SNP. To identify members of a blood group system a number of critical SNPs have to be taken into account. The platforms which are currently used to perform molecular diagnostics are mostly gel-based, requiring time-consuming multiple manual steps. To implement molecular methods in transfusion medicine in the future the development of higher-throughput SNP genotyping non-gel-based platforms which allow a rapid, cost-effective screening are essential. Because of its potential for automation, high throughput and cost effectiveness the special focus of this paper is a relative new technique: SNP genotyping by MALDI-TOF MS analysis.
RESUMO
Peyer's patches (PPs) are typical gut-associated lymphoid tissues that are located along the wall of the small intestine and that serve as the major sites for generation of immunity to intestinal antigens. Their unique micro-organization is crucial for the generation of the immune response. Although many studies have been reported on the functional anatomy of PP, most investigations have relied on the random sampling of these organs, a procedure that is insufficient for the systemic scanning of the whole tissue or organ. By combining a variety of methods, we have accomplished 3D reconstructions of Peyer's patch. The complex reconstruction procedure includes several steps. First, the PP are serially sectioned at a thickness of 10 microm with a cryostat; (b) the serial sections are stained with haematoxylin-eosin; (c) multiple images from the PP are acquired with an automatic microscope and stitched together with Image Pro Plus to generate a composite image for the whole organ; (d) the serial images are reconstructed with Image J, Reconstruct and 3D Studio Max. The combinational approaches that we present here should be of value when extrapolated to the reconstruction of other tissues or organs. Moreover, the 3D model that we have created and our stereological analysis should be extremely helpful for further in vivo microscopic studies of PP with respect to the immune response.
Assuntos
Crioultramicrotomia/métodos , Imageamento Tridimensional/métodos , Nódulos Linfáticos Agregados/citologia , Animais , Feminino , Histocitoquímica , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The first step towards the three-dimensional (3D) reconstruction of histological structures from serial sectioned tissue blocks is the proper alignment of microscope image sequences. We have accomplished an automatic rigid registration program, named Image-Reg, to align serial sections from mouse lymph node and Peyer's patch. Our approach is based on the calculation of the pixel-correlation of objects in adjacent images. The registration process is mainly divided into two steps. Once the foreground images have been segmented from the original images, the first step (primary alignment) is performed on the binary images of segmented objects; this process includes rotation by using the moments and translation through the X, Y axes by using the centroid. In the second step, the matching error of two binary images is calculated and the registration results are refined through multi-scale iterations. In order to test the registration performance, Image-Reg has been applied to an image and its transformed (rotated) version and subsequently to an image sequence of three serial sections of mouse lymph node. In addition, to compare our algorithm with other registration methods, three other approaches, viz. manual registration with Reconstruct, semi-automatic landmark registration with Image-Pro Plus and the automatic phase-correlation method with Image-Pro Plus, have also been applied to these three sections. The performance of our program has been also tested on other two-image data sets. These include: (a) two light microscopic images acquired by the automatic microscope (stitched with other software); (b) two images fluorescent images acquired by confocal microscopy (tiled with other software). Our proposed approach provides a fast and accurate linear alignment of serial image sequences for the 3D reconstruction of tissues and organs.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Linfonodos/citologia , Microscopia/métodos , Nódulos Linfáticos Agregados/citologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia ConfocalRESUMO
The function of lymph nodes is greatly influenced by their unique microanatomy, in which distinct subpopulations of cells are compartmentalized by a meshwork of reticular cells and fibres, specialized blood and lymphatic vessels and nerves. Using antibodies against extracellular matrix (ECM) proteins (fibronectin, collagen IV and laminin), proteoglycan (perlecan), and a fibroblastic marker (ERTR-7), the distribution and molecular organization of the system of reticular fibres was investigated by three-dimensional (3D) reconstruction methods. Fibronectin, collagen IV and laminin are restricted to reticular fibres and have a similar distribution pattern, whereas perlecan is limited to the vascular system of the lymph node. Various compartments of the lymph node, such as the B-cell follicle, paracortex (including the high endothelial venules and paracortical cord), and medulla have been reconstructed to visualize their vasculature with respect to B and T cells. Since the morphology of lymph nodes may change significantly in pathological conditions, different compartments of reactive lymph node (after low-dose Listeria monocytogenes infection), especially germinal centres, were also investigated. The data presented here should facilitate our understanding of the 3D organization of non-immune cell components of lymph nodes, which is crucial for cell adhesion, migration, activation, and differentiation in normal and pathological conditions.
Assuntos
Proteínas da Matriz Extracelular/análise , Linfonodos , Microcirculação/citologia , Microtomia/métodos , Reticulina/análise , Animais , Biomarcadores/análise , Modelos Animais de Doenças , Feminino , Fibronectinas , Técnica Indireta de Fluorescência para Anticorpo , Proteoglicanas de Heparan Sulfato/análise , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Listeriose/patologia , Linfonodos/irrigação sanguínea , Linfonodos/química , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Livres de Patógenos EspecíficosRESUMO
Image stitching is the process of combining multiple images to produce a panorama or larger image. In many biomedical studies, including those of cancer and infection, the use of this approach is highly desirable in order to acquire large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we describe the application of Autostitch, viz. software that is normally used for the generation of panoramas in photography, in the seamless stitching of microscope images. First, we tested this software on image sets manually acquired by normal light microscopy and compared the performance with a manual stitching approach performed with Paint Shop Pro. Secondly, this software was applied to an image stack acquired by an automatic microscope. The stitching results were then compared with that generated by a self-programmed rectangular tiling macro integrated in Image J. Thirdly, this program was applied in the image stitching of images from electron microscopy. Thus, the automatic stitching program described here may find applications in convenient image stitching and virtual microscopy in the biomedical research.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica/métodos , Microscopia/métodos , Software , Animais , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Linfonodos/anatomia & histologia , Linfonodos/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia/estatística & dados numéricos , Microscopia Eletrônica/estatística & dados numéricos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Varredura/estatística & dados numéricos , Nódulos Linfáticos Agregados/anatomia & histologiaRESUMO
Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.
Assuntos
Listeria monocytogenes , Listeriose/patologia , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Complexo CD3/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Listeriose/microbiologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Baço/imunologia , Baço/patologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Multi-colour imaging of immunofluorescently labelled tissue using confocal microscopy was accomplished by using colour addition theory. This new technique includes several improvements for immunolabelling: (1) the co-localization of two or more markers on one cell for the identification of specific cell populations; (2) the co-localization of two fluorescent dyes from secondary reagents for the identification of the cells; (3) a multi-step staining protocol with two primary antibodies originating from the same host species or with two or three biotin-conjugated primary antibodies. After image acquisition, colour segmentation/unmixing are applied to the single multi-colour image to generate multi-pseudo-channels for individual or co-localized fluorescent dyes. With this new technique, we have been able to visualize six cell populations simultaneously in the mouse lymph node and intestine. The efficiency of this method has also been demonstrated in the three-dimensional reconstruction of thick sections from mouse ileum. Our method is simple, efficient, and may be indispensable in experimental cell and tissue studies requiring multiple immunolabelling.
Assuntos
Cor , Diagnóstico por Imagem/métodos , Corantes Fluorescentes , Imuno-Histoquímica/métodos , Tecido Linfoide/anatomia & histologia , Animais , Anticorpos/análise , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia ConfocalRESUMO
Here, we report the identification of the ubiquitin-like gene UBD as a downstream element of FOXP3 in human activated regulatory CD4(+)CD25(hi) T cells (T(reg)). Retroviral transduction of UBD in human allo-reactive effector CD4(+) T helper (T(h)) cells upregulates CD25 and mediates downregulation of IL4 and IL5 expression similar to overexpression of FOXP3. Moreover, UBD impairs T(h) cell proliferation without upregulation of FOXP3 and impairs calcium mobilization. In the presence of ionomycin, overexpression of UBD in T(h) cells leads to the induction of IL1R2 that resemble FOXP3-transduced T(h) cells and naturally derived T(reg) cells. A comparison of the transcriptome of FOXP3- and UBD-transduced T(h) cells with T(reg) cells allowed the identification of the gene LGALS3. However, high levels of LGALS3 protein expression were observed only in human CD4(+)CD25(hi) derived T(reg) cells and FOXP3-transduced T(h) cells, whereas little was induced in UBD-transduced T(h) cells. Thus, UBD contributes to the anergic phenotype of human regulatory T cells and acts downstream in FOXP3 induced regulatory signaling pathways, including regulation of LGALS3 expression. High levels of LGALS3 expression represent a FOXP3-signature of human antigen-stimulated CD4(+)CD25(hi) derived regulatory T cells.
Assuntos
Fatores de Transcrição Forkhead/imunologia , Galectina 3/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Ubiquitinas/imunologia , Biomarcadores/metabolismo , Antígenos CD4/imunologia , Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Galectina 3/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-4/imunologia , Interleucina-4/metabolismo , Interleucina-5/imunologia , Interleucina-5/metabolismo , Ionomicina/farmacologia , Ionóforos/farmacologia , Receptores de Interleucina-1/biossíntese , Receptores Tipo II de Interleucina-1 , Receptores de Interleucina-2/imunologia , Retroviridae/genética , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Transdução Genética , Ubiquitinas/genéticaRESUMO
Murine listeriosis is a paradigm to understand host pathogen interactions. Airway infections with Listeria monocytogenes, although representing a serious problem in early onset neonatal listeriosis, has not been investigated in detail in animal models so far. Here, the susceptibility of BALB/c, DBA/2 and C57BL/6 mice towards an intratracheal (i.t.) infection with virulent L. monocytogenes EGDe and the attenuated variant L. monocytogenes EGD hlyW491A(pERL3-CMVGFP) is reported. The course of infection was characterized by determination of bacterial numbers in the organs and assessment of the health condition of the mice. The distribution and cellular localization of Listeria in the airways was assessed by immunocytochemistry and confocal and electron microscopy. The differential susceptibility of inbred mouse strains to airway infections with L. monocytogenes could be assigned to the major virulence factor listeriolysin O. Resistant C57BL/6 mice were not affected by the two listerial strains. In contrast, BALB/c and DBA/2 mice showed differential susceptibility towards L. monocytogenes EGDe and attenuated bacteria, with all the mice being killed by the wild-type bacteria but rarely by the variant that secretes a listeriolysin of only 10% activity of that of the wild-type toxin. Thus, listeriolysin is a decisive factor for differential susceptibility against Listeria. After i.t. application, bacteria were predominantly localized in the peribronchiolar space and invaded alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into the deep organs started almost immediately after application, although a pulmonary bacterial reservoir remained during the first 4 days.
Assuntos
Toxinas Bacterianas/metabolismo , Modelos Animais de Doenças , Proteínas de Choque Térmico/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/fisiopatologia , Pneumopatias/fisiopatologia , Animais , Contagem de Colônia Microbiana , Suscetibilidade a Doenças , Proteínas Hemolisinas , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeriose/imunologia , Listeriose/microbiologia , Pulmão/microbiologia , Pulmão/ultraestrutura , Pneumopatias/imunologia , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Eletrônica de Transmissão , Especificidade da EspécieRESUMO
Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Toxinas Shiga/farmacologia , Apoptose/efeitos dos fármacos , Quimiocinas/biossíntese , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Primers do DNA/genética , Endotélio Vascular/citologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli/química , Escherichia coli/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Síndrome Hemolítico-Urêmica/fisiopatologia , Humanos , Imuno-Histoquímica , Receptores de Superfície Celular/metabolismo , Toxinas Shiga/toxicidade , Triexosilceramidas/metabolismo , Veias Umbilicais/citologiaRESUMO
Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions.