Kristel Demeyere

In Search for Cross-Reactivity to Immunophenotype Equine Mesenchymal Stromal Cells by Multicolor Flow Cytometry

During recent years, cell‐based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross‐reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC‐II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood‐derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine‐marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7‐aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC‐II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine‐marker panel can be used for the adequate immunophenotyping of equine MSC.

Validation of immunoassays for the candidate renal markers C-reactive protein, immunoglobulin G, thromboxane B2 and retinol binding protein in canine urine

The study of early markers for glomerular and tubular dysfunction in dogs with renal diseases holds promise to gain new insights in the pathogenesis of canine nephropathies. However, the validation of such markers in canine urine is largely lacking. Therefore, immunoassays for the quantification of a set of four urinary markers, C-reactive protein (CRP), immunoglobulin G (IgG), thromboxane B(2) (TXB(2)) and retinol binding protein (RBP), were validated by determining their sensitivity, reproducibility, precision and accuracy in a large patient group. The results show that the immunoassays are appropriate for analysis of urinary CRP, IgG, TXB(2) and RBP in dogs. Furthermore, the significant differences in urinary concentrations of the selected glomerular and tubular markers between healthy (H) dogs and dogs with several types of nephropathies (R) support their future application in both clinical settings and research models.

Technical note: Flow cytometric identification of bovine milk neutrophils and simultaneous quantification of their viability

The aim of the present study was to develop a flow cytometric procedure for the quantification of the proportion of viable, apoptotic, and necrotic polymorphonuclear neutrophilic leukocytes (PMNL) isolated from both low- and high-somatic-cell-count quarter milk samples. Milk PMNL were differentiated from other cells by indirect fluorescent labeling using a primary anti-bovine granulocyte monoclonal antibody (CH138A) and an Alexa 647-labeled secondary antibody. Polymorphonuclear neutrophilic leukocytes were identified flow cytometrically based on their cytoplasmic granularity and CH138A-positivity. Additional labeling with annexin-V-fluorescein isothiocyanate and propidium iodide was used to determine milk PMNL viability. Thirty milk samples were run in parallel to assess the repeatability of the immunoassay and 6 repeated measurements per sample were performed to assess the instrument stability. Fluorescence microscopic verification of the CH138A staining pattern showed both a high sensitivity (90.9%) and specificity (92.3%). The combination of the side-scatter properties of granulated PMNL and CH138A-Alexa 647 positivity allows the distinction of labeled PMNL from other milk cells and particles that may bind nonspecifically, and from autofluorescent particles present in milk. Quantification of the proportion of PMNL and viable, apoptotic, and necrotic subpopulations in parallel samples gave repeatable results with concordance correlation coefficients varying between 0.93 and 0.99. The average coefficient of variation for repeated measurements in identical samples ranged between 4.2 and 9.7%. In conclusion, this is the first flow cytometric method suited for the simultaneous quantification of viable, apoptotic, and necrotic bovine milk PMNL in a straightforward manner.

Optimization of the Isolation, Culture, and Characterization of Equine Umbilical Cord Blood Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) represent a promising population for supporting new clinical concepts in cellular therapy. A wide diversity of isolation procedures for MSC from umbilical cord blood (UCB) has been described for humans. In contrast, a few data are available in horses. In the current study, a sedimentation method using hydroxyethyl starch and a method based on the lysis of red blood cells using ammonium chloride (NH(4)Cl) were compared with two density gradient separation methods (Ficoll-Paque and Percoll). Adherent cell colonies could be established using all four isolation methods. The mononuclear cell recovery after Percoll separation, however, resulted in significantly more putative MSC colonies; and, therefore, this isolation method was used for all further experiments. Culture conditions such as cell density and medium or serum coating of the wells did not significantly affect putative MSC recovery. Isolated MSC using Percoll were subsequently differentiated toward the osteogenic, chondrogenic, and adipogenic lineage. In addition, MSC were phenotyped by multicolor flow cytometry based on their expression of different cell protein markers. Cultured MSC were CD29, CD44, and CD90-positive and CD79α, Macrophage/Monocyte and MHC II-negative. In conclusion, this study reports optimized protocols to isolate, culture, and characterize solid equine MSC from UCB.

Heifer and quarter characteristics associated with periparturient blood and milk neutrophil apoptosis in healthy heifers and in heifers with subclinical mastitis

Polymorphonuclear neutrophilic leukocytes (PMNL) play an important role in the first line cell-mediated immune defense of the body in general and of the mammary gland against mastitis pathogens in particular. Reduced viability of PMNL close to parturition may explain the high incidence of infectious diseases and the high prevalence of intramammary infections (IMI) in periparturient dairy heifers. Apoptosis of blood PMNL 1 wk before the expected calving date and of blood and milk PMNL at 1 to 4 d in milk was determined using flow cytometry. Information on heifer and gland characteristics was collected before calving and in early lactation. Data were analyzed using multivariable, multilevel regression analysis. Supplementation of a commercial mineral/vitamin mix before calving was associated with less blood (14.4 +/- 2.9 vs. 22.4 +/- 2.1%) and milk PMNL apoptosis (19.0 +/- 1.1 vs. 26.4 +/- 0.9%) near calving, presumably related to higher blood selenium concentrations. Both blood and milk PMNL apoptosis showed seasonal variation with the highest proportion of apoptotic cells between January and March (32.0 +/- 6.1 and 34.6 +/- 2.7%, respectively) and April and June (31.3 +/- 5.7 and 37.8 +/- 2.3%, respectively). Heifers losing 0.25 points or more of their body condition in the periparturient period had higher proportions of apoptotic blood PMNL in early lactation compared with heifers losing less than 0.25 points (24.0 +/- 2.8 vs. 16.6 +/- 1.7%). Milk PMNL apoptosis was less pronounced in quarters having teat orifices colonized with non-aureus staphylococci before calving (18.9 +/- 1.0 vs. 29.4 +/- 1.0%). The variation in blood PMNL apoptosis before and after calving mainly resided at the heifer level (71.4 and 98.4% of the total variation, respectively), whereas the variation in milk PMNL apoptosis mainly resided at the heifer (45.7% of the total variation) and quarter levels (45.5% of the total variation). These data imply that the impaired blood and milk PMNL viability in periparturient heifers can be reduced by optimization of certain heifer management practices such as supplementation of minerals/vitamins, and pasture and feeding strategies.

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

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-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.

Dose-response effect of fish oil substitution in parturition feed on erythrocyte membrane characteristics and sow performance.

The present study aimed to investigate whether n-3 polyunsaturated fatty acids (PUFA) incorporate into erythrocyte membranes of peripartal sows in a dose-responsive manner and whether the altered fatty acid profile affects the cell membrane characteristics. At day 109 of gestation (day 0), 51 sows were divided into five treatment groups. Each group received a diet with a different ratio of fish oil to pork lard for nine consecutive days. Blood samples were taken at day 0 and 10 days later. The fatty acid profile of erythrocytes was determined, as well as the osmotic fragility and oxidative stability of erythrocytes. Thiobarbituric acid reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) were determined in plasma samples. Finally, reproductive and performance parameters of both sows and piglets were recorded until weaning. Supplementation of fish oil during the peripartal period changed the fatty acid profile of erythrocyte membranes in a dose-responsive manner. Although the n-3 PUFA content of erythrocyte membranes increased with increasing amounts of fish oil in the diet, no significant effect on erythrocyte osmotic fragility could be recorded. In contrast, oxidative stability of erythrocytes decreased linearly with increasing amounts of fish oil in the diet. Similarly, both TBARS and FRAP linearly increased with increasing percentages of fish oil in the diet. Neither piglet nor sow performance was influenced by dietary treatments, except for a decrease of both piglet survival and weaning weight with increasing quantities of fish oil supplemented. It is concluded that changes in dietary lipid sources can affect the membranes fatty acid profile within days, and mainly influences oxidative stability of the cells.

Non-classical ProIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.

Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation. In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1β and interleukin-6 was subsequently optimized on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture

Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1β and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1β production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1β-dependent pyroptosis reported for macrophages.