Real-time high resolution 3D imaging of the lyme disease spirochete adhering to and escaping from the vasculature of a living host.

Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme borreliosis. They navigate efficiently through dense extracellular matrix and cross the blood-brain barrier by unknown mechanisms. Due to their slender morphology, spirochetes are difficult to visualize by standard light microscopy, impeding studies of their behavior in situ. We engineered a fluorescent infectious strain of Borrelia burgdorferi, the Lyme disease pathogen, which expressed green fluorescent protein (GFP). Real-time 3D and 4D quantitative analysis of fluorescent spirochete dissemination from the microvasculature of living mice at high resolution revealed that dissemination was a multi-stage process that included transient tethering-type associations, short-term dragging interactions, and stationary adhesion. Stationary adhesions and extravasating spirochetes were most commonly observed at endothelial junctions, and translational motility of spirochetes appeared to play an integral role in transendothelial migration. To our knowledge, this is the first report of high resolution 3D and 4D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.

Continuous thoracic epidural anesthesia improves gut mucosal microcirculation in rats with sepsis.

Microcirculatory dysfunction contributes significantly to tissue hypoxia and multiple organ failure in sepsis. Ischemia of the gut and intestinal hypoxia are especially relevant for the evolution of sepsis because the mucosal barrier function may be impaired, leading to translocation of bacteria and toxins. Because sympathetic blockade enhances intestinal perfusion under physiologic conditions, we hypothesized that thoracic epidural anesthesia (TEA) may attenuate microcirculatory perturbations during sepsis. The present study was designed as a prospective and controlled laboratory experiment to assess the effects of continuous TEA on the mucosal microcirculation in a cecal ligation and perforation model of sepsis in rats. Anesthetized Sprague-Dawley rats underwent laparotomy and cecal ligation and perforation to induce sepsis. Subsequently, either bupivacaine 0.125% (n = 10) or isotonic sodium chloride solution (n = 9) was continuously infused via the thoracic epidural catheter for 24 h. In addition, a sham laparotomy was carried out in eight animals. Intravital videomicroscopy was then performed on six to ten villi of ileum mucosa. The capillary density was measured as areas encircled by perfused capillaries, that is, intercapillary areas. The TEA accomplished recruitment of microcirculatory units in the intestinal mucosa by decreasing total intercapillary areas (1,317 +/- 403 vs. 1,001 +/- 236 microm2) and continuously perfused intercapillary areas (1,937 +/- 512 vs. 1,311 +/- 678 microm2, each P < 0.05). Notably, TEA did not impair systemic hemodynamic variables beyond the changes caused by sepsis itself. Therefore, sympathetic blockade may represent a therapeutic option to treat impaired microcirculation in the gut mucosa resulting from sepsis. Additional studies are warranted to assess the microcirculatory effects of sympathetic blockade on other splanchnic organs in systemic inflammation.

HIV-1 gp41 ectodomain enhances Cryptococcus neoformans binding to HBMEC.

Cryptococcus neoformans infection has significantly increased recently, particularly in AIDS patients and immunocompromised individuals. C. neoformans has a predilection to the brain, resulting in devastating meningoencephalitis. We have previously shown the invasion of C. neoformans into the human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. Here, we demonstrated that C. neoformans invasion of HBMEC was enhanced by HIV-1 gp41 protein. Peptide mapping defined its functional domain around the disulfide-bond linkage of gp41 molecule (a.a. 579-611). Recombinant protein gp41-I90 (a.a. 550-639) can also enhance the binding activity. The enhancement of C. neoformans binding to HBMEC is a strain-independent manner, suggesting that gp41 ectodomain peptide exerts its function directly on HBMEC. Importantly, the enhancement could be observed in mouse animal model. Our results suggest that HIV-1 gp41 ectodomain and C. neoformans may follow a similar invasion mechanism, possibly actin reorganization and/or membrane activation, during pathogen infections on HBMEC.

Interaction of a neurotropic strain of Borrelia turicatae with the cerebral microcirculation system.

Relapsing fever (RF) is a spirochetal infection characterized by relapses of a febrile illness and spirochetemia due to the sequential appearance and disappearance of isogenic serotypes in the blood. The only difference between isogenic serotypes is the variable major outer membrane lipoprotein. In the absence of specific antibody, established serotypes cause persistent infection. Studies in our laboratory indicate that another consequence of serotype switching in RF is a change in neuroinvasiveness. As the next step to elucidate this phenomenon, we studied the interaction of the neurotropic Oz1 strain of the RF agent Borrelia turicatae with the cerebral microcirculation. During persistent infection of antibody-deficient mice, we found that serotype 1 entered the brain in larger numbers and caused more severe cerebral microgliosis than isogenic serotype 2. Microscopic examination revealed binding of B. turicatae to brain microvascular endothelial cells in vivo. In vitro we found that B. turicatae associated with brain microvascular endothelial cells (BMEC) significantly more than with fibroblasts or arachnoidal cells. The binding was completely eliminated by pretreatment of BMEC with proteinase K. Using transwell chambers with BMEC barriers, we found that serotype 1 crossed into the lower compartment significantly better than serotype 2. Heat killing significantly reduced BMEC crossing but not binding. We concluded that the interaction of B. turicatae with the cerebral microcirculation involves both binding and crossing brain microvascular endothelial cells, with significant differences among isogenic serotypes.

Bacterial translocation, microcirculation injury and sepsis.

Sepsis is the result from a complex bacterial-host interaction, which is an often-fatal response when host protective molecular mechanisms designed to fight invading bacteria surpass the beneficial intensity to the point of causing injury to the host. Increasing evidences have implicated the bacterial translocation (BT) as the main source for the induction of sepsis, although the beneficial effect of BT process has been related to the development of the intestinal immune response by physiological interaction between bacteria and host. In this article, we examined evolving concepts concerning to BT and discussed about its potential role in the promotion of microcirculation injury, moreover, its possible participation in the sepsis induction. According to our data obtained from in-vivo BT animal-model, both bacterial overgrowth and bacterial pathogenic determinants seem to be major predisposing factors for the induction of BT. Besides, translocation of luminal bacteria through the lymphatic via elicits the activation of the GALT inflammatory response contributing to microcirculation injuries, and the haematological via of BT was responsible to the systemic bacterial spread. On other hand, the combination of BT process to the pre-existing host systemic infection played a crucial role in the worsening of the clinical outcome. In our understanding, studies concerning to intestinal immune response and the pathophysiology of bacterial-host interaction, under normal and disease conditions, seems to be the key elements to the development of therapeutic approaches towards sepsis.

CPS1, a homolog of the Streptococcus pneumoniae type 3 polysaccharide synthase gene, is important for the pathobiology of Cryptococcus neoformans.

The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37 degrees C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Delta strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans.

Approaches to bacterial RNA isolation and purification for microarray analysis of Escherichia coli K1 interaction with human brain microvascular endothelial cells.

We established a protocol for isolation of microarray-grade bacterial RNA from Escherichia coli K1 interacting with human brain microvascular endothelial cells. The extracted RNA was free of human RNA contamination. More importantly, microarray analysis demonstrated that no bias was introduced in the gene expression pattern during the RNA isolation procedure.

Escherichia coli K1 RS218 interacts with human brain microvascular endothelial cells via type 1 fimbria bacteria in the fimbriated state.

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

Capsule structure changes associated with Cryptococcus neoformans crossing of the blood-brain barrier.

Cryptococcus neoformans is a yeast responsible for disseminated meningoencephalitis in patients with cellular immune defects. The major virulence factor is the polysaccharide capsule. We took advantage of a relevant murine model of disseminated meningoencephalitis to study the early events associated with blood-brain barrier (BBB) crossing. Mice were sacrificed at 1, 6, 24, and 48 hours post-intravenous inoculation, and classical histology, electron microscopy, and double immunofluorescence were used to study tissues and yeasts. Crossing of the BBB occurred early after inoculation, did not involve the choroid plexus but instead occurred at the level of the cortical capillaries, and caused early and severe damage to the structure of the microvessels. Seeding of the leptomeninges was not the primary event but occurred secondary to leakage of cortical pseudocysts. Organ invasion was associated with changes in cryptococcal capsule structure and cell size, which differed in terms of magnitude and kinetics, depending on both the organs involved, and potentially, on the bed structure of the local capillary. The rapid changes in capsule structure could contribute to inability of the host immune response to control cryptococcal infection in extrapulmonary spaces.

Intravital fluorescence microscopy: a novel tool for the study of the interaction of Staphylococcus aureus with the microvascular endothelium in vivo.

BACKGROUND: The ability of Staphylococcus aureus to adhere to endothelial cells is a major prerequisite for the tissue-invasive stage of bacterial infection. METHODS: To develop a model for the study of endothelial attachment and detachment kinetics of S. aureus within the host's microvasculature in vivo, we labeled inactivated staphylococci with fluorescein isothiocyanate and investigated their interaction with the vascular endothelium of arterioles, capillaries, and venules in the dorsal skin-fold chamber of untreated and tumor necrosis factor (TNF)-alpha-treated hamsters by use of intravital fluorescence microscopy. RESULTS: During the first 20 min after injection, >99% of the bacteria were removed from the microvascular bloodstream. In parallel, single bacteria and bacterial clusters adhered to the endothelial lining of postcapillary venules and to nutritive capillaries. Bacterial adherence to the endothelium of arterioles was only rarely observed. TNF-alpha treatment significantly accelerated bacterial clearance and resulted in a significant increase of venular, but not arteriolar and capillary, bacterial adherence, indicating the venular endothelium to be the target structure for bacterial recruitment. CONCLUSION: The insights into host-pathogen interaction gained with this new in vivo model offer highly promising novel aspects of the understanding of infections caused by S. aureus.

Nitric oxide as a mediator of increased microvascular permeability during acute rickettsioses.

Rickettsiae primarily infect the microvascular endothelium, leading to changes in microvascular permeability that result in potentially severe pulmonary and cerebral edema. The mechanisms responsible for these changes are not well understood. One potential mechanism of increased vascular permeability is the anti-rickettsial nitric oxide response described by Walker and colleagues. We hypothesized that anti-rickettsial levels of nitric oxide adversely affects microvascular permeability in vitro. To this end we sought to describe the effects of exogenous nitric oxide on the proliferation of intracellular rickettsiae while monitoring the transendothelial electrical resistance as a measure of endothelial barrier integrity. It was determined that the addition of the NO-donor DETA NONOate at certain levels results in a dose-dependent change in electrical resistance across the monolayer while effectively limiting the number of intracellular rickettsiae in human microvascular endothelial cells. The data presented support the idea that nitric oxide produced by infected endothelial cells may be contributing to the changes in vascular permeability that occur during acute rickettsioses. Future experiments aim to elaborate on these results in a model that more clearly depicts the in vivo response as well as to describe the changes that occur with respect to interendothelial junctions.

Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.

Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain. The mechanism(s) by which C. neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to C. neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of C. neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of C. neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h. C. neoformans was found in the brain parenchyma away from the vessels by 22 h. Association of C. neoformans with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that C. neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.

Human intestinal microvascular endothelial cells express Toll-like receptor 5: a binding partner for bacterial flagellin.

Bacterial flagellin has recently been identified as a ligand for Toll-like receptor 5 (TLR5). Human sites known to specifically express TLR5 include macrophages and gastric and intestinal epithelium. Because infection of intestinal epithelial cells with Salmonella leads to an active transport of flagellin to the subepithelial compartment in proximity to microvessels, we hypothesized that human intestinal endothelial cells functionally express TLR5, thus enabling an active inflammatory response upon binding of translocated flagellin. Endothelial expression of TLR5 in human macro- and microvascular endothelial cells was examined by RT-PCR, immunoblot analysis, and immunofluorescence. Endothelial expression of TLR5 in vivo was verified by immunohistochemistry. Endothelial modulation of ICAM-1 expression was quantitated using flow cytometry, and leukocyte transmigration in vitro was assessed by an endothelial transmigration assay. Epithelial-endothelial cellular interactions upon infection with viable Salmonella were investigated using a coculture system in vitro. We found that Salmonella-infected intestinal epithelial cells induce endothelial ICAM-1 expression in cocultured human endothelial cells. Both macro- (HUVEC) and microvascular endothelial cells derived from human skin (human dermal microvascular endothelial cell 1) and human colon (human intestinal microvascular endothelial cells) were found to express high constitutive amounts of TLR5 mRNA and protein. These findings were paralleled by strong immunoreactivity for TLR5 of normal human colonic microvessels in vivo. Furthermore, incubation of human dermal microvascular endothelial cells with flagellin from clinical isolates of Escherichia and Salmonella strains led to a marked up-regulation of ICAM-1, as well as to an enhanced leukocyte transendothelial cell migration. These results suggest that endothelially expressed TLR5 might play a previously unrecognized role in the innate immune response toward bacterial Ags.

Invasion of porcine brain microvascular endothelial cells by Streptococcus suis serotype 2.

Streptococcus suis is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of S. suis infection has not been completely defined. However, in order to cause meningitis, S. suis has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of S. suis serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European S. suis serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of S. suis seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable S. suis could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by S. suis required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by S. suis.

Antibodies present in normal human serum inhibit invasion of human brain microvascular endothelial cells by Listeria monocytogenes.

Listeria monocytogenes causes meningitis and encephalitis in humans and crosses the blood-brain barrier by yet unknown mechanisms. The interaction of the bacteria with different types of endothelial cells was recently analyzed, and it was shown that invasion into, but not adhesion to, human brain microvascular endothelial cells (HBMEC) depends on the product of the inlB gene, the surface molecule InlB, which is a member of the internalin multigene family. In the present study we analyzed the role of the medium composition in the interaction of L. monocytogenes with HBMEC, and we show that invasion of HBMEC is strongly inhibited in the presence of adult human serum. The strong inhibitory activity, which is not present in fetal calf serum, does not inhibit uptake by macrophage-like J774 cells but does also inhibit invasion of Caco-2 epithelial cells. The inhibitory component of human serum was identified as being associated with L. monocytogenes-specific antibodies present in the human serum. Human newborn serum (cord serum) shows only a weak inhibitory activity on the invasion of HBMEC by L. monocytogenes.