In vitro effects of nonesterified fatty acids on bovine neutrophils oxidative burst and viability

An in vitro study was conducted to examine the influence of nonesterified fatty acids (NEFA) on bovine polymorphonuclear leukocytes (PMN). Eight healthy, midlactating Holstein cows were used as blood donors. Blood PMN were isolated and incubated with a mixture of NEFA, reflecting composition of bovine plasma NEFA at concentrations that were intended to mimic those found in blood of cows undergoing high, moderate, or low lipomobilization intensity (2, 1, 0.5, 0.25, 0.125, and 0.0625 mM). Control samples were incubated in absence of NEFA. Phagocytosis and oxidative burst activities were assessed by a 2-color flow cytometric method, which was based on oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123. Oxidative burst products were generated by incubating PMN with Staphylococcus aureus labeled with propidium iodide. A flow cytometric technique was used to detect PMN viability, necrosis, and apoptosis using fluorescein isothiocyanate-labeled annexin-V and propidium iodide. Phagocytic activity was not affected by NEFA. The highest concentration of NEFA (2 mM) was associated with a dramatic increase of phagocytosis-associated oxidative burst activities with a reduction in cell viability (48.0 vs. 97.5% in control samples) and with a marked increase of necrosis (49.4 vs. 0.5% in control samples). Conversely, the mixture of NEFA did not affect the occurrence of apoptosis. Enhancement of the oxidative burst associated with the highest concentration of NEFA might explain the reduced viability and higher percentage of necrosis observed under the same conditions. This study demonstrated a substantial resistance of bovine PMN to an overload of fatty acids. However, observation that the highest concentration of NEFA regulated some PMN functions encourages the possibility of in vivo studies to assess the relationships between intensity of lipomobilization, plasma NEFA, and bovine PMN functions

Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in plasma of lipopolysaccharide-challenged pigs

Introduction : Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and IL-6. In research, multiplex assays currently are a very popular tool for the simultaneous detection of biomarkers of infection and inflammation. Specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single factor analysis, yet for multi-parameter analyses, this approach is time-consuming and expensive. Cytometric bead array (CBA) is a flexible, bead-based flow cytometric application for the simultaneous detection of various soluble proteins of interest. The aim of the present study was to develop and validate a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6. The results were compared to commercial ELISA kits. Materials and Methods : Four pigs with a mean body weight (BW) of 24.9 kg were intravenously challenged with 15 µg ultrapure LPS/kg BW (Escherichia coli serotype O111:B4). Plasma was isolated and stored at -70 °C until analysis. Capture antibodies were covalently coupled to the surface of beads with unique fluorescence intensities (Becton Dickinson Biosciences). Detection antibodies were conjugated with R-Phycoerythrin (R-PE). A mixture of beads was firstly incubated with an appropriate standard mixture. Subsequently, a mixture of detection antibodies, either directly or indirectly conjugated to R-PE, was added to accomplish the desired sandwich format. The samples were finally analyzed on a BD FACSArrayTM Bioanalyzer. ELISAs were purchased from R&D Systems. Results : Table 1 shows the validation parameters of the developed CBA 3-plex assay and the commercial ELISAs. Following an in vivo LPS challenge, similar plasma concentration-time profiles were observed for all cytokines with CBA and ELISA. Discussion : This is the first CBA study describing a validated multiplex protocol for the simultaneous measurement of the major porcine pro-inflammatory cytokines TNF-α, IL-1β and IL-6. In research, ELISAs are still considered as the gold standard for determination of secreted proteins in serum or plasma, however, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of cytokine profiles in small porcine plasma volumes

Urinary chitinase 3-like protein 1 for early diagnosis of acute kidney injury : a prospective cohort study in adult critically ill patients

Background: Acute kidney injury (AKI) occurs frequently and adversely affects patient and kidney outcomes, especially when its severity increases from stage 1 to stages 2 or 3. Early interventions may counteract such deterioration, but this requires early detection. Our aim was to evaluate whether the novel renal damage biomarker urinary chitinase 3-like protein 1 (UCHI3L1) can detect AKI stage >= 2 more early than serum creatinine and urine output, using the respective Kidney Disease vertical bar Improving Global Outcomes (KDIGO) criteria for definition and classification of AKI, and compare this to urinary neutrophil gelatinase-associated lipocalin (UNGAL). Methods: This was a translational single-center, prospective cohort study at the 22-bed surgical and 14-bed medical intensive care units (ICU) of Ghent University Hospital. We enrolled 181 severely ill adult patients who did not yet have AKI stage >= 2 based on the KDIGO criteria at time of enrollment. The concentration of creatinine (serum, urine) and CHI3L1 (serum, urine) was measured at least daily, and urine output hourly, in the period from enrollment till ICU discharge with a maximum of 7 ICU-days. The concentration of UNGAL was measured at enrollment. The primary endpoint was the development of AKI stage >= 2 within 12 h after enrollment. Results: After enrollment, 21 (12 %) patients developed AKI stage >= 2 within the next 7 days, with 6 (3 %) of them reaching this condition within the first 12 h. The enrollment concentration of UCHI3L1 predicted the occurrence of AKI stage >= 2 within the next 12 h with a good AUC-ROC of 0.792 (95 % CI: 0.726-0.849). This performance was similar to that of UNGAL (AUC-ROC of 0.748 (95 % CI: 0.678-0.810)). Also, the samples collected in the 24-h time frame preceding diagnosis of the 1st episode of AKI stage >= 2 had a 2.0 times higher (95 % CI: 1.3-3.1) estimated marginal mean of UCHI3L1 than controls. We further found that increasing UCHI3L1 concentrations were associated with increasing AKI severity. Conclusions: In this pilot study we found that UCHI3L1 was a good biomarker for prediction of AKI stage >= 2 in adult ICU patients

Preconditioning with lipopolysaccharide or lipoteichoic acid protects against Staphylococcus aureus mammary infection in mice

Staphylococcus aureus is one of the most causative agents of mastitis and is associated with chronic udder infections. The persistency of the pathogen is believed to be the result of an insufficient triggering of local inflammatory signaling. In this study, the preclinical mastitis model was used, aiming to evaluate if lipopolysaccharide (LPS) or lipoteichoic acid (LTA) preconditioning could aid the host in more effectively clearing or at least limiting a subsequent S. aureus infection. A prototypic Gram-negative virulence factor, i.e., LPS and Gram-positive virulence factor, i.e., LTA were screened whether they were able to boost the local immune compartment. Compared to S. aureusinduced inflammation, both toxins had a remarkable high potency to efficiently induce two novel selected innate immunity biomarkers i.e., lipocalin 2 (LCN2) and chitinase 3-like 1 (CHI3L1). When combining mammary inoculation of LPS or LTA prior to a local S. aureus infection, we were able to modulate the innate immune response, reduce local bacterial loads, and induce either LCN2 or CHI3L1 at 24 h post-infection. Clodronate depletion of mammary macrophages also identified that macrophages contribute only to a limited extend to the LPS/LTA-induced immunomodulation upon S. aureus infection. Based on histological neutrophil influx evaluation, concomitant local cytokine profiles and LCN2/CHI3L1 patterns, the macrophage-independent signaling plays a major role in the LPS-or LTA-pretreated S. aureus-infected mouse mammary gland. Our results highlight the importance of a vigilant microenvironment during the innate immune response of the mammary gland and offer novel insights for new approaches concerning effective immunomodulation against a local bacterial infection

Flow cytometry, a powerful novel tool to rapidly assess bacterial viability in metal working fluids : proof-of-principle

Metalworking fluids (MWF) are water-or oil-based liquids to cool and lubricate tools, work pieces and machines, inhibit corrosion and remove swarf. One of the major problems in the MWF industry is bacterial growth as bacterial enzymes can cause MWF degradation. In addition, bacteria can form biofilms which hamper the functioning of machines. Last but not least, some bacterial by-products are toxic (e.g. endotoxins) and present potential health risks for metalworking machine operators via the formation of aerosols. Therefore, a novel fast yet accurate analytical method to evaluate and predict the antibacterial capacity of MWF would be an important asset. As such a tool is currently lacking, the present study aimed to develop a protocol based on flow cytometry (FCM) to assess the antibacterial potential of newly developed MWF independent of bacterial growth. Results of this novel method were compared to a biochallenge test currently used in MWF industry and also to traditional plate counts. Our results represent a proof-of-principle that FCM can reliably predict the antibacterial capacity of MWF already within one day of incubation with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis, being substantially faster than the current growth-based methods

Flow cytometric assessment of the viability and functionality of uterine polymorphonuclear leukocytes in postpartum dairy cows

Postpartum dairy cows experience impaired peripheral polymorphonuclear leukocyte (PMN) functionality, which has been associated with reproductive tract inflammatory diseases. However, it has not been elucidated yet whether endometrial PMN functionality is (equally) impaired. We developed a method for endometrial PMN isolation and flow cytometric assessment of their viability and functionality. We also evaluated PMN immunolabeling, using a specific bovine granulocyte marker, CH138A. Blood and endometrial cytobrush samples were collected in duplicate from seventeen clinically healthy Holstein-Friesian cows between 9 and 37 days in milk. The proportion of viable, apoptotic, and necrotic PMN in endometrial samples roughly ranged from 10 to 80%, indicating highly dynamic endometrial PMN populations in the postpartum uteri. Endometrial PMN functionality testing revealed that PMN immunolabeling increased the accuracy, although this protocol might influence the median fluorescence intensity of the sample. Phagocytosis seemed the most stable and reliable endometrial PMN function and could be assessed satisfactorily without prior CH138A immunolabeling. However, the interpretation of oxidative burst and intracellular proteolysis tests remains challenging. The correlation between peripheral and endometrial PMN functionality was poor. Further research is warranted to unravel the role of uterine PMN viability and functionality in bovine uterine health

Effect of subinhibitory exposure to quaternary ammonium compounds on the ciprofloxacin susceptibility ofEscherichia colistrains in animal husbandry

Background Quaternary ammonium compound based disinfectants are commonly used in pig and poultry husbandry to maintain farm hygiene. However, studies have shown that subinhibitory concentrations of these disinfectants may increase antibiotic resistance. Investigation of antibiotic susceptibility is usually assessed via the microbroth dilution method, although this conventional culture-based technique only provides information on the bacteriostatic activity of an antimicrobial agent. Therefore, experiments were performed to investigate the effect of prior benzalkonium chloride (BKC) exposure on the viability of subsequent ciprofloxacin (CIP) treatedEscherichia coli. Results Following CIP treatment, bacterial cell counts were significantly higher after exposure to a subinhibitory BKC concentration than without BKC exposure. The flow cytometric results suggested a BKC-dependent onset of membrane damage and loss of membrane potential. Conclusion Our results indicate a lower bactericidal effect of CIP treatment on BKC-exposedE. coliisolates compared to unexposedE. coliisolates