Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections.

PURPOSE: Fracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery. METHODS: Extracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar. RESULTS: Importantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection. CONCLUSION: Here, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections.

Transient von Willebrand factor-mediated platelet influx stimulates liver regeneration after partial hepatectomy in mice.

BACKGROUND & AIMS: In addition to their function in thrombosis and haemostasis, platelets play an important role in the stimulation of liver regeneration. It has been suggested that platelets deliver mitogenic cargo to the regenerating liver, and accumulation of platelets in the regenerating liver has been demonstrated. We studied kinetics of platelet influx in the regenerating liver and investigated the signal that initiates platelet influx. METHODS: We visualized platelets in the liver remnant after partial hepatectomy in mice using intravital microscopy and assessed liver regeneration by examination of liver/body weight ratio and the number of proliferating hepatocytes examined by immunohistochemistry. RESULTS: We demonstrated rapid but transient platelet influx into the liver remnant after a partial liver resection. Liver regeneration in thrombocytopenic mice was substantially impaired as evidenced by a reduced liver-to-body weight ratio and decreased numbers of proliferating hepatocytes at day 3 compared to mice with normal platelet counts. In contrast, liver regeneration was only mildly impaired when thrombocytopaenia was induced 2 hours after partial liver resection. Platelet influx into the liver remnant was virtually absent in the presence of an antibody to von Willebrand factor (VWF) suggesting that VWF release from liver sinusoidal endothelial cells mediates platelet influx. Additionally, liver regeneration in mice deficient in VWF was markedly impaired. CONCLUSIONS: A rapid but transient VWF-dependent platelet influx into the liver remnant drives platelet-mediated liver regeneration.

Immunolabeling artifacts and the need for live-cell imaging.

Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

CLSM as quantitative method to determine the size of drug crystals in a solid dispersion.

PURPOSE: To test whether confocal laser scanning microscopy (CLSM) can be used as an analytical tool to determine the drug crystal size in a powder mixture or a crystalline solid dispersion. METHODS: Crystals of the autofluorescent drug dipyridamole were incorporated in a matrix of crystalline mannitol by physical mixing or freeze-drying. Laser diffraction analysis and dissolution testing were used to validate the particle size that was found by CLSM. RESULTS: The particle size of the pure drug as determined by laser diffraction and CLSM were similar (D(50) of approximately 22 µm). CLSM showed that the dipyridamole crystals in the crystalline dispersion obtained by freeze-drying of less concentrated solutions were of sub-micron size (0.7 µm), whereas the crystals obtained by freeze-drying of more concentrated solutions were larger (1.3 µm). This trend in drug crystal size was in agreement with the dissolution behavior of the tablets prepared from these products. CONCLUSION: CLSM is a useful technique to determine the particle size in a powder mixture. Furthermore, CLSM can be used to determine the drug crystal size over a broad size distribution. A limitation of the method is that the drug should be autofluorescent.

Particle Bombardment of Ex Vivo Skin to Deliver DNA and Express Proteins.

Particle bombardment of gold microparticles coated with plasmids, which are accelerated to high velocity, is used for transfection of cells within tissue. Using this method, cDNA encoding proteins of interest introduced into ex vivo living human skin enables studying of proteins of interest in real time. Here, technical aspects of particle bombardment of ex vivo skin are described using green fluorescent protein (GFP) as readout for efficiency. This method can be applied on numerous tissues, including in living model animals.

Entry of PIP3-containing polyplexes into MDCK epithelial cells by local apical-basal polarity reversal.

The polarized architecture of epithelium presents a barrier to therapeutic drug/gene carriers, which is mainly due to a limited (apical) internalization of the carrier systems. The bacterium Pseudomonas aeruginosa invades epithelial cells by inducing production of apical phosphatidylinositol-3, 4, 5-triphosphate (PIP3), which results in the recruitment of basolateral receptors to the apical membrane. Since basolateral receptors are known receptors for gene delivery vectors, apical PIP3 may improve the internalization of such vectors into epithelial cells. PIP3 and nucleic acids were complexed by the cationic polymer polyethylenimine (PEI), forming PEI/PIP3 polyplexes. PEI/PIP3 polyplexes showed enhanced internalization compared to PEI polyplexes in polarized MDCK cells, while basolateral receptors were found to redistribute and colocalize with PEI/PIP3 polyplexes at the apical membrane. Following their uptake via endocytosis, PEI/PIP3 polyplexes showed efficient endosomal escape. The effectiveness of the PIP3-containing delivery system to generate a physiological effect was demonstrated by an essentially complete knock down of GFP expression in 30% of GFP-expressing MDCK cells following anti-GFP siRNA delivery. Here, we demonstrate that polyplexes can be successfully modified to mimic epithelial entry mechanisms used by Pseudomonas aeruginosa. These findings encourage the development of pathogen-inspired drug delivery systems to improve drug/gene delivery into and across tissue barriers.

How antibodies alter the cell entry pathway of dengue virus particles in macrophages.

Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact.

Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells.

BACKGROUND: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31-1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. RESULTS: Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter. CONCLUSION: The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism.

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.

Destruction of tissue, cells and organelles in type 1 diabetic rats presented at macromolecular resolution.

Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution. The resource includes six datasets, totalling 25.000 micrographs, annotated for cellular and ultrastructural changes during autoimmune diabetes. Granulocytes are attracted to the endocrine tissue, followed by extravasation of a pleiotrophy of leukocytes. Subcellullar changes in beta cells include endoplasmic reticulum stress, insulin degranulation and glycogen accumulation. Rare findings include erythrocyte extravasation and nuclear actin-like fibers. While we focus on a rat model of autoimmune diabetes, our approach is general applicable.

Nonviral gene delivery vectors use syndecan-dependent transport mechanisms in filopodia to reach the cell surface.

Lipoplexes and polyplexes, that is, assemblies of cationic lipids and polymers with nucleic acids, respectively, are popular nanocarriers for delivery of genes or siRNA into cells for therapeutic or cell biological purposes. Although endocytosis represents a major mechanism for their cellular entry, very little is known about parameters that govern early events in the initial interaction of such delivery devices with the cell surface. Here, we demonstrate that prior to entry, poly- and lipoplexes are captured by thin, actin-rich filopodial extensions, protruding from the cell surface. Subsequent additional recruitment and local clustering of filopodia-localized syndecans, presumably driven by multivalent interactions with the polycationic nanocarriers, appear instrumental in their processing to the cell body. Detailed microscopic analyses reveal that the latter relies on either directional surfing along or retraction of the filopodia. By interfering with actin polymerization or inhibiting the motor protein myosin II, localized at the base of filopodia, our data reveal that the binding of the nanocarriers to and subsequent clustering of syndecans initiates actin retrograde flow, which moves the syndecan-bound nanocarriers to the cell body. At the present experimental conditions, inhibition of this process inhibits nanocarrier-mediated transfection by 50-90%. The present findings add novel insight to our understanding of the mechanism of nanocarrier-cell surface interaction, which may be instrumental in further improving delivery efficiency. In addition, the current experimental approach may also be of relevance to improving our understanding of cellular infection by viruses and pathogenic bacteria, given a striking parallel in filopodia-mediated processing of these infectious particles and nanocarriers.

Correlated light microscopy and electron microscopy.

Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals selected biomolecules or organelles but not the (ultra)structural context, as can be examined by electron microscopy (EM). LM and EM of the same cells, so-called correlative (or correlated) light and electron microscopy (CLEM), allow examining rare or dynamic events first by LM, and subsequently by EM. Here, we review progress in CLEM, with focus on matching the areas between different microscopic modalities. Moreover, we introduce a method that includes a virtual overlay and automated large-scale imaging, allowing to switch between most microscopes. Ongoing developments will revolutionize and standardize CLEM in the near future, which thus holds great promise to become a routine technique in cell biology.

Proteinuria triggers renal lymphangiogenesis prior to the development of interstitial fibrosis.

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet. Therefore, we evaluated the time course of lymph vessel (LV) formation in relation to proteinuria and interstitial damage in a rat model of chronic unilateral adriamycin nephrosis. Proteinuria and kidneys were evaluated up to 30 weeks after induction of nephrosis. LVs were identified by podoplanin/VEGFR3 double staining. After 6 weeks proteinuria was well-established, without influx of interstitial macrophages and myofibroblasts, collagen deposition, osteopontin expression (tubular activation) or LV formation. At 12 weeks, a ∼3-fold increase in cortical LV density was found (p<0.001), gradually increasing over time. This corresponded with a significant increase in tubular osteopontin expression (p<0.01) and interstitial myofibroblast numbers (p<0.05), whereas collagen deposition and macrophage numbers were not yet increased. VEGF-C was mostly expressed by tubular cells rather than interstitial cells. Cultured tubular cells stimulated with FCS showed a dose-dependent increase in mRNA and protein expression of VEGF-C which was not observed by human albumin stimulation. We conclude that chronic proteinuria provoked lymphangiogenesis in temporal conjunction with tubular osteopontin expression and influx of myofibroblasts, that preceded interstitial fibrosis.

Differential effects of TNF (TNFSF2) and IFN-γ on intestinal epithelial cell morphogenesis and barrier function in three-dimensional culture.

BACKGROUND: The cytokines TNF (TNFSF2) and IFNγ are important mediators of inflammatory bowel diseases and contribute to enhanced intestinal epithelial permeability by stimulating apoptosis and/or disrupting tight junctions. Apoptosis and tight junctions are also important for epithelial tissue morphogenesis, but the effect of TNF and IFNγ on the process of intestinal epithelial morphogenesis is unknown. METHODS/PRINCIPAL FINDINGS: We have employed a three-dimensional cell culture system, reproducing in vivo-like multicellular organization of intestinal epithelial cells, to study the effect of TNF on intestinal epithelial morphogenesis and permeability. We show that human intestinal epithelial cells in three-dimensional culture assembled into luminal spheres consisting of a single layer of cells with structural, internal, and planar cell polarity. Exposure of preformed luminal spheres to TNF or IFNγ enhanced paracellular permeability, but via distinctive mechanisms. Thus, while both TNF and IFNγ, albeit in a distinguishable manner, induced the displacement of selected tight junction proteins, only TNF increased paracellular permeability via caspase-driven apoptosis and cell shedding. Infliximab and adalumimab inhibited these effects of TNF. Moreover, we demonstrate that TNF via its stimulatory effect on apoptosis fundamentally alters the process of intestinal epithelial morphogenesis, which contributes to the de novo generation of intestinal epithelial monolayers with increased permeability. Also IFNγ contributes to the de novo formation of monolayers with increased permeability, but in a manner that does not involve apoptosis. CONCLUSIONS: Our study provides an optimized 3D model system for the integrated analysis of (real-time) intestinal epithelial paracellular permeability and morphogenesis, and reveals apoptosis as a pivotal mechanism underlying the enhanced permeability and altered morphogenesis in response to TNF, but not IFNγ.

Thialkalivibrio nitratireducens sp. nov., a nitrate-reducing member of an autotrophic denitrifying consortium from a soda lake.

Strain ALEN 2(T) was isolated from a mixed culture capable of complete autotrophic denitrification with thiosulfate as electron donor at pH 10; the mixed culture was enriched from sediment from Lake Fazda (Wadi Natrun, Egypt), a hypersaline alkaline lake. The isolate had large, non-motile, coccoid or barrel-shaped cells with intracellular sulfur globules. The bacterium was obligately chemolithoautotrophic. It grew with reduced sulfur compounds aerobically and anaerobically with nitrate as electron acceptor, nitrate being reduced to nitrite. It was moderately halophilic and obligately alkaliphilic. On the basis of genetic analysis and its unique phenotype, strain ALEN 2(T) (=DSM 14787(T)=UNIQEM 213(T)) is proposed as the type strain of a novel species of the genus Thialkalivibrio, Thialkalivibrio nitratireducens.

Thioalkalispira microaerophila gen. nov., sp. nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake.

An anaerobic enrichment medium (pH 10) with thiosulfate as electron donor and nitrate as electron acceptor was inoculated with sediment from soda lake Fazda (Wadi Natrun, Egypt); a novel strain, ALEN 1(T), was isolated from the subsequent enrichment culture. Cells of strain ALEN 1(T) had a spiral morphology (0.3-0.45 x 1-4 microm), were motile and had a single polar flagellum. Sphaeroplasts were formed by the cells and were rapidly lysed during prolonged aerobic incubation of cultures. Cells of strain ALEN 1(T) contained a membrane-associated yellow pigment. The metabolism of this novel organism was obligately chemolithoautotrophic, and thiosulfate or sulfide were utilized as electron donors. Washed cells of strain ALEN 1(T) oxidized thiosulfate, sulfide, polysulfide and elemental sulfur to sulfate. Best growth was observed when the strain was grown under micro-oxic conditions (1-2% O2 in gas phase), whereas growth was inhibited under fully oxic conditions. Nitrate was reduced to nitrite without growth of the novel organism, but other nitrogen oxides were not utilized as electron acceptors. Strain ALEN 1(T) was alkaliphilic and moderately halophilic. It grew between pH 8 and 10.4 (optimum around pH 10) with a salt concentration of between 0.3 and 1.5 M Na+ (optimum 0-5 M). The maximum growth rate (0.08 h(-1)) of the organism was achieved in a thiosulfate-limited micro-oxic continuous culture (pH 10). Phylogenetic analyses of the 16S rDNA sequences of strain ALEN 1(T) and its closest relatives demonstrated that this strain formed a deep branch within the gamma-Proteobacteria, with no obvious association to any described cluster of species/genera. On the basis of its unique physiological properties and distinct phylogenetic position, it is proposed that strain ALEN 1(T) (= DSM 14786(T) = UNICEM 212(T)) represents a novel genus within the gamma-Proteobacteria, for which the name Thioalkalispira is proposed. It is also proposed that the type species of this novel genus be named Thioalkalispira microaerophila.