Antibacterial apple cider vinegar eradicates methicillin resistant Staphylococcus aureus and resistant Escherichia coli.

Methicillin-resistant Staphylococcus aureus (MRSA) and resistant Escherichia coli (rE.coli) infections can spread rapidly. Further they are associated with high morbidity and mortality from treatment failure. Therapy involves multiple rounds of ineffective antibiotics alongside unwanted side effects, alternative treatments are crucial. Apple cider vinegar (ACV) is a natural, vegan product that has been shown to have powerful antimicrobial activity hence we investigated whether ACV could ameliorate these resistant bacteria. The minimum dilution of ACV required for growth inhibition was comparable for both bacteria (1/25 dilution of ACV liquid and ACV tablets at 200 µg/ml were effective against rE. coli and MRSA). Monocyte co-culture with microbes alongside ACV resulted in an increase in monocyte phagocytosis by 21.2% and 33.5% compared to non-ACV treated but MRSA or rE. coli stimulated monocytes, respectively. Label free quantitative proteomic studies of microbial protein extracts demonstrated that ACV penetrated microbial cell membranes and organelles, altering the expression of key proteins. This resulted in significant reductions in total protein expression, moreover we could only detect ribosomal proteins; 50 s 30 s, enolase, phosphenol pyruvate and the ATP synthase subunit in rE. coli. Elongation factor iNOS and phosphoglycerate kinase OS were the only proteins present in MRSA samples following ACV treatment.

Urate crystals induce macrophage PAF­AH secretion which is differentially regulated by TGFß1 and hydrocortisone.

The aim of the present study was to establish the role of platelet­activating factor acetyl hydrolase (PAF­AH) in the resolution phase of gout using an established in vitro mononuclear cell model. The effects of signalling pathway inhibitors on PAF­AH secretion, as well as the effects of the common treatments hydrocortisone and colchicine, an antibody against the anti­inflammatory cytokine transforming growth factor ß1 (TGFß1), and the transcriptional inhibitor actinomycin D, were also investigated. The effect of recombinant PAF­AH on cytokine secretion by these cells was also determined. Human peripheral blood­derived monocytes were isolated and differentiated into macrophages. Monocytes and macrophages were stimulated with monosodium monohydrate urate (MSU) crystals or lipopolysaccharide in the presence or absence of AEG3482 [a c­Jun N­terminal kinase (JNK) inhibitor], MG132 (a proteasome inhibitor), hydrocortisone or colchicine. Cultures were then analysed for PAF­AH secretion using ELISA. A 6­fold upregulation of PAF­AH secretion was observed following macrophage exposure to MSU crystals for 24 h (29.3±6 vs. 5.4±0.3 ng/ml unstimulated; P<0.05). Following 72 h, PAF­AH levels decreased significantly (11.1±1.8; P<0.01). Secretion was further enhanced following pre­treatment with the JNK protein kinase inhibitor AEG3482 prior to MSU crystal stimulation (P<0.05) and was abrogated when cells were preincubated with actinomycin D or the proteasome inhibitor MG132 (50, 100 and 200 µM). The addition of recombinant PAF­AH (2.5­10 ng/ml) to MSU crystal­stimulated immature monocyte cultures significantly decreased pro­inflammatory interleukin (IL)­1ß (unstimulated 687±124 vs. stimulated 113±30 pg/ml) and IL­6 secretion (unstimulated 590±50 vs. stimulated 182±19 pg/ml). Treatment of MSU crystal­stimulated macrophages with hydrocortisone (2 µM) also significantly decreased PAF­AH release (P<0.05). Neutralising anti­TGFß1 addition decreased PAF­AH dose­dependently with the highest inhibition observed at 1 µg/ml (P<0.05). The results implicated that PAF­AH may have an anti­inflammatory role in the resolution phase of gout.

Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Macrophage derived platelet activating factor implicated in the resolution phase of gouty inflammation.

Human blood derived in vitro differentiated monocytes or macrophages are a population of cells which have been investigated over the years to determine the role these cells play in the resolution phase of gout. Macrophages are able to phagocytose monosodium urate monohydrate (MSU) crystals without releasing inflammatory factors. This study analysed macrophage platelet activating factor secretion and its possible role in the pathway of gout resolution. Analysis of sunatants from in vitro differentiated macrophages stimulated with MSU crystals revealed the secretion of platelet activating factor (PAF) 1.54 ± 0.10 mean ± SEM; ng/mL per 10(6) cells. This secretion was absent in immature monocytes treated similarly. When these monocytes were pretreated with recombinant human PAF-acetylhydrolase (rhuPAF-AH) and MSU crystals resulted in TNFα suppression. Addition of WEB2086, a platelet activating factor (PAF) antagonist, to differentiated macrophages with MSU crystals unmasked TNFα secretion 0.7 ± 0.06 mean ± SEM; ng/mL per 10(6) cells. This study identifies a role for PAF and the PAF receptor antagonist in the pathway by which macrophages ingest MSU crystals and resolve the concomitant inflammation.

Macrophage release of transforming growth factor beta1 during resolution of monosodium urate monohydrate crystal-induced inflammation.

OBJECTIVE: It has previously been shown that as monocytes differentiate into macrophages, they lose the ability to secrete proinflammatory cytokines in response to monosodium urate monohydrate (MSU) crystals. The purpose of this study was to investigate whether MSU crystals induce macrophages to secrete antiinflammatory factor instead. METHODS: Human monocyte or macrophage isolates were prepared from samples obtained from healthy volunteer donors either by differentiation of blood monocytes in vitro or by collecting cells from skin blisters during the early or late phase of the dermal inflammatory response to cantharidin. Monocyte or macrophage isolates were then incubated with MSU crystals for 24 hours, and culture supernatants were assayed for candidate antiinflammatory mediators (by enzyme-linked immunosorbent assay) and for the capacity to activate or suppress endothelial cell E-selectin expression and secondary neutrophil recruitment under shear flow. RESULTS: Analysis of supernatants from in vitro-differentiated macrophages revealed that transforming growth factor beta1 (TGFbeta1) was induced following MSU crystal stimulation (mean +/- SEM 1.50 +/- 0.24 ng/ml/10(6) cells), but there was no evidence of interleukin-10 (IL-10), IL-1 receptor antagonist, or tumor necrosis factor (TNF) receptor p55 release. Macrophage TGFbeta1 significantly suppressed endothelial cell E-selectin expression and secondary neutrophil capture on endothelial monolayers stimulated with supernatants from MSU-treated monocytes. Leukocytes isolated from resolving (40-hour) skin blisters similarly elaborated TGFbeta1 when challenged with MSU crystals (0.66 +/- 1.3 ng/ml/10(5) CD14+ cells). In contrast, cells isolated from acute (16-hour) skin blisters secreted TNFalpha (0.49 +/- 0.08 ng/ml/10(5) CD14+ cells) but no detectable TGFbeta1. CONCLUSION: These data provide further support for the concept that differentiated macrophages play a protective role in the pathophysiology of gout, and they identify macrophage TGFbeta1 as a mediator of paracrine suppression during the resolution phase of inflammation.

Monocyte Fc gamma receptor expression in patients undergoing coronary artery bypass grafting.

BACKGROUND: Cardiopulmonary bypass is associated with an inflammatory response with potential deleterious effects. The white cell subpopulation mostly investigated so far is the neutrophil. To date very little has been investigated regarding the role of the monocyte/macrophage. This study focuses on the expression of Fc gamma receptors I, II, and III by monocytes in patients undergoing cardiopulmonary bypass. METHODS: We studied the surface expression of Fc gamma receptors I, II, and III by flow cytometry on gated monocyte subpopulations in the whole blood of adult patients undergoing elective coronary artery bypass grafting. Blood samples were drawn preoperatively and at 15 minutes, 1, 2, 4, 24, 48, and 72 hours, and 6 days postoperatively. A second group of patients undergoing lung resection surgery were studied in a similar fashion. RESULTS: Neither Fc receptor I nor receptor II expression were significantly changed throughout the time points studied. Fc receptor III expression was reduced at 2 and 4 hours (p = 0.016 and 0.002) and increased at 24, 48, and 72 hours after commencement of CPB on a selected subpopulation (15%-35%) of monocytes (p = 0.004, < 0.001, and < 0.001, respectively). This expression returned to preoperative levels by the sixth postoperative day. There were no statistically significant changes in the lung resection group. CONCLUSIONS: Our study demonstrated that cardiopulmonary bypass is associated with a biphasic Fc gamma receptor III expression on a subpopulation of peripheral blood monocytes up to 3 days postoperatively.

Safe disposal of inflammatory monosodium urate monohydrate crystals by differentiated macrophages.

OBJECTIVE: Although monosodium urate monohydrate (MSU) crystals have been recognized since the 18th century as the etiologic agent of gout, it is still unknown why certain hyperuricemic individuals remain asymptomatic, and how an acute attack of gout spontaneously resolves. We hypothesized that mononuclear phagocytes hold the key to these questions, and that the state of monocyte/macrophage differentiation is critical. METHODS: Human peripheral blood monocytes were differentiated for 1-7 days in vitro and examined with respect to 1) uptake of MSU crystals, 2) expression of macrophage, dendritic cell, and activation markers, 3) secretion of tumor necrosis factor alpha (TNFalpha), interleukin 1beta (IL-1beta), IL-6, and IL-10, 4) activation of endothelial E-selectin expression, and 5) enhancement of secondary neutrophil recruitment by endothelial cells. RESULTS: MSU crystals induced TNFalpha, IL-1beta, and IL-6 (but not IL-10) secretion in undifferentiated monocytes, which in turn promoted endothelial cell E-selectin expression and secondary neutrophil capture under shear flow. In contrast, differentiation over 3-5 days led to development of a noninflammatory phenotype characterized by a lack of proinflammatory cytokine secretion, lack of endothelial cell activation, and lack of secondary neutrophil recruitment. Acquisition of the noninflammatory phenotype correlated with expression of macrophage antigen but not with expression of dendritic cell marker or activation marker. Monocytes and macrophages were similarly phagocytic, and a control particle, zymosan, elicited secretion of the full panel of cytokines in both cell types. However, coincubation with MSU led to a significant suppression of zymosan-induced TNFalpha secretion (P = 0.009) from macrophages but not monocytes. CONCLUSION: These findings imply that differentiated macrophages provide a safe-disposal mechanism for the removal of inflammatory urate crystals. This may be of clinical relevance to the maintenance of asymptomatic hyperuricemia and the resolution of acute gout.