Evaluation of a fluorochrome assay for assessing the bactericidal activity of neutrophils in human phagocyte dysfunctions

Bioactive compounds present in fruit oils have an antioxidant activity, which may be related to an immunomodulatory effect, which in turn can result in a microbicidal activity. Hence, the main aim of the current study is to evaluate the immunomodulatory effect of buriti oil (Mauritia Flexuosa L.) against enteropathogenic Escherichia coli (EPEC). Therefore, buriti pulp oil was extracted and characterized for saponification, peroxide and acidity index, total carotenoid content, fatty acid profile, hydrophilic and lipophilic fraction antioxidant capacity, cell viability, and phagocytosis of EPEC by mononuclear (MN) cells. The heat extraction process did not cause hydrolytic rancidity and oxidative rancidity in the oil, and the result is an acid value of 17.44 mg KOH/g of oil and a low peroxide value (0.062 meq peroxide/1000 g of oil). However, the saponification index was elevated (239.79 mg KOH/g of oil) due to the presence of low molecular weight fatty acid. As concerns the profile of the fatty acid, the oil is composed mainly of oleic acid (72.23 %) and palmitic acid (22.18 %). In addition, buriti oil presented a high content of carotenoids (760.5 ± 46.4 μg of β-carotene/g of oil), related to its antioxidant capacity. Moreover, buriti oil did not show toxicity to human blood MN phagocytes and increased the rate of cellular phagocytosis in EPEC and its microbicide index (69.3 ± 6.6 %), when compared with the negative control group. (48.1 ± 5.4 %).

The aim of this study was to investigate changes in phagocytic activity of neutrophils of type 2 diabetic patients with foot infections over short treatment courses. The potential utility of the phagocytic index in determining the efficacy of treatment modalities and it's relationship with metabolic control parameters were evaluated.

The phagocytic activity of neutrophils was determined in blood samples of 38 type 2 diabetic patients with foot infections (14 women and 24 men). Mean age and mean duration of diabetes were 66.3 ± 9.4 and 19.1 ± 11.2 (yrs), respectively. All patients received standard treatment (intensive insulin therapy, antibiotherapy, hyperbaric oxygen therapy and surgical debridement). Phagocytic activity of neutrophils was determined by a standard method. Phagocytic activity of neutrophils, acute phase proteins (C-reactive protein) and glycosylated haemoglobin was determined before therapy and two weeks later.

The phagocytic index before and after therapy were 47.7 ± 11.4 and 62.5 ± 15.6, respectively (p < 0.05). There was a significant correlation between phagocytic index and both CRP and HbA1c (r = 0.52, p < 0.05 and r = −0.41, p < 0.05, respectively).

Derangement of carbohydrate metabolism may underlie the impairment of bactericidal activity of neutrophils of poorly controlled diabetic patients. These data reveal that phagocytic activity improves during short-course standard therapy and might enable monitoring of efficacy of treatment modalities in diabetic patients with foot infections.

This study was carried out prospectively to determine the effect on prognosis of phagocytic activity index (PAI) and intracellular killing activity (IKA) of polymorphonuclear leukocytes (PMNL), and the levels of interleukin-1 beta (IL-1β) on prognosis in patients with diabetic foot infection (DFI).

The evaluation of PAI and IKA in PMNL and the levels of IL-1β were performed at the beginning and in the second and fourth weeks of therapy in all diabetic patients, who were categorized into a healing group (HG) and a non-healing group (NHG) on the basis of therapy results.

Sixty-six cases (38 diabetic patients and 28 non-diabetic controls) were included in the study. Full recovery was observed in 23 HG patients, whereas 15 (NHG) patients were unresponsive to treatment and nine patients were subjected to amputation at the end. At the baseline, PAI, IKA and IL-1β levels in HG were not significantly different compared to those of NHG, but at weeks 2 and 4, PAI and IKA levels were significantly higher and IL-1β levels were significantly lower than those in NHG. On the other hand, at the baseline, PAI and IKA values in HG were significantly lower and IL-1β levels were significantly higher in comparison with the controls. However, no significant difference was observed at week 2 or 4.

Our results suggest that the PMNL functions and IL-1β regulation deteriorated in patients with DFI, and that such deteriorations might indicate inefficient therapeutic responses in patients with diabetes mellitus.

Activated phagocytes oxidize the hormone melatonin to N¹-acethyl-N²-formyl-5-methoxykynuramine (AFMK) in a superoxide anion- and myeloperoxidase-dependent reaction. We examined the effect of melatonin, AFMK and its deformylated-product N-acetyl-5-methoxykynuramine (AMK) on the phagocytosis, the microbicidal activity and the production of hypochlorous acid by neutrophils. Neither neutrophil and bacteria viability nor phagocytosis were affected by melatonin, AFMK or AMK. However these compounds affected the killing of Staphylococcus aureus. After 60 min of incubation, the percentage of viable bacteria inside the neutrophil increased to 76% in the presence of 1 mM of melatonin, 34% in the presence of AFMK and 73% in the presence of AMK. The sole inhibition of HOCl formation, expected in the presence of myeloperoxidase substrates, was not sufficient to explain the inhibition of the killing activity. Melatonin caused an almost complete inhibition of HOCl formation at concentrations of up to 0.05 mM. Although less effective, AMK also inhibited the formation of HOCl. However, AFMK had no effect on the production of HOCl. These findings corroborate the present view that the killing activity of neutrophils is a complex phenomenon, which involves more than just the production of reactive oxygen species. Furthermore, the action of melatonin and its oxidation products include additional activities beyond their antioxidant property. The impairment of the neutrophils' microbicidal activity caused by melatonin and its oxidation products may have important clinical implications, especially in those cases in which melatonin is pharmacologically administered in patients with infections.

This study investigated the ability of sub-MICs of gemifloxacin to interfere with the bacterial virulence parameters of adhesiveness, haemagglutination, hydrophobicity and motility, as well as their interactions with host neutrophilic defences such as phagocytosis, killing and respiratory bursts. The adhesiveness of both Escherichia coli and Staphylococcus aureus was significantly reduced to a subinhibitory concentration of 1/32 MIC. Indirect fimbriation parameters, such as hydrophobicity and haemagglutination were significantly reduced at a concentration of 1/8 MIC, as was migration (swarming). Phagocytosis and the respiratory burst measured by means of chemiluminescence were not affected, but killing was significantly increased from 1/2 to 1/8 MIC. The interpolation of these pharmacodynamic findings with pharmacokinetic curves indicates that sub-MIC concentrations of gemifloxacin can prolong antimicrobial effects on virulence determinants up to 27 h after the antimicrobial concentration has fallen below the MIC value.

The ingestion and killing of bacteria by phagocytic cells is an important step in the sequence of interactions between invading microorganisms and host defense systems and may be affected by antibiotics. We investigated the effects of gatifloxacin on the phagocytosis, killing and oxidative bursts of human polymorphonuclear neutrophils (PMNs). The percentage phagocytosis and the phagocytosis index were unaffected by exposure of Escherichia coli strains to sub-MICs of gatifloxacin to a 1/64 dilution. However a significant increase in percentage intraphagocytic killing and the killing index occurred in one E. coli strain at 1/32 MIC and in two strains at 1/16 MIC. The incubation of PMNs with sub-MICs and supra-MICs of gatifloxacin (to 32 MIC) did not affect the oxidative bursts.