RESUMO
A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate-poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of the bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (STR; 1-L scale). Two proprietary T lymphocyte culture media as well as a standard RPMI-based medium were used. As before, encapsulation coincided with the presence of only a low fraction of activated T lymphocytes (peripheral blood T cells) in the total population. Unexpectedly, growth rates were lower in well-mixed reactors than those in cultivations under static conditions, that is, in T-flasks. Switching the STR to low oxygen conditions (40% air saturation) improved the growth rates to the level of the static cultures and thus forms the potential basis for efficient scale-up of T lymphocyte expansion.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células , Proliferação de Células , Células Imobilizadas/metabolismo , Linfócitos T/metabolismo , Células Imobilizadas/citologia , Meios de Cultura/química , Humanos , Linfócitos T/citologiaRESUMO
Smart polymers are a valuable platform to protect and control the activity of biological agents over a wide range of conditions, such as low pH, by proper encapsulation. Such conditions are present in olive oil mill wastewater with phenol as one of the most problematic constituents. We show that elastic and pH-responsive diblock copolymer fibers are a suitable carrier for Corynebacterium glutamicum, i.e., bacteria which are known for their ability to degrade phenol. Free C. glutamicum does not survive low pH conditions and fails to degrade phenol at low pH conditions. Our tea-bag like biohybrid system, where the pH-responsive diblock copolymer acts as a protecting outer shell for the embedded bacteria, allows phenol degradation even at low pH. Utilizing a two-step encapsulation process, planktonic cells were first encapsulated in poly(vinyl alcohol) to protect the bacteria against the organic solvents used in the second step employing coaxial electrospinning.
Assuntos
Biotecnologia/métodos , Corynebacterium glutamicum/metabolismo , Fenol/metabolismo , Álcool de Polivinil/química , Águas Residuárias/química , Purificação da Água/métodos , BiotransformaçãoRESUMO
In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103âºCX3CR1â»CD11c⺠dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.
Assuntos
Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Células Dendríticas/microbiologia , Linfonodos/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Carga Bacteriana/efeitos dos fármacos , Ceco , Diarreia/tratamento farmacológico , Diarreia/imunologia , Diarreia/microbiologia , Farmacorresistência Bacteriana , Lipopolissacarídeos/farmacologia , Linfonodos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Fenótipo , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/crescimento & desenvolvimentoRESUMO
The mammalian intestine is colonized by a dense microbial community, the microbiota. Homeostatic and symbiotic interactions facilitate the peaceful co-existence between the microbiota and the host, and inhibit colonization by most incoming pathogens ('colonization resistance'). However, if pathogenic intruders overcome colonization resistance, a fierce, innate inflammatory defense can be mounted within hours, the adaptive arm of the immune system is initiated, and the pathogen is fought back. The molecular nature of the homeostatic interactions, the pathogen's ability to overcome colonization resistance, and the triggering of native and adaptive mucosal immune responses are still poorly understood. To study these mechanisms, the streptomycin mouse model for Salmonella diarrhea is of great value. Here, we review how S. Typhimurium triggers mucosal immune responses by active (virulence factor elicited) and passive (MyD88-dependent) mechanisms and introduce the S. Typhimurium mutants available for focusing on either response. Interestingly, mucosal defense turns out to be a double-edged sword, limiting pathogen burdens in the gut tissue but enhancing pathogen growth in the gut lumen. This model allows not only studying the molecular pathogenesis of Salmonella diarrhea but also is ideally suited for analyzing innate defenses, microbe handling by mucosal phagocytes, adaptive secretory immunoglobulin A responses, probing microbiota function, and homeostatic microbiota-host interactions. Finally, we discuss the general need for defined assay conditions when using animal models for enteric infections and the central importance of littermate controls.
Assuntos
Diarreia/imunologia , Diarreia/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella/imunologia , Fatores de Virulência/imunologia , Animais , Diarreia/etiologia , Diarreia/fisiopatologia , Modelos Animais de Doenças , Humanos , Imunidade nas Mucosas , Metagenoma/genética , Metagenoma/imunologia , Camundongos , Salmonella/genética , Infecções por Salmonella/complicações , Infecções por Salmonella/fisiopatologia , Estreptomicina/administração & dosagem , Estreptomicina/efeitos adversos , VirulênciaRESUMO
An understanding of how pathogens colonize their hosts is crucial for the rational design of vaccines or therapy. While the molecular factors facilitating the invasion and systemic infection by pathogens are a central focus of research in microbiology, the population biological aspects of colonization are still poorly understood. Here, we investigated the early colonization dynamics of Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in the streptomycin mouse model for diarrhea. We focused on the first step on the way to systemic infection -- the colonization of the cecal lymph node (cLN) from the gut -- and studied roles of inflammation, dendritic cells and innate immune effectors in the colonization process. To this end, we inoculated mice with mixtures of seven wild type isogenic tagged strains (WITS) of S. Tm. The experimental data were analyzed with a newly developed mathematical model describing the stochastic immigration, replication and clearance of bacteria in the cLN. We estimated that in the beginning of infection only 300 bacterial cells arrive in the cLN per day. We further found that inflammation decreases the net replication rate in the cLN by 23%. In ccr7(-/-) mice, in which dendritic cell movement is impaired, the bacterial migration rate was reduced 10-fold. In contrast, cybb(-/-) mice that cannot generate toxic reactive oxygen species displayed a 4-fold higher migration rate from gut to cLN than wild type mice. Thus, combining infections with mixed inocula of barcoded strains and mathematical analysis represents a powerful method for disentangling immigration into the cLN from replication in this compartment. The estimated parameters provide an important baseline to assess and predict the efficacy of interventions.
Assuntos
Translocação Bacteriana , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Imunidade Inata , Linfonodos/microbiologia , Intoxicação Alimentar por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ceco , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Linfonodos/imunologia , Linfonodos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Intoxicação Alimentar por Salmonella/metabolismo , Intoxicação Alimentar por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Organismos Livres de Patógenos Específicos , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.
Assuntos
Adesinas Bacterianas/metabolismo , Bartonella henselae/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Adesinas Bacterianas/genética , Animais , Aderência Bacteriana/fisiologia , Clonagem Molecular , Colágeno/química , Colágeno/metabolismo , Escherichia coli/patogenicidade , Larva/microbiologia , Mariposas/microbiologia , Mutação , Estrutura Terciária de Proteína , Proteínas RecombinantesRESUMO
Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin Bartonella adhesin A (BadA) which is modularly constructed and consists of a head, a long and repetitive neck-stalk module with 22 repetitive neck/stalk repeats and a membrane anchor. The BadA head is crucial for bacterial adherence to host cells, binding to several extracellular matrix proteins and for the induction of vascular endothelial growth factor (VEGF) secretion. Here, we analysed the biological role of the BadA stalk in the infection process in greater detail. For this purpose, BadA head-bearing and headless deletion mutants with different lengths (containing one or four neck/stalk repeats in the neck-stalk module) were produced and functionally analysed for their ability to bind to fibronectin, collagen and endothelial cells and to induce VEGF secretion. Whereas a head-bearing short version (one neck/stalk element) of BadA lacks exclusively fibronectin binding, a substantially truncated headless BadA mutant was deficient for all of these biological functions. The expression of a longer headless BadA mutant (four neck/stalk repeats) restored fibronectin and collagen binding, adherence to host cells and the induction of VEGF secretion. Our data suggest that (i) the stalk of BadA is exclusively responsible for fibronectin binding and that (ii) both the head and stalk of BadA mediate adherence to collagen and host cells and the induction of VEGF secretion. This indicates overlapping functions of the BadA head and stalk.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Bartonella henselae/patogenicidade , Adesinas Bacterianas/genética , Células Cultivadas , Colágeno/metabolismo , Análise Mutacional de DNA , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Deleção de SequênciaRESUMO
Trimeric autotransporter adhesins (TAAs) are important virulence factors of Gram-negative bacteria responsible for adherence to extracellular matrix (ECM) and host cells. Here, we analyzed three different TAAs (Bartonella adhesin A [BadA] of Bartonella henselae, variably expressed outer membrane proteins [Vomps] of Bartonella quintana, and Yersinia adhesin A [YadA] of Yersinia enterocolitica) for mediating bacterial adherence to ECM and endothelial cells. Using static (cell culture vials) and dynamic (capillary flow chambers) experimental settings, adherence of wild-type bacteria and of the respective TAA-negative strains was analyzed. Under static conditions, ECM adherence of B. henselae, B. quintana, and Y. enterocolitica was strongly dependent on the expression of their particular TAAs. YadA of Y. enterocolitica did not mediate bacterial binding to plasma or cellular fibronectin under either static or dynamic conditions. TAA-dependent host cell adherence appeared more significant under dynamic conditions although the total number of bound bacteria was diminished compared to the number under static conditions. Dynamic models expand the methodology to perform bacterial adherence experiments under more realistic, bloodstream-like conditions and allow dissection of the biological role of TAAs in ECM and host cell adherence under static and dynamic conditions.
Assuntos
Adesinas Bacterianas/fisiologia , Aderência Bacteriana , Bartonella henselae/fisiologia , Bartonella quintana/fisiologia , Células Endoteliais/microbiologia , Yersinia enterocolitica/fisiologia , Adesinas Bacterianas/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bartonella henselae/patogenicidade , Bartonella quintana/patogenicidade , Junções Célula-Matriz , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/microbiologia , Imunofluorescência , Interações Hospedeiro-Patógeno , Humanos , Microscopia Eletrônica de Transmissão , Veias Umbilicais , Fatores de Virulência/metabolismo , Yersinia enterocolitica/patogenicidadeRESUMO
Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium) is a frequent cause of diarrhea worldwide. It employs 2 type III secretion systems (TTSS) to elicit mucosal inflammation via the TTSS-1-dependent 'classical' or the TTSS-2-dependent 'alternative' pathway. If TTSS-1 is defective (in invG or invC mutants), the pathogen is confined to the alternative pathway; transits the epithelium in a dendritic cell-dependent fashion, relocalizes from CD11c(+) dendritic cells to CD11c(-) cells, and elicits inflammation by day 3 post infection (p.i.). It has remained unclear whether other virulence factors may also contribute to this process. Here, we used the streptomycin mouse model to analyze whether spvB and spvC, virulence factors known to affect the pathogen-phagocyte interaction at systemic sites, might contribute to triggering colitis. By 12h p.i., spvBC mutants elicited wild-type levels of gut inflammation and mucosal cytokine induction via the classical pathway. However, spvBC mutants confined to the alternative pathway triggered reduced levels of gut inflammation by day 3 p.i. (S. tm(ΔinvGΔspvBC) vs. S. tm(ΔinvG)). Detailed analyses using spvB or spvC mutants (e.g. S. tm(ΔinvCΔspvB)) revealed that spvB was required for efficient lamina propria colonization and suggested that this was attributable to defective relocalization from dendritic- to CD11c(-) cells. This establishes a novel virulence phenotype for spvB in the alternative pathway of S. Typhimurium colitis.
Assuntos
ADP Ribose Transferases/metabolismo , Carbono-Oxigênio Liases/metabolismo , Colite/microbiologia , Colite/patologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/metabolismo , Animais , Carga Bacteriana , Colo/microbiologia , Colo/patologia , Citocinas/metabolismo , Histocitoquímica , Humanos , Inflamação/microbiologia , Inflamação/patologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Índice de Gravidade de Doença , Fatores de TempoRESUMO
After 2 decades of Bartonella research, knowledge on transmission and pathology of these bacteria is still limited. Bartonella spp. have emerged to be important pathogens in human and veterinary medicine. For humans, B. henselae is considered to represent the most relevant zoonotic Bartonella species and is responsible for cat scratch disease, bacillary angiomatosis, and other disorders. Over the years, many Bartonella species have been isolated from humans, cats, dogs, and other mammals, and infections range from an asymptomatic state (e.g., animal-specific species) to even life-threatening diseases (e.g., Oroya fever). It is obvious that the analysis of pathogenicity mechanisms underlying Bartonella infections is needed to increase our understanding of how these pathogens adapt to their mammalian hosts resulting in acute or chronic diseases.
Assuntos
Infecções por Bartonella/patologia , Infecções por Bartonella/veterinária , Bartonella/patogenicidade , Zoonoses/epidemiologia , Zoonoses/transmissão , Animais , Doenças Assintomáticas , Infecções por Bartonella/diagnóstico , Infecções por Bartonella/epidemiologia , Humanos , Zoonoses/microbiologiaRESUMO
Adhesion to host cells represents the first step in the infection process and one of the decisive features in the pathogenicity of Bartonella spp. B. henselae and B. quintana are considered to be the most important human pathogenic species, responsible for cat scratch disease, bacillary angiomatosis, trench fever and other diseases. The ability to cause vasculoproliferative disorders and intraerythrocytic bacteraemia are unique features of the genus Bartonella. Consequently, the interaction with endothelial cells and erythrocytes is a focus in Bartonella research. The genus harbours a variety of trimeric autotransporter adhesins (TAAs) such as the Bartonella adhesin A (BadA) of B. henselae and the variably expressed outer-membrane proteins (Vomps) of B. quintana, which display remarkable variations in length and modular construction. These adhesins mediate many of the biologically-important properties of Bartonella spp. such as adherence to endothelial cells and extracellular matrix proteins and induction of angiogenic gene programming. There is also significant evidence that the laterally acquired Trw-conjugation systems of Bartonella spp. mediate host-specific adherence to erythrocytes. Other potential adhesins are the filamentous haemagglutinins and several outer membrane proteins. The exact molecular functions of these adhesins and their interplay with other pathogenicity factors (e.g., the VirB/D4 type 4 secretion system) need to be analysed in detail to understand how these pathogens adapt to their mammalian hosts.
Assuntos
Adesinas Bacterianas/fisiologia , Bartonella/fisiologia , Bartonella/patogenicidade , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Angiomatose Bacilar/etiologia , Animais , Bartonella/genética , Infecções por Bartonella/etiologia , Infecções por Bartonella/microbiologia , Doença da Arranhadura de Gato/etiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Peliose Hepática/etiologia , Febre das Trincheiras/etiologia , Virulência/fisiologiaRESUMO
Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders (e.g. bacillary angiomatosis). Expression of Bartonella adhesin A (BadA) is crucial for bacterial autoagglutination, adhesion to host cells, binding to extracellular matrix proteins and proangiogenic reprogramming via activation of hypoxia inducible factor (HIF)-1. Like the prototypic Yersinia adhesin A, BadA belongs to the class of trimeric autotransporter adhesins and is constructed modularly consisting of a head, a long and repetitive neck-stalk module and a membrane anchor. Until now, the exact biological role of these domains is not known. Here, we analysed the function of the BadA head by truncating the repetitive neck-stalk module of BadA (B. henselae badA(-)/pHN23). Like B. henselae Marseille wild type, B. henselae badA(-)/pHN23 showed autoagglutination, adhesion to collagen and endothelial cells and activation of HIF-1 in host cells. Remarkably, B. henselae badA(-)/pHN23 did not bind to fibronectin (Fn) suggesting a crucial role of the deleted stalk domain in Fn binding. Additionally, the recombinantly expressed BadA head adhered to human umbilical vein endothelial cells and to a lesser degree to epithelial (HeLa 229) cells. Our data suggest that the head represents the major functional domain of BadA responsible for host adhesion and angiogenic reprogramming.
Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Bartonella henselae/patogenicidade , Interações Hospedeiro-Patógeno , Conformação Proteica , Adesinas Bacterianas/genética , Angiomatose Bacilar/metabolismo , Animais , Aderência Bacteriana/fisiologia , Bartonella henselae/genética , Bartonella henselae/fisiologia , Gatos , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Living Micrococcus luteus (M. luteus) and Escherichia coli (E. coli) are encapsulated in poly(vinyl alcohol), poly(vinylpyrrolidone), hydroxypropyl cellulose, and gelatin by high-temperature spray drying. The challenge is the survival of the bacteria during the standard spray-drying process at temperatures of 150 °C (M. luteus) and 120 °C (E. coli). Raman imaging and transmission electron microscopy indicate encapsulated bacteria in hollow composite microparticles. The versatility of the spray-dried polymer bacteria microparticles is successfully proved by standard polymer solution-processing techniques such as electrospinning, even with harmful solvents, to water-insoluble polyacrylonitrile, polystyrene, poly(methyl methacrylate), and poly(vinyl butyrate) nanofiber nonwovens, which opens numerous new opportunities for novel applications.
Assuntos
Células Imobilizadas , Escherichia coli , Viabilidade Microbiana , Micrococcus luteus , Nanofibras/química , Polímeros/química , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Micrococcus luteus/citologia , Micrococcus luteus/metabolismoRESUMO
Human- and animal-pathogenic Bartonella species are fastidious and slow-growing bacteria difficult to isolate and cultivate. We describe a novel, easy-to-prepare liquid medium for the fast and reliable growth of several Bartonella spp. that does not affect bacterial protein expression patterns or interactions with host cells.
Assuntos
Técnicas Bacteriológicas/métodos , Bartonella/crescimento & desenvolvimento , Meios de Cultura , Animais , Células Cultivadas , Insetos/microbiologia , Fatores de TempoRESUMO
Biocomposites, i.e., materials consisting of metabolically active microorganisms embedded in a synthetic extracellular matrix, may find applications as highly specific catalysts in bioproduction and bioremediation. 3D constructs based on fibrous biocomposites, so-called "artificial biofilms," are of particular interest in this context. The inability to produce biocomposite fibers of sufficient mechanical strength for processing into bioactive fabrics has so far hindered progress in the area. Herein a method is proposed for the direct wet spinning of microfibers suitable for weaving and knitting. Metabolically active bacteria (either Shewanella oneidensis or Nitrobacter winogradskyi (N. winogradskyi)) are embedded in these fibers, using poly(vinyl alcohol) as matrix. The produced microfibers have a partially crystalline structure and are stable in water without further treatment, such as coating. In a first application, their potential for nitrite removal (N. winogradskyi) is demonstrated, a typical challenge in potable water treatment.
Assuntos
Fibras Minerais/análise , Nitritos/isolamento & purificação , Nitrobacter/química , Shewanella/química , Têxteis/análise , Poluentes da Água/isolamento & purificação , Biodegradação Ambiental , Biofilmes/crescimento & desenvolvimento , Água Potável/química , Humanos , Teste de Materiais , Membranas Artificiais , Nitrobacter/metabolismo , Álcool de Polivinil/química , Shewanella/metabolismoRESUMO
The ex vivo expansion of primary human T cells is of considerable interest. Current protocols call for the addition of massive amounts of stimuli. This study presents as alternative the expansion of such cells in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (SCS/PDADMAC) polyelectrolyte microcapsules, which supports at least six cell divisions and results in >40 × 106 cells mLcapsule-1 within less than 10 d. Inside the microcapsules, the T cells are suspended in a viscous SCS-solution. The low molecular weight cut off (<10 000 Da) of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained, while nutrients and metabolites can pass. Expensive additives, such as interleukin-2 (IL-2), can be coencapsulated. Expansion then no longer requires specialized T-cell media. Moreover, these results suggest that an SCS with a low degree of sulfation has biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate.
Assuntos
Materiais Biomiméticos/química , Cápsulas/química , Proliferação de Células , Microambiente Celular , Linfócitos T/efeitos dos fármacos , Materiais Biomiméticos/farmacologia , Biomimética/métodos , Celulose/análogos & derivados , Humanos , Polietilenos , Compostos de Amônio Quaternário , Linfócitos T/fisiologiaRESUMO
Integration of electrogenic microorganisms remains a challenge in biofuel cell technology. Here, synthetic biocomposites ("artificial biofilms") are proposed. Bacteria (Shewanella oneidensis) are embedded in a hydrogel matrix (poly(vinyl alcohol)) via wet- and electrospinning, creating fibers and nonwoven gauzes. The bacteria remain viable and metabolically active. The performance is compared to S. oneidensis suspension cultures and "natural" biofilms. While lower than with the suspension cultures, the power output from the fuel cells with the artificial biofilms is higher than with the natural one. Handling, reproducibility, and stability are also better. Artificial biofilms can therefore contribute to resolving fundamental issues of design, scale up, and monosepsis in biofuel cell technology.
Assuntos
Fontes de Energia Bioelétrica/microbiologia , Biofilmes/crescimento & desenvolvimento , Hidrogéis/química , Álcool de Polivinil/química , Shewanella/fisiologiaRESUMO
In vivo, antibiotics are often surprisingly inefficient at eliminating bacterial pathogens. In the case of ciprofloxacin therapy in a Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, S. Tm) mouse infection model, this has been traced to tolerant bacterial cells surviving in lymph node monocytes (i.e., classical dendritic cells). To analyze the growth characteristics of these persisters, we have developed a population dynamics approach using mixtures of wild-type isogenic tagged strains (WITS) and a computational model. Here, we are providing a detailed description of the inoculum, the infection experiments, the computational analysis of the WITS data, and a computer simulation for assessing the quality of the growth parameters of the persistent S. Typhimurium cells. This approach is generic. It may be adapted to any organ infected and to any bacterial pathogen, provided that tools exist for generating, retrieving, and quantifying isogenic tagged strains.
Assuntos
Ciprofloxacina/administração & dosagem , Diarreia/genética , Dinâmica Populacional , Salmonella typhimurium/efeitos dos fármacos , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/microbiologia , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Modelos Animais de Doenças , Humanos , Linfonodos/efeitos dos fármacos , Camundongos , Monócitos/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidadeRESUMO
Herein, we report on composite materials of biologically active microorganisms placed in a synthetic polymer matrix. These so-called "living composites" were utilized for gold sequestration (Micrococcus luteus) and bioremediation of nitrite (Nitrobacter winogradskyi) to demonstrate functionality. For the preparation of the living composites the bacteria were first encased in a water-soluble polymer fiber (poly(vinyl alcohol), PVA) followed by coating the fibers with a shell of hydrophobic poly(p-xylylene) (PPX) by chemical vapor deposition (CVD). The combination of bacteria with polymer materials assured the stability and biologically activity of the bacteria in an aqueous environment for several weeks.
Assuntos
Biodegradação Ambiental , Micrococcus luteus/química , Nitrobacter/química , Purificação da Água , Biomimética , Micrococcus luteus/metabolismo , Nitrobacter/metabolismo , Polímeros/química , Álcool de Polivinil/química , Água/química , Xilenos/químicaRESUMO
BACKGROUND: Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model. RESULTS: Upon S. Tm(wt) infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This "fast cycling" through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. CONCLUSION: In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut.