Gum Arabic Increases Phagocytosis of Escherichia coli by Blood Leukocytes of Young and Old Healthy Volunteers.

BACKGROUND: Gum arabic, a polysaccharide exudate from Acacia senegal (L.) Willdenow trees, has already been used by African native people in natural medicine. METHODS: Using whole-blood samples from young (20-35 years) and older (>80 years) healthy volunteers (each group n = 10), the effect of an aqueous solution of GA on phagocytosis of Escherichia coli was examined with a gentamicin protection assay. Whole-blood samples of each volunteer were stimulated with GA and as a control with CpG oligodeoxynucleotides (Toll-like receptor -9 agonists) for 2 h, then co-incubated with E. coli for 30 min and thereafter treated with gentamicin for up to 240 min to kill extracellular bacteria. Then, whole-blood cells were lysed with distilled water, and colony-forming units were counted by quantitative plating. Cytokine enzyme-linked immunosorbent assay for the detection of TNF-α and IL-6 was performed using the blood supernatant. RESULTS: The GA concentration tested (20 mg/mL) did not affect the viability of eukaryotic cells. Phagocytosis of E. coli by whole-blood leukocytes derived from young (p = 0.008) and older (p = 0.004) healthy volunteers was increased by 120.8% (young) and 39.2% (old) after stimulation with GA. In contrast, CpG only stimulated the bacterial phagocytosis by cells derived from young volunteers (p = 0.004). Stimulation of whole blood with GA increased the intracellular killing of E. coli in young (p = 0.045) and older volunteers (p = 0.008) and induced a TNF-α release in whole blood collected from older volunteers but not from younger ones (p = 0.008). CONCLUSIONS: These data encourage the isolation of active compounds of GA and the initiation of clinical trials addressing the preventive effect of GA on bacterial infections.

Antimicrobial and Immunomodulatory Effect of Gum Arabic on Human and Bovine Granulocytes Against Staphylococcus aureus and Escherichia coli.

Gum arabic (GA) is a traditional herbal medicine from Acacia Senegal (L.) Willdenow trees, which consist of a complex mixture of polysaccharides and glycoproteins. It is used in daily applications for several diseases and is considered to protect against bacterial infections. The detailed mechanisms behind these observations are still unclear. In this study, we investigated the direct antibacterial activity of GA water and ethanol extracts against Staphylococcus (S.) aureus or Escherichia (E.) coli and the immunomodulating properties of those extracts on granulocytes as a first line of defense against bacteria. Firstly, the direct antimicrobial effect of GA was tested on three different S. aureus strains and two E. coli strains. The growth of bacteria was analyzed in the presence of different GA concentrations over time. GA water as well as ethanol extracts showed a significant growth inhibition in a concentration-dependent manner in the case of S. aureus Newman, S. aureus Rd5, and E. coli 25922, but not in the case of S. aureus USA300 and E. coli K1. Transmission electron microscopic analysis confirmed an antibacterial effect of GA on the bacteria. Secondly, the immunomodulatory effect of GA on the antimicrobial activity of bovine or human blood-derived granulocytes was evaluated. Interestingly, water and ethanol extracts enhanced antimicrobial activity of granulocytes by the induction of intracellular ROS production. In line with these data, GA increased the phagocytosis rate of E. coli. No effect was seen on neutrophil extracellular trap (NET) formation that mediates killing of extracellular bacteria such as S. aureus. In conclusion, we show that GA exhibits a direct antibacterial effect against some S. aureus and E. coli strains. Furthermore, GA boosts the antimicrobial activities of granulocytes and increases intracellular ROS production, which may lead to more phagocytosis and intracellular killing. These data might explain the described putative antimicrobial activity of GA used in traditional medicine.

Comparison Between K3EDTA and Lithium Heparin as Anticoagulant to Isolate Bovine Granulocytes From Blood.

Granulocytes play a key role in the defense against invading pathogens. To study granulocyte functions, the isolation of a pure and active cell population from fresh blood is required. Anticoagulants and red blood cells (RBCs) lysis used in the isolation procedure may influence cell harvest, cell marker expression, and pre-activation of cells. In this study, the influence of the anticoagulants K3EDTA or lithium heparin and the effect of different RBCs lysis methods on bovine granulocyte population from fresh blood of healthy cows after density gradient centrifugation were investigated. Venous blood from healthy cows was collected in K3EDTA and lithium heparin tubes. Density gradient centrifugation to separate granulocytes from other cells was conducted using Biocoll. Then, RBCs were lysed with hypotonic water or 0.2% sodium chloride (NaCl). Immediately after isolation, harvest, viability, size, granularity, purity, and CD11b expression as a marker for granulocytes was analyzed by flow cytometry. In addition, as a marker for activation and reactivity of the granulocytes, we stimulated cells with phorbol-myristate-acetate to evaluate the release of reactive oxygen species. Furthermore, extracellular trap (ET) formation was investigated by confocal immunofluorescence microscopy in untreated control cells and cells treated with the cholesterol-depleting agent methyl-ß-cyclodextrin. We did not find a significant difference in percentage of dead cells when comparing the two anticoagulants or the different RBCs lysis methods. However, the percentage of granulocytes in the harvested population was significantly less using lithium heparin blood as anticoagulant compared to K3EDTA. The granulocytes harvested from lithium heparin blood and water lysis exhibited higher clumping and pre-activation of unstimulated control cells as indicated by isolation of doublet cells, increased CD11b expression, and increased oxidative burst and higher amount of ET-releasing cells. Furthermore, the combination of K3EDTA as anticoagulant and NaCl as RBCs lysis method revealed the lowest variability and highest difference between untreated and methyl-ß-cyclodextrin-treated cells when quantifying ET formation. In conclusion, density gradient centrifugation of K3EDTA blood resulted in higher purity of bovine granulocytes compared to lithium heparin blood. In contrast to water lysis, NaCl lysis method is recommended to avoid pre-activation of cells which may occur during hypotonic water lysis.