Vibrio cholerae-induced inflammation in the neonatal mouse cholera model.

Vibrio cholerae is the causative agent of the acute diarrheal disease of cholera. Innate immune responses to V. cholerae are not a major cause of cholera pathology, which is characterized by severe, watery diarrhea induced by the action of cholera toxin. Innate responses may, however, contribute to resolution of infection and must be required to initiate adaptive responses after natural infection and oral vaccination. Here we investigated whether a well-established infant mouse model of cholera can be used to observe an innate immune response. We also used a vaccination model in which immunized dams protect their pups from infection through breast milk antibodies to investigate innate immune responses after V. cholerae infection for pups suckled by an immune dam. At the peak of infection, we observed neutrophil recruitment accompanied by induction of KC, macrophage inflammatory protein 2 (MIP-2), NOS-2, interleukin-6 (IL-6), and IL-17a. Pups suckled by an immunized dam did not mount this response. Accessory toxins RtxA and HlyA played no discernible role in neutrophil recruitment in a wild-type background. The innate response to V. cholerae deleted for cholera toxin-encoding phage (CTX) and part of rtxA was significantly reduced, suggesting a role for CTX-carried genes or for RtxA in the absence of cholera toxin (CTX). Two extracellular V. cholerae DNases were not required for neutrophil recruitment, but DNase-deficient V. cholerae caused more clouds of DNA in the intestinal lumen, which appeared to be neutrophil extracellular traps (NETs), suggesting that V. cholerae DNases combat NETs. Thus, the infant mouse model has hitherto unrecognized utility for interrogating innate responses to V. cholerae infection.

Fasting before Intra-Gastric Dosing with Antigen Improves Intestinal Humoral Responses in Syrian Hamsters.

Oral vaccines, unlike injected, induce intestinal secretory immunoglobulin A (sIgA) mimicking our natural defense against gut pathogens. We previously observed sIgA responses after administering the Clostridioides difficile colonisation factor CD0873 orally in enteric capsules to hamsters. Enteric-coated capsules are designed to resist dissolution in the stomach and disintegrate only at the higher pH of the small intestine. However, the variable responses between animals led us to speculate suboptimal transit of antigens to the small intestine. The rate of gastric emptying is a controlling factor in the passage of oral drugs for subsequent availability in the small intestine for absorption. Whilst in humans, food delays gastric emptying, in rats, capsules can empty quicker from fed stomachs than from fasted. To test in hamsters if fasting improves the delivery of antigens to the small intestine, as inferred from the immune responses generated, 24 animals were dosed intragastrically with enteric capsules containing recombinant CD0873. Twelve hamsters were fasted for 12 h prior to each dose and the other 12 fed. Significantly higher sIgA titres, with significantly greater bacterial-adherence-blocking activity, were detected in small intestinal lavages in the fasted group. We conclude that fasting in hamsters improves intestinal delivery leading to more robust responses.

Immunization of mice with vibrio cholerae outer-membrane vesicles protects against hyperinfectious challenge and blocks transmission.

BACKGROUND: Vibrio cholerae excreted by cholera patients is "hyperinfectious" (HI), which can be modeled by passage through infant mice. Immunization of adult female mice with V. cholerae outer-membrane vesicles (OMVs) passively protects suckling mice from challenge. Although V. cholerae is unable to colonize protected pups, the bacteria survive passage and have the potential to be transmitted to susceptible individuals. Here, we investigated the impact of OMV immunization and the HI state on V. cholerae transmission. METHODS: Neonatal mice suckled by OMV- or sham-immunized dams were challenged with HI V. cholerae. The infectivity of spatially and temporally separate V. cholerae populations obtained from infected naive or protected pups was tested. Recombination-based in vivo expression technology was used to assess virulence gene expression within these populations. RESULTS: OMV immunization significantly reduced colonization of neonates challenged with HI V. cholerae. Vibrio cholerae that had colonized the naive host was HI, whereas V. cholerae excreted by neonates born to OMV-immunized dams, although viable, was hypoinfectious and failed to fully induce virulence gene expression. CONCLUSIONS: OMV immunization can significantly reduce the V. cholerae burden upon challenge with HI V. cholerae and can also block transmission from immune mice by reducing the infectivity of shed bacteria.

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation.

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae.

Mucosal immunization with Vibrio cholerae outer membrane vesicles provides maternal protection mediated by antilipopolysaccharide antibodies that inhibit bacterial motility.

Vibrio cholerae is the causative agent of cholera, a severe diarrheal disease that remains endemic in many parts of the world and can cause outbreaks wherever sanitation and clean water systems break down. Prevention of disease could be achieved through improved sanitation and clean water provision supported by vaccination. V. cholerae serogroup O1 is the major cause of cholera; O1 serotypes Inaba and Ogawa have similar disease burdens, while O139 is the only non-O1 serogroup to cause epidemics. We showed previously that immunization of adult female mice with purified V. cholerae outer membrane vesicles (OMVs) elicits an antibody response that protect neonates from oral V. cholerae challenge and that suckling from an immunized dam accounts for the majority of protection from V. cholerae colonization. Here we report that lipopolysaccharide (LPS) is the major OMV protective antigen. Mucosal immunization with OMVs from Inaba or Ogawa provides significant cross-serotype protection from V. cholerae colonization, although serotype-specific antigens are dominant. OMVs from O1 or O139 do not provide cross-serogroup protection, but by immunization with a mixture of O1 and O139 OMVs, cross-serogroup protection was achieved. Neonatal protection is not associated with significant bacterial death but may involve inhibition of motility, as antibodies from OMV-immunized mice inhibit V. cholerae motility in vitro, with trends that parallel in vivo protection. Motility assays also reveal that a higher antibody titer is required to immobilize O139 compared to O1, a phenotype that is O139 capsule dependent.

Effects of fibroblast growth factors on the differentiation of the pulmonary progenitors from murine embryonic stem cells.

The fibroblast growth factors (FGFs) play an important role in the development of embryonic lung. In this study, we investigated the effects of mainly FGF 1, 2, and 10 at concentrations selected on the basis of data obtained from previous in vitro culture on the derivation of the pulmonary progenitors from murine embryonic stem cells cultured on gelatin or Matrigel-coated plates. For cells cultured on a gelatin-coated plate, high concentrations of FGF1 were found to enhance the expression of mRNAs for SPC and CC10, markers of distal airway epithelium, while high levels of FGF2 decreased the expression of RNAs for not only SPC, CC10 but also for the additional markers SPD and aquaporin 5. FGF10 at all tested concentrations was found to have no effect on the differentiation of pneumocytes when ESCs were grown on gelatin-coated plates. However, when differentiation was performed on Matrigel-coated plates, the addition of 60 ng/ml FGF10 enhanced the expression of pneumocyte markers, suggesting a synergic effect of FGF10 and extracellular matrix. In conclusion, growth factors were proven to be effective in the differentiation of pulmonary progenitors from mESCs. The need of signals from extracellular matrix proteins depends on the growth factors supplemented.

Complexity of rice-water stool from patients with Vibrio cholerae plays a role in the transmission of infectious diarrhea.

At the International Centre for Diarrhoeal Disease Research, Bangladesh, one-half of the rice-water stool samples that were culture-positive for Vibrio cholerae did not contain motile V. cholerae by standard darkfield microscopy and were defined as darkfield-negative (DF(-)). We evaluated the host and microbial factors associated with DF status, as well as the impact of DF status on transmission. Viable counts of V. cholerae in DF(-) stools were three logs lower than in DF(+) stools, although DF(-) and DF(+) stools had similar direct counts of V. cholerae by microscopy. In DF(-) samples, non-V. cholerae bacteria outnumbered V. cholerae 10:1. Lytic V. cholerae bacteriophage were present in 90% of DF(-) samples compared with 35% of DF(+) samples, suggesting that bacteriophage may limit culture-positive patients from producing DF(+) stools. V. cholerae in DF(-) and DF(+) samples were found both planktonically and in distinct nonplanktonic populations; the distribution of organisms between these compartments did not differ appreciably between DF(-) and DF(+) stools. This biology may impact transmission because epidemiological data suggested that household contacts of a DF(+) index case were at greater risk of infection with V. cholerae. We propose a model in which V. cholerae multiply in the small intestine to produce a fluid niche that is dominated by V. cholerae. If lytic phage are present, viable counts of V. cholerae drop, stools become DF(-), other microorganisms bloom, and cholera transmission is reduced.

Characterization of Vibrio cholerae outer membrane vesicles as a candidate vaccine for cholera.

Outer membrane vesicles (OMVs) offer a new approach for an effective cholera vaccine. We recently demonstrated that immunization of female mice with OMVs induces a long-lasting immune response and results in protection of their neonatal offspring from Vibrio cholerae intestinal colonization. This study investigates the induced protective immunity observed after immunization with OMVs in more detail. Analysis of the stomach contents and sera of the neonates revealed significant amounts of anti-OMV immunoglobulins (Igs). Swapping of litters born to immunized and nonvaccinated control mice allowed us to distinguish between prenatal and neonatal uptakes of Igs. Transfer of Igs to neonates via milk was sufficient for complete protection of the neonates from colonization with V. cholerae, while prenatal transfer alone reduced colonization only. Detection of IgA and IgG1 in the fecal pellets of intranasally immunized adult mice indicates an induced immune response at the mucosal surface in the gastrointestinal tract, which is the site of colonization by V. cholerae. When a protocol with three intranasal immunizations 14 days apart was used, the OMVs proved to be efficacious at doses as low as 0.025 microg per immunization. This is almost equivalent to OMV concentrations found naturally in the supernatants of LB-grown cultures of V. cholerae. Heterologous expression of the periplasmic alkaline phosphatase (PhoA) of Escherichia coli resulted in the incorporation of PhoA into OMVs derived from V. cholerae. Intranasal immunization with OMVs loaded with PhoA induced a specific immune response against this heterologous antigen in mice. The detection of an immune response against this heterologously expressed protein is a promising step toward the potential use of OMVs as antigen delivery vehicles in vaccine design.

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles.

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5' region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, sigma(E), suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract.

The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.

Cell attachment properties and infectivity of host-adapted and environmentally adapted Citrobacter rodentium.

Citrobacter rodentium belongs to a family of extracellular enteric pathogens that include enterohaemorrhagic and enteropathogenic Escherichia coli, which colonises the gastrointestinal mucosa by the attaching and effacing (A/E) mechanism. We previously described the appearance of a 'hyper-infectious' state after passage of C. rodentium through the murine gastrointestinal tract. Here we report that host-adapted C. rodentium is able to efficiently adhere and trigger actin polymerisation on cultured epithelial cells. Consistent with these observations we recorded higher levels of expression of genes carried on the LEE pathogenicity island and type III secretion system effector genes carried on prophages compared with in vitro-grown bacteria; importantly, the level of ler gene expression was unchanged. These phenotypes were lost after shed C. rodentium was adapted to the external environment. Upon exposure of C57Bl/6 mice, environmentally adapted C. rodentium was no longer infectious at the low doses associated with host-adapted bacteria and the bacteria were found to be localised in the caecal patch in a similar way to C. rodentium cultured in laboratory media. Thus, the 'hyper-infectious' host-adapted state, allowing efficient transmission and colonisation of naive hosts, is transient in nature and gradually lost after shedding into the environment.

Effects of growth factors on the differentiation of murine ESC into type II pneumocytes.

We have previously shown that embryonic stem cells (ESC) can be directed to differentiate into alveolar type II cells by provision of a serum-free medium designed for in vitro maintenance of mature alveolar epithelial cells (small airway growth medium: SAGM), although the target cell yield was low. SAGM comprises a basal serum-free medium (SABM) plus a series of defined supplements. In order to try increase the proportion of pneumocytes in differentiated cultures, we aimed in this study to determine the effects on murine ESC of each of the individual growth factors in SAGM. In accordance with our previous reports, expression of surfactant protein C (SPC) and its mRNA was used to monitor differentiation of type II pneumocytes. Surprisingly, we found that addition of each factor separately to SABM decreased the expression of SPC mRNA when compared with the effect of SABM alone. Thus, it seems that the observed enhancement by SAGM of pneumocyte differentiation from murine ESC can, in fact, be attributed to the provision of a serum-free environment.

Pulmonary epithelium.

Repair or regeneration of defective lung epithelium would be of great therapeutic potential. Cellular sources for such repair have long been searched for within the lung, but the identification and characterization of stem or progenitor cells have been hampered by the complexity and cellular heterogeneity of the organ. In recent years, various pulmonary cells have been identified that meet the criteria for stem cells but it remains to be seen how far manipulation of these tissue-specific cell pools can upregulate epithelial repair. The initial excitement that greeted the results of animal experiments showing cells of bone marrow origin in murine lung has been tempered by more recent data suggesting that the cells do not repair pulmonary epithelium. However, there are reports of engraftment of bone marrow-derived cells in human lung, albeit at a low level, so the administration of cell therapy via the circulation, for repair and/or gene delivery, needs further investigation. The potential of human embryonic stem cells to generate any cell, tissue, or organ on demand for tissue repair or replacement is promising to revolutionize the treatment of human disease. Although some headway has been made into making pulmonary epithelium from these stem cells, human embryonic stem cell technology is still in its infancy and many technical, safety, and ethical hurdles must be cleared before clinical trials can begin. This chapter focuses on the potential role of stem cells in future approaches to lung repair and regeneration.

Upregulated genes in sporadic, idiopathic pulmonary arterial hypertension.

BACKGROUND: To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH). METHODS: Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test. RESULTS: We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase. CONCLUSION: Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.

Antibiotics reduce the growth rate and differentiation of embryonic stem cell cultures.

Embryonic stem cells (ESCs) are being investigated increasingly for their potential as a cell source for tissue engineering. Antibiotics are regularly used in ESC culture media to control contamination, although they can be cytotoxic and interfere with protein synthesis. Our aim was to examine the effects of the frequently used antibiotics gentamicin and combined penicillin and streptomycin on ESC culture using differentiation of murine ESC into type II pneumocytes as a model. Antibiotics reduced the expression of the specific marker for type II pneumocytes, SPC mRNA, by up to 60%. We also identified an adverse effect on the growth rate of differentiating embryoid bodies, causing a significant ( p < 0.05) reduction of up to 40%, and an increase in population doubling time of up to 48%. No contamination was seen in any of the cultures. Our findings suggest that the routine use of antibiotics in ESC culture should be avoided as it may reduce the efficiency of the culture system.

Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment.

We have previously induced differentiation of embryonic stem cells (ESC) to specific phenotypes by manipulating the culture conditions, including the use of indirect co-culture. In this study, we hypothesized that co-culture with primary chondrocytes can induce human embryonic stem cells (hESC) to differentiate towards the chondrocyte lineage. Co-cultures of hESC and chondrocytes were established using well inserts, with control comprising hESC grown alone or with fibroblasts. After 28 days, after removal of the chondrocyte inserts, hESC differentiation was assessed, by morphology, immunocytochemistry, and reverse transcription polymerase chain reaction. hESC, co-cultured or grown alone, were also implanted into SCID mice on a poly-D, L-lactide scaffold, harvested 35 days later and assessed in the same way. hESC co-cultured with chondrocytes formed colonies and secreted extracellular matrix containing glycosaminoglycans (GAG). Quantitative assay showed increased synthesis of sulfated GAG in co-culture as compared with control hESC grown alone for the same period (p < 0.0001). In addition, co-cultured hESC expressed Sox 9 and collagen type II, unlike control hESC. Co-culture with fibroblasts did not induce chondrogenic differentiation. The implanted constructs with co-cultured hESC contained significantly more type II collagen (p < 0.01), type I collagen (p < 0.05), total collagen (p < 0.01), and GAG (p < 0.01) than those with hESC grown alone. Thus, we show for the first time differentiation of hESC to chondrocytes. Our results confirm the potential of the culture micro-environment to influence ESC differentiation and could provide the basis for future generation of chondrogenic cells for use in tissue repair and increase our understanding of the mechanisms that direct differentiation.

Derivation of distal airway epithelium from human embryonic stem cells.

The pluripotency of embryonic stem cells (ESC) is offering new opportunities in tissue engineering and cell therapy. We have shown previously that alveolar epithelial cells, specifically type II pneumocytes, can be derived from murine ESC and hypothesized that a similar protocol could be used successfully on human ESC. Undifferentiated human ESC were induced to form embryoid bodies that were transferred into adherent culture conditions and grown in a medium designed for the maintenance of mature small airway epithelium. On inverted microscopy, the generated cells showed the cobblestone-like morphology of epithelium. The presence of surfactant protein C, a specific marker of type II pneumocytes, and its corresponding RNA were demonstrated by immunostaining and reverse transcription polymerase chain reaction, respectively. Electron microscopy revealed frequent cells with the typical ultrastructure of type II pneumocytes. This study provides evidence for in vitro induction of the differentiation from human ESC of alveolar type II cells, which have the potential for therapeutic use or construction of an in vitro model of human lung.

Stem cells and tissue engineering: past, present, and future.

Tissue engineering is an interdisciplinary field that brings together the principles of the life sciences and medicine with those of engineering. The increase in its development over the past decade has resulted from a variety of factors; advances in genomics and proteomics, the advent of new biomaterials as potential templates for tissue growth, improvements in bioreactor design, and increased understanding of healing processes. Possibly the greatest contribution has come from our increased knowledge and understanding of stem cell biology, which is paving the way for the generation of unlimited cells of specific phenotypes for incorporation into engineered tissue constructs. Thus, tissue engineering approaches for expanding and engrafting the differentiated progeny of embryonic, fetal, or adult stem cells have major potential for tissue repair and will make a major contribution to medicine in the 21st century.

Derivation and characterization of alveolar epithelial cells from murine embryonic stem cells in vitro.

We present a protocol that has been developed for induction of the differentiation of murine embryonic stem (ES) cells to alveolar type II cells. With this protocol, undifferentiated murine ES cells are induced to form embryoid bodies (EBs). The 10-d-old EBs are transferred to adherent culture conditions and are fed with high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum, 2 mM L-glutamine, and 0.1 mM 2-mercaptoethanol for 20 d without splitting. Then, the cells are fed with a medium designed for the maintenance and growth of mature distal airway epithelial cells (small airway growth medium, SAGM) for 3 d. Characterization of the alveolar type II cells was done using real-time reverse transcriptase polymerase chain reaction detection of surfactant protein C mRNA and immunocytochemical detection of prosurfactant protein C. Real-time reverse transcriptase polymerase chain reaction revealed that SAGM increases the mRNA expression level of SPC by a factor of 8 when compared to that of cells grown in supplemented high-glucose DMEM (p < 0.05, Student t-test). Immunocytochemistry revealed that proSPC-expressing cells comprised 2.8 +/- 0.23% of the total cell population in SAGM-treated samples and 0.5 +/- 0.1% in samples treated with supplemented high-glucose DMEM (p < 0.05, chi2 test).