Bacterial Ingestion Research Articles

Poly(ethylene glycol)-polyacrylate copolymers modified to control adherent monocyte-macrophage physiology: interactions with attaching Staphylococcus epidermidis or Pseudomonas aeruginosa bacteria.

The ability of various surface modifications of poly(ethylene glycol)-graft-polyacrylate (PEG-g-PA) copolymers (tethered adhesion peptides and fragments of monoclonal antibodies) to modulate monocyte-macrophage cell interactions with surface colonizing bacteria is reported. The PEG-g-PA copolymers were made to inhibit nonspecific protein and cellular adhesion. The copolymers were then covalently modified with either cell adhesion peptides (YRGDS, YEILDV, or YRGES) or fragments of antibodies to monocyte-macrophage integrin receptors (anti-VLA4, anti-beta(1), anti-beta(2), and anti-CD64), which are known to enhance macrophage adhesion and perhaps modulate their activation. Cytokine expression and phagocytosis response by surface adherent monocyte-macrophages to Staphylococcus epidermidis and Pseudomonas aeruginosa bacteria were quantified. The cytokine expression (interleukins 6 and 1 beta) of adherent macrophages in response to the modified polymers only and to bacterial challenges were quantified by dynamic ELISA assays. The adherent macrophage phagocytic response (oxidative burst) to various materials is compared to oxidative responses to both opsonized and nonopsonized S. epidermidis and P. aeruginosa bacteria. The efficiency of adherent macrophages to ingest and kill both species was determined using radiolabeled and fluorescent labeled bacterial cell ingestion studies as a function of the PEG-g-PA surface modification. Materials modified with adhesion peptides marginally enhanced (2x) macrophage attachment versus controls but, upon bacterial challenges, these materials predisposed adherent macrophages to overexpress proinflammatory cytokines and to exhibit a significant phagocytic response. Conversely, PEG-g-PA materials modified by fragments of monoclonal antibodies significantly enhanced (7x) macrophage adhesion but, upon bacterial challenge, "per cell" cytokine expression levels were reduced compared to peptide modified materials. Macrophages adhering to antibody fragment modified surfaces also exhibited sustained enhanced phagocytic response and higher bacterial killing efficiencies when compared with peptide modified materials.

Effects of prey abundance and light intensity on nutrition of a mixotrophic flagellate and its competitive relationship with an obligate heterotroph

The mixotroph Poterioochromonas malhamensis was grown in batch and semicontinuous cultures in order to examine the dependence of phagotrophy versus phototrophy at bacterial densities similar to those found in oligo- and mesotrophic lakes in combination with differing light regimes. In addition, the growth rates and biomass accumulation of the mixotroph were compared to those of the heterotroph Spumella elongata grown under similar conditions. In P. malhamensis, primary production rates and cell-specific chlorophyll a concentrations were significantly higher when grown at low bacterial densities, and the carbon cell-(1) gained by photosynthesis corresponded to 79% of the carbon cell-(1) gained by bacterial ingestion. Furthermore, due to a longer exponential growth phase, cell densities and biomass of P. malhamensis were significantly higher when the mixotroph was cultured in high light than in darkness. As a result, the cumulative biomass of the mixotroph was significantly higher in the light treatments than the biomass of the heterotroph at similar bacterial densities. These results suggest that photosynthesis may contribute more to the nutrition of P. Malhamensis in lakes with low bacterial abundances than previously suggested, and that the mixotroph's capacity for photosynthesis enables it to dominate in terms of biomass over the heterotroph.

Taxon-specific and seasonal variations in flagellates grazing on heterotrophic bacteria in the oligotrophic Lake Annecy – importance of mixotrophy

We investigated the taxonomic composition of flagellate assemblages and taxon-specific bacterial grazing rates of heterotrophic and mixotrophic flagellates in the oligotrophic Lake Annecy (France). The comparison of bacterial grazing rates to bacterial production demonstrated a high transfer efficiency from the bacterial compartment up to flagellates. Per capita grazing rates ranged from 1.2×10 3 to 5.1×10 6 bacteria l −1 h −1 for heterotrophic flagellates, and from 4.8×10 6 to 6.8×10 7 bacteria l −1 h −1 for mixotrophic flagellates. The main bacterial grazers were Katablepharis within heterotrophic flagellates and Dinobryon within mixotrophic flagellates. Our results show that bacterial ingestion by a given flagellate taxon changed seasonally and could vary up to 30-fold. We also provide evidence that mixotrophic flagellates represent an important link in the flux of materials through planktonic food webs in Lake Annecy, suggesting that the introduction of mixotrophs within functional groups could improve our understanding of carbon flux pathways.

Diurnal and seasonal variations in grazing by bacterivorous mixotrophs in an oligotrophic clearwater lake

The biomass and bacterial ingestion rates of mixotrophic phytoflagellates were examined on diurnal and seasonal scales in oligotrophic Lake Skarlen (S. Sweden). Ingestion rates were measured using fluorescent microspheres in situ on three depths every 6(th) hour during 24 hours in summer in order to quantify diurnal variations and once at midday during autumn, winter and spring. Mixotrophic phytoflagellates composed a large part of the phytoplankton biomass (18-31 %) throughout the year. Nonetheless, calculations suggest that the mixotrophs only consumed between 0.5 and 4.4 % of the bacterial carbon production, with the highest consumption percentage in winter under ice. Two groups of algae, Cryptomonas spp. and Dinobryon spp., were commonly found with ingested prey and both exhibited significant diurnal differences in bacterial ingestion rates with the highest mean rates measured in late afternoon (0.50 and 0.27 bacteria cell(-1) hour(-1), respectively) and very low rates during night (0.09 and 0.06 bacteria cell(-1) hour). In Cryptomonas spp., ingestion rates also varied.somewhat over the year and bacterial ingestion was highest during winter (0.57 bacteria cell(-1) hour), possibly as a result of the lengthy impaired light climate. (Less)

Bacteria Ingestion by Blowfly Larvae: An in vitro Study

Background: Maggot debridement therapy is the medical use of live fly larvae for cleaning chronic and infected wounds, removing devitalized tissue and decreasing the risk of infection. Maggot-derived proteins are able to kill bacteria, and proteolytic enzymes are responsible for the liquefying of necrotic tissue. Objective: The aim of this study is to investigate bacterial ingestion by larvae roaming free on bacterial agar, compared to those larvae contained within vinyl bags. Methods: Free-roaming sterile larvae of Lucilia sericata and larvae contained in vinyl bags were fed on Escherichia coli producing green fluorescent protein (GFP). The time interval to the onset of fluorescent maggots was determined. At different time intervals, maggots were sacrificed, washed in sterile saline, sagittally cut in frozen sections and examined under a microscope with UV light. Results: After feeding on GFP-labelled E. coli, maggots roaming free on bacterial lawn agar demonstrated fluorescence after 3 min, maggots entrapped in vinyl bags after 25 min. In the sagittal frozen sections, the highest fluorescent intensity was detected in the larvae’s rostral part of the alimentary tract, the crop and the anterior midgut. Conclusion: In an in vitro setting, digestion and ingestion of whole or disintegrated bacteria is accomplished within minutes. The vinyl bag’s permeable membrane clearly causes a delay of this process.

Safety Aspects ofLactobacillusandBifidobacteriumSpecies Originating from Human Oro-gastrointestinal Tract or from Probiotic Products

Probiotics are live microbial preparations, which have documented health benefits for the consumers by maintaining or improving their intestinal microbiota balance. Established probiotic effects include improved lactose digestion, modulation of gut microbiota, immune modulation, reduced duration of rotavirus diarrhoea, changes in biomarkers such as harmful faecal enzyme activities, alleviation of atopic dermatitis symptoms in babies, and positive effects against superficial bladder cancer and cervical cancer. Most bacteria that have probiotic properties belong to the genera Lactobacillus and Bifidobacterium . Since probiotic consumption involves ingestion of large numbers of viable bacterial cells (daily dosage between log 9 and log 11 CFU) safety aspects of probiotic consumption are of utmost importance. Knowledge on the survival of probiotics in the GI-tract, their translocation and colonisation properties and the fate of probiotic-derived active components is important for the evaluation of possible negative and positive effects of probiotic consumption. Assessing the risks of probiotic consumption can be a very expensive and time-consuming task. While considering the risk of probiotic consumption we have to keep in mind that lactic acid bacteria have been globally consumed in a myriad of fermented food varieties (milk, meat, vegetable and cereal products) for a very long time without an indication that they could be generally harmful to the consumers’ health. In Finland lactobacilli strains isolated from bacteremia have been systematically characterised. Although the yearly consumption of probiotic products containing lactobacilli has increased during the last 10 years, the incidence of lactobacillemia has not increased. There is one local infection case with an indistinguishable isolate to L. rhamnosus GG. The extensive follow-up period indicates that the risk of serious infection by one single probiotic strain is very low. Simultaneously, it is difficult to estimate all the health benefits the same probiotic strain has implemented, also in immunocompromised patients. However, no live bacterium can be guaranteed for a zero risk in each individual host since the outcome of bacterial ingestion (passage through GI-tract, colonisation, infection) is determined both by the host and the bacterium. Keywords: Lactobacillus , Bifidobacterium , infection, bacteremia, sepsis, endocarditis.

Salmonella enterica serovar typhi modulates cell surface expression of its receptor, the cystic fibrosis transmembrane conductance regulator, on the intestinal epithelium.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an epithelial receptor mediating the translocation of Salmonella enterica serovar Typhi to the gastric submucosa. Since the level of cell surface CFTR is directly related to the efficiency of serovar Typhi translocation, the goal of this study was to measure CFTR expression by the intestinal epithelium during infection. CFTR protein initially present in the epithelial cell cytoplasm was rapidly trafficked to the plasma membrane following exposure to live serovar Typhi or bacterial extracts. CFTR-dependent bacterial uptake by epithelial cells increased (>100-fold) following CFTR redistribution. The bacterial factor which triggers CFTR redistribution is heat and protease sensitive. These data suggest that serovar Typhi induces intestinal epithelial cells to increase membrane CFTR levels, leading to enhanced bacterial ingestion and submucosal translocation. This could be a key, early step in the infectious process leading to typhoid fever.

Safety Aspects of Lactobacillus and Bifidobacterium Species Originating from Human Oro-gastrointestinal Tract or from Probiotic Products

Probiotics are live microbial preparations, which have documented health benefits for the consumers by maintaining or improving their intestinal microbiota balance. Established probiotic effects include improved lactose digestion, modulation of gut microbiota, immune modulation, reduced duration of rotavirus diarrhoea, changes in biomarkers such as harmful faecal enzyme activities, alleviation of atopic dermatitis symptoms in babies, and positive effects against superficial bladder cancer and cervical cancer. Most bacteria that have probiotic properties belong to the genera Lactobacillus and Bifidobacterium . Since probiotic consumption involves ingestion of large numbers of viable bacterial cells (daily dosage between log 9 and log 11 CFU) safety aspects of probiotic consumption are of utmost importance. Knowledge on the survival of probiotics in the GI-tract, their translocation and colonisation properties and the fate of probiotic-derived active components is important for the evaluation of possible negative and positive effects of probiotic consumption. Assessing the risks of probiotic consumption can be a very expensive and time-consuming task. While considering the risk of probiotic consumption we have to keep in mind that lactic acid bacteria have been globally consumed in a myriad of fermented food varieties (milk, meat, vegetable and cereal products) for a very long time without an indication that they could be generally harmful to the consumers’ health. In Finland lactobacilli strains isolated from bacteremia have been systematically characterised. Although the yearly consumption of probiotic products containing lactobacilli has increased during the last 10 years, the incidence of lactobacillemia has not increased. There is one local infection case with an indistinguishable isolate to L. rhamnosus GG. The extensive follow-up period indicates that the risk of serious infection by one single probiotic strain is very low. Simultaneously, it is difficult to estimate all the health benefits the same probiotic strain has implemented, also in immunocompromised patients. However, no live bacterium can be guaranteed for a zero risk in each individual host since the outcome of bacterial ingestion (passage through GI-tract, colonisation, infection) is determined both by the host and the bacterium. Keywords: Lactobacillus , Bifidobacterium , infection, bacteremia, sepsis, endocarditis.

Nutrient Acquisition and Population Growth of a Mixotrophic Alga in Axenic and Bacterized Cultures.

Axenic growth of a mixotrophic alga, Ochromonas sp., was compared in several inorganic and organic media, and in the presence of live bacteria under nutrient-replete and low-nutrient conditions. Axenic growth in the light was negligible in inorganic media with or without the addition of glucose. Addition of vitamins increased growth rate, but average cell size declined, resulting in no net increase in biomass. Supplementing axenic cultures with a more complex organic substrate resulted in moderate growth and higher maximal abundance (and biomass) than in the inorganic media with added vitamins. The absence of light did not greatly affect population growth rate in the presence of complex dissolved organic compounds, although cell size was significantly greater in the light than in the dark. The highest growth rates for the alga (up to 2.6 d-1) were measured in treatments containing live bacteria. Increases in cell number of Ochromonas sp. in the presence of bacterial prey were similar in the light and dark, although chloroplast and cell sizes differed. Bacterial abundance was reduced and dissolved phosphorus and ammonia were rapidly released in bacterized cultures in the light and dark, indicating high rates of bacterial ingestion and suggesting an inability of the alga to store or utilize N and P in excess of the quantities required for heterotrophic growth. Low-nutrient conditions in the presence of bacteria were promoted by adding glucose to stimulate bacterial growth and the uptake of N and P released by algal phagotrophy. Subsequent decreases in dissolved N and P following the addition of glucose corresponded to a second period of rapid growth of the alga in both light and dark. This result, combined with evidence for slow axenic growth of this strain, indicated that nutrient acquisition for this species in the presence of bacteria was accomplished primarily via ingestion of bacteria.

Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates.

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.

Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance

We investigated the taxonomic composition of the heterotrophic nanoflagellate (HNF) assemblage and its taxon-specific bacterial grazing rates in Lake Constance (Germany) over the course of 1 yr. Bacterial grazing rates were measured using natural fluorescently labelled bacteria (FLB) and compared to bacterial production estimated by the uptake of 14 C-leucine incorporation. Glutaraldehyde-fixed, DAPI-stained flagellates were counted using epifluorescence microscopy. Based on annual averages, small species such as Spumella sp. (2 to 6 µm) were the most numerous HNF and the dominant bacterivores. Larger flagellates such as Kathablepharis sp. contributed sig- nificantly to total HNF biomass, in particular during spring, but were relatively unimportant as bac- terial grazers. The HNF community structure changed during the transition from the phytoplankton spring bloom to the clearwater phase, with small flagellates such as heterokonts, kinetoplastids and choanoflagellates becoming increasingly abundant. The flagellate community composition was more diverse during summer and autumn than in spring. Per capita ingestion rates ranged from 0 to 31 bac- teria HNF -1 h -1 and changed seasonally up to 10-fold within a given taxon. Mixotrophic species con- tributed little to total bacterivory. We provide evidence that the relative significance of bacterial ingestion by a given flagellate taxon may change seasonally. Based upon our experimental results, we discuss potential shortcomings inherent in the FLB technique.

Diel changes in phagotrophy by Cryptomonas in Lake Biwa

Diel changes in bacterial ingestion by a mixotrophic flagellate, Cryptomonas sp., and heterotrophic nanoflagellates (HNF) were examined in situ at 4‐h intervals for 2 d in the epilimnion and metalimnion of Lake Biwa using bacteria‐sized fluorescent microspheres as a tracer food. Clearance rates of HNF for the microspheres ranged between 1.3 and 4.5 nl cell−1 h−1, but the average rate did not differ between day and night. In contrast, clear diel changes were observed in the clearance rate of Cryptomonas sp. in the epilimnion from <0.5 nl cell−1 h−1 at midnight to >3 nl cell−1 h−1 at noon. In the metalimnion where light intensity was lower, however, the clearance rate of Cryptomonas sp. was always <0.5 nl cell−1 h−1 through the study period. Thus, bacterial ingestion of Cryptomonas sp. is not to acquire supplementary energy or carbon at low phototrophic activities. During the study period, both inorganic phosphorus and nitrogen concentrations were less than or close to the detection limits (10 nM P and 1 µM N) in the epilimnion, but much higher in the metalimnion. The results strongly support the idea that Cryptomonas sp. utilizes N and P from bacteria as substitutable nutrients when photosynthesis takes place under conditions of nutrient depletion. To assess the grazing effect of mixotrophic algae on bacterial populations, it is essential to consider diel changes in their phagotrophic mode of nutrition that are induced by light regime and nutrient concentrations in ambient water.

Temporal variation in body composition and lipid storage of the overwintering, subarctic copepod Neocalanus plumchrus in the Strait of Georgia, British Columbia (Canada)

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 192:239-247 (2000) - doi:10.3354/meps192239 Temporal variation in body composition and lipid storage of the overwintering, subarctic copepod Neocalanus plumchrus in the Strait of Georgia, British Columbia (Canada) Melissa Evanson1,*, Elizabeth A. Bornhold2, Robert H. Goldblatt3, Paul J. Harrison3, Alan G. Lewis3 1Department of Zoology, University of Guelph, Guelph, Ontario N1G 2W1, Canada 2Ocean Science and Productivity, Institute of Ocean Sciences, 9860 West Saanich Rd, Sidney, British Columbia V8L 4B2, Canada 3Department of Earth and Ocean Sciences (Oceanography), University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada *E-mail: mevanson@uoguelph.ca ABSTRACT: Neocalanus plumchrus (Marukawa) was sampled between October 1996 and August 1997 in the upper 400 m of the water column in Strait of Georgia (49° 15' N, 123° 45' W), a large, partly enclosed basin in British Columbia, Canada. The overwintering vertical stage distribution of N. plumchrus is described together with the temporal variations in total body length, dry weight, lipid and body composition in copepodite stage V (CV) and adult females. Adult males and females were found at depths of less than 200 m from January to March. Regressions of total body length to total dry weight showed significant correlations for CVs and females. Carbon and nitrogen isotopic analysis (δ15N and δ13C) indicated no temporal increase, suggesting a lack of feeding in CVs (October to December) and females (January to March) at depth and a dependency on stored lipid reserves accumulated during times of high primary production in the euphotic zone. Total lipid content per individual copepod showed significant decreases from 0.5 to 0.2 mg during the time of adult female gonadal development between January and February. A decrease in total dry mass per copepod from 0.9 to 0.4 mg also occurred during this time period. Lipid composition showed preferential retention of long-chained fatty acids until the onset of egg release in March. Significant decreases in carbon and nitrogen from January to February and, to a lesser extent, between February and March were observed, corresponding to periods of gonad maturation and egg release, respectively. Significant increase in nitrogen content suggests the possibility of bacterial ingestion by late moulting CVs. The significant decrease in C:N ratio, from 8.7 to 4.1, suggests a depletion of lipid reserves and resultant reliance on proteins as an energy source for egg release. Variations in organic content verify that the CVs remain in a state of dormancy in which minimal lipid reserves are depleted, while the onset of maturation and reproduction is the major energy consumer. KEY WORDS: Neocalanus plumchrus · Lipid · Body composition · Vertical distribution · Temporal variation Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 192. Publication date: January 31, 2000 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 2000 Inter-Research.

Inhibition of bacterial adherence to host tissue does not markedly affect disease in the murine model of Pseudomonas aeruginosa corneal infection.

The prevention of bacterial infections by the inhibition of binding to host tissues is an oft-touted approach, but few studies with appropriate models of infection have tested its feasibility. Pseudomonas aeruginosa causes severe corneal infections in mice after inoculations with low doses, and infection is thought to depend upon an initial adherence of the bacteria to corneal cells. In vitro, adherence to corneal cells is mediated to a large degree by the complete-outer-core oligosaccharide of the bacterial lipopolysaccharide (LPS). However, bacteria adhering to tissues in vivo are difficult to differentiate from nonadherent bacteria. Since a direct correlate of P. aeruginosa adherence to corneal epithelial cells is the degree to which these cells internalize P. aeruginosa, the level of adherence in vivo can be approximated by measuring P. aeruginosa ingestion by cells by using gentamicin exclusion assays. To determine the degree to which inhibition of the corneal cell adherence affects the course of infection and disease in the murine model, we evaluated the ability of LPS-outer-core oligosaccharide to inhibit bacterial association and entry into corneal cells and to modulate the development of disease. Mice were anesthetized, and their corneas were scratched and inoculated with virulent P. aeruginosa 6294 or PAO1, along with either 50 microg of oligosaccharide derived from LPS from P. aeruginosa PAC557 (complete outer core but no O side chains) or oligosaccharide derived from LPS of P. aeruginosa PAC1RalgC::tet (incomplete-core oligosaccharide). After 4 h, there were no differences between groups in the counts of infecting and internalized bacteria. At 24 h, the complete-core oligosaccharide decreased the levels of bacteria per eye by 70 to 99.7% compared with the levels achieved by including the incomplete-core oligosaccharide in the infectious inoculum. Epithelial cell ingestion of bacteria was comparably affected. However, the effect on disease was modest and only evident at lower challenge doses that elicited mild disease in controls and when the bacterial association and ingestion were inhibited by >99%. Overall, it appears that in the murine model of P. aeruginosa corneal infection at challenge doses of bacteria 10-fold or greater than the minimal amount needed to cause disease, the absolute level of inhibition of bacterial adherence is insufficient to reduce the bacterial counts below that which elicits disease.

Utilization of Substitutable Carbon and Phosphorus Sources by the Mixotrophic Chrysophyte Ochromonas Sp

In laboratory experiments, I studied the influences of bacterial density and light on the ingestion and growth rates, pigment contents, and the carbon and phosphorus turnover rates of the mixotrophic flagellate Ochromonas sp. The investigated strain is a bacterivorous flagellate that can enhance its photosynthetic apparatus and grow phototrophically when bacterial densities are low. This was also evident from significantly higher chlorophyll $a$ contents during active photosynthetic growth phases. Moderate phototrophic growth should be possible even if bacteria were absent. Bacterial ingestion rates increased hyperbolically with bacterial density, and there was no difference between light— and dark—adapted cells. Ochromonas released soluble reactive phosphorus (SRP) when growth was predominantly phagotrophic, but it took up SRP when growth was phototrophic. The mixotrophic strategy in Ochromonas appears to be bound up with costs and trade—offs: Ochromonas needs high bacterial densities to reach maximum growth rates, its basic metabolic costs are higher than for obligately phagotrophic flagellates, and its phototrophic growth rates are lower than for obligately phototrophic phytoplankton of comparable size.

Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections.

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the delta F508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Bacterial ingestion, tumor necrosis factor-alpha, and heat induce programmed cell death in activated neutrophils.

Resolution of acute inflammation requires the removal of sequestered neutrophils (PMN) from the inflammatory site by apoptosis and ingestion by tissue macrophages; however, sequestered PMN are prevented from undergoing programmed cell death by some of the mediators of the acute inflammatory process, including lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor, and interleukin 2. This delay in apoptosis could lead to necrosis resulting in tissue damage. Tumor necrosis factor-alpha (TNF-alpha), Escherichia coli ingestion resulting in a respiratory burst, and heat have been shown to induce PMN apoptosis. The effects of TNF-alpha, E. coli ingestion, and heat shock on the one hand and LPS on the other, on PMN apoptosis are unknown. The aim of this study was to determine if TNF-alpha, E. coli ingestion, and heat shock, which have been shown to induce PMN apoptosis, could override the delay in apoptosis associated with LPS. PMN (10(6)) isolated from 10 healthy volunteers were cultured in either medium alone or PMN cultured with LPS (10 ng/mL/1 h). PMN activation was assessed subsequently by phagocytosis of E. coli and CD11b expression. PMN were then further studied under four culture conditions: medium alone, TNF-alpha (100 U/mL), E. coli (1:25, PMN:E. coli), and heat shock (42 degrees C for 45 min). Apoptosis was assessed over time by propidium iodide staining of DNA and Fc gamma RIII receptor expression. The results demonstrate, for the first time, that the mechanisms by which LPS delays PMN apoptosis are overridden by the mechanisms by which TNF-alpha, E. coli ingestion, and heat shock induce programmed cell death. Factors regulating PMN apoptosis have an important role to play in the resolution of acute inflammation. Identification of these factors and their interaction have important implications for the development of therapeutic strategies aimed at modulating the acute inflammatory response.

Gastroduodenal mucosal defense

The observation that a significant proportion of patients who are infected with Helicobacter pylori or who consume nonsteroidal anti-inflammatory drugs on a regular basis do not develop gastric or duodenal ulcers suggests that differences in host responses to these agents play an important role in the pathogenesis of the gastroduodenal injury. In the past year, significant progress has been made in understanding the contribution of various components of the “mucosal defense network” to resistance to injury. For example, advances have been made in understanding the mechanisms through which epithelial cells maintain their intracellular pH near neutrality in the face of significant luminal acid loads. The importance of nitric oxide and prostaglandins in the regulation of mucosal blood flow and other components of mucosal defense is becoming more clearly understood. Advances have been made in understanding the contribution of the mucosal immune system and the mediators and cytokines it produces in the responses of the mucosa to bacterial infection or ingestion of noxious substances. The role of the sensory afferent neurons in modulating mucosal defense has been the subject of a number of studies, as has been the mechanisms underlying restitution of the gastroduodenal epithelium, factors influencing healing of ulcers, and factors contributing to the adaptation of the stomach to repeated administration of nonsteroidal anti-inflammatory drugs.

Measurement of Bacterial Ingestion and Killing by Macrophages

This unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.