Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Heinzelmann, Michael; Gardner, Sarah; Mercer-Jones, Mark; Roll, Amy J.; Polk, Hiram C.

doi:10.1111/j.1348-0421.1999.tb02435.x

Cited by 36 publications

(31 citation statements)

References 22 publications

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“…The neutrophil isolation from healthy volunteers and the bacterial labeling with fluorescein isothiocyanate (FITC) were performed as previously described (7,11). The mixture of the labeled bacteria, the neutrophil suspension, the pooled normal human serum, and the phosphate-buffered saline (PBS; pH 7.4) was incubated for 0 and 10 min in a shaking 37°C water bath.…”

Section: Methodsmentioning

confidence: 99%

Capsular Serotype K1 or K2, Rather than magA and rmpA , Is a Major Virulence Determinant for Klebsiella pneumoniae Liver Abscess in Singapore and Taiwan

Kurup²,

et al. 2007

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Capsular serotypes, magA, and rmpA have been documented in high prevalence for Klebsiella pneumoniae liver abscess. To investigate the regional difference and the correlation of capsular serotype, magA, and rmpA with virulence, 73 isolates were collected in Singapore and Taiwan. Capsular serotypes were determined by countercurrent immunoelectrophoresis, the presence of magA and rmpA was determined by PCR, and virulence was determined by phagocytosis and mouse inoculation. Isolates from Singapore were similar to those from Taiwan in genomic heterogeneity, prevalence of serotype, and the presence of magA and rmpA. The most common serotype was K1 (34/73; 46.6%), followed by K2 (15/73; 20.5%). magA was restricted to serotype K1. All K1 or K2 isolates and 66.7% (16/24) of isolates that were neither serotype K1 nor serotype K2 (non-K1/K2) carried rmpA. Serotype K1 or K2 isolates demonstrated significantly more phagocytic resistance and virulence than did rmpA-positive and -negative groups of non-K1/K2 isolates. In the non-K1/K2 group, the virulence profiles of rmpA-positive strains from Taiwan and Singapore were different by phagocytosis assay and in the mouse model, indicating that factors other than rmpA contributed to virulence. The characteristics of K. pneumoniae liver abscess in Singapore and Taiwan are similar. Capsular serotype K1 or K2 plays a more important role than magA and rmpA in determining virulence in K. pneumoniae liver abscess.

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Section: Methodsmentioning

confidence: 99%

Capsular Serotype K1 or K2, Rather than magA and rmpA , Is a Major Virulence Determinant for Klebsiella pneumoniae Liver Abscess in Singapore and Taiwan

Kurup²,

et al. 2007

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“…Phagocytosis was measured by using a standard assay (20). A FACscan (Becton Dickinson Immunocytometry Systems, San Jose, CA) was used to measure the phagocytic rate.…”

Section: Analysis Of Ompsmentioning

confidence: 99%

Klebsiella pneumoniae Outer Membrane Porins OmpK35 and OmpK36 Play Roles in both Antimicrobial Resistance and Virulence

Tsai

Fung

Lin

et al. 2011

Antimicrob Agents Chemother

278

228

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OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (⌬ompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (⌬ompK35) had no significant effect. A double deletion of ompK35 and ompK36 (⌬ompK35/36) further increased these MICs to high-level resistance and led to 8-and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ⌬ompK35 mutation resulted in a significant (>4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ⌬ompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ⌬ompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ⌬ompK36 and ⌬ompK35/36 mutants. In a mouse peritonitis model, the ⌬ompK35 mutant showed no change in virulence, and the ⌬ompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ⌬ompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

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“…To investigate the nature of the interaction between fimbriated or nonfimbriated E. coli and neutrophils, the bacteria were labeled with FITC, and the interaction was analyzed by both microscopy and flow cytometry (19). Bacterial aliquots were resuspended in an FITC solution (0.1 mg of FITC/ml in 0.1 M carbonate buffer, pH 9.6), incubated for 1 h at room temperature with rotation, washed, and then resuspended in RPMI medium.…”

Section: Reagentsmentioning

confidence: 99%

UropathogenicEscherichia coliTriggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide

2004

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Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose-and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of D-mannose. These results suggest that LPS is required for E. coli to exert both proand antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.

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Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Cited by 36 publications

References 22 publications

Capsular Serotype K1 or K2, Rather than magA and rmpA , Is a Major Virulence Determinant for Klebsiella pneumoniae Liver Abscess in Singapore and Taiwan

Capsular Serotype K1 or K2, Rather than magA and rmpA , Is a Major Virulence Determinant for Klebsiella pneumoniae Liver Abscess in Singapore and Taiwan

Klebsiella pneumoniae Outer Membrane Porins OmpK35 and OmpK36 Play Roles in both Antimicrobial Resistance and Virulence

UropathogenicEscherichia coliTriggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide

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