Evelyne Meyer

Markers of stemness in equine mesenchymal stem cells: a plea for uniformity.

Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79α or CD19 and MHC class II antigens. Thirdly, MSC must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. Successful isolation and differentiation of equine MSC from different sources such as bone marrow, fat tissue, umbilical cord blood, Whartons Jelly or peripheral blood has been widely reported. However, their unequivocal immunophenotyping is hampered by the lack of a single specific marker and the limited availability of monoclonal anti-horse antibodies, which are two major factors complicating successful research on equine MSC. Detection of gene expression on mRNA level is hereby a valuable alternative, although the need still exists to test several antibody clones in search for cross-reactivity. To date, commercial antibodies recognizing equine epitopes are only available for CD13, CD44 and MHC-II. Moreover, as the expression of certain adult stem cell markers may differ between species, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of equine MSC.

Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation.

The non-stimulated and phorbol 12-myristate 13-acetate (PMA)-stimulated luminol-augmented cellular chemiluminescence (CL) response and viability of milk and blood polymorphonuclear leukocytes (PMN) were determined in lactating dairy cows during different stages of lactation. In the first study, ten healthy cows each in early, mid and late lactation were compared. In a second study, the same measurements as in the first study were evaluated longitudinally in 12 cows during 1 month following parturition. The CL activity and myeloperoxidase (MPO) content of milk PMN and macrophages (M) were also compared. Milk M did not possess MPO activity and were devoid of any luminol-enhanced CL. The CL activity of milk and blood PMN was significantly lower in early lactation than in mid and late lactation (P < 0.001). Whereas little changes were observed in viability of blood PMN, the viability of milk PMN was lower in early lactation than in mid and late lactation (P < 0.001). The percentage of PMN in isolated milk cells was also lower during early lactation than during mid and late lactation (P < 0.001). The CL activity in response to PMA during early, mid and late lactation increased 13, 59 and 42-fold in blood PMN and 1.7, 2.6 and 2.4-fold in milk PMN, respectively, in comparison with non-stimulated PMN. The CL activity, both in milk and blood PMN. the milk PMN viability and the percentage of milk PMN were lowest between 3 d and 11 d post partum. These observed changes immediately after calving could contribute to a higher susceptibility to mastitis in that period.

Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution

The mammary gland is subjected to major morphological and biochemical changes during the lactation cycle. It is therefore not surprising that this dynamic process is strictly controlled. The importance of the sex steroid hormones 17beta-estradiol and progesterone for normal development of the mammary gland was recognized several decades ago and has been unequivocally confirmed since. Furthermore, it is now also established that the influence of sex steroids is not restricted to mammogenesis, but that these hormones also control involution. Another important regulatory role is played by growth factors that have been shown to modulate survival (epidermal growth factor, amphiregulin, transforming growth factor alpha, insulin like growth factor, and tumor necrosis factor alpha) or apoptosis (tumor necrosis factor alpha, transforming growth factor beta) of mammary cells. However, the molecular mechanism underlying the influence of sex steroid hormones and/or growth factors on the development and function of the mammary gland remains largely unknown to date. Also scarce is information on the interaction between both groups of modulators. Nevertheless, based on the current indications compiled in this review, an important functional role for sex steroid hormones in the lactation cycle in co-operation with growth factors can be suggested.

Simultaneous Targeting of IL-1 and IL-18 Is Required for Protection against Inflammatory and Septic Shock

RATIONALE Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1β, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS Interestingly, deficiency of both IL-1β and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1β and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

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.

Urinary Markers in Healthy Young and Aged Dogs and Dogs with Chronic Kidney Disease

BACKGROUND Blood urea nitrogen and serum creatinine concentrations only detect a decrease of > 75% of renal functional mass. Therefore, there is a need for markers that allow early detection and localization of renal damage. HYPOTHESIS Urinary albumin (uALB), C-reactive protein (uCRP), retinol binding protein (uRBP), and N-acetyl-beta-D-glucosaminidase (uNAG) concentrations are increased in dogs with chronic kidney disease (CKD) compared with healthy controls and in healthy older dogs compared with young dogs. ANIMALS Ten dogs with CKD, 10 healthy young dogs (age 1-3 years), and 10 healthy older dogs (age > 7 years) without clinically relevant abnormalities on physical examination, hematology, biochemistry, and urinalysis. METHODS Urinary markers were determined using an ELISA (uALB, uCRP, and uRBP) or a colorimetric test (uNAG). Results were related to urinary creatinine (c). The fixed effects model or the Wilcoxon rank sum test were used to compare the different groups of dogs. RESULTS uALB/c, uRBP/c, and uNAG/c were significantly higher in CKD dogs than in healthy dogs. No significant difference was found for uCRP, which was not detectable in the healthy dogs and only in 3 of the CKD dogs. Between the healthy young and older dogs, no significant difference was detected for any of the markers. CONCLUSION The urinary markers uALB/c, uRBP/c, and uNAG/c were significantly increased in dogs with CKD compared with healthy controls. Additional studies are needed to evaluate the ability of these markers to detect renal disease before the onset of azotemia.

Chitinase-like Proteins are Candidate Biomarkers for Sepsis-induced Acute Kidney Injury

Sepsis-induced acute kidney injury (AKI) is a frequent complication of critically ill patients and leads to high mortality rates. The specificity of currently available urinary biomarkers for AKI in the context of sepsis is questioned. This study aimed to discover urinary biomarkers for septic AKI by contemporary shotgun proteomics in a mouse model for sepsis and to validate these in individual urine samples of mice and human septic patients with and without AKI. At 48 h after uterine ligation and inoculation of Escherichia coli, aged mice (48 weeks) became septic. A subgroup developed AKI, defined by serum creatinine, blood urea nitrogen, and renal histology. Separate pools of urine from septic mice with and without AKI mice were collected during 12 h before and between 36–48 h after infection, and their proteome compositions were quantitatively compared. Candidate biomarkers were validated by Western blot analysis of urine, plasma, and renal tissue homogenates from individual mice, and a limited number of urine samples from human septic patients with and without AKI. Urinary neutrophil gelatinase-associated lipocalin, thioredoxin, gelsolin, chitinase 3-like protein 1 and -3 (CHI3L3) and acidic mammalian chitinase were the most distinctive candidate biomarkers selected for septic AKI. Both neutrophil gelatinase-associated lipocalin and thioredoxin were detected in urine of septic mice and increased with severity of AKI. Acidic mammalian chitinase was only present in urine of septic mice with AKI. Both urinary chitinase 3-like protein 1 and -3 were only detected in septic mice with severe AKI. The human homologue chitinase 3-like protein 1 was found to be more excreted in urine from septic patients with AKI than without. In summary, urinary chitinase 3-like protein 1 and -3 and acidic mammalian chitinase discriminated sepsis from sepsis-induced AKI in mice. Further studies of human chitinase proteins are likely to lead to additional insights in septic AKI.

Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis

Abstract. Objective and design: To investigate the effect of enrofloxacin on endotoxin resorption during bovine Escherichia coli mastitis.¶Animals: 12 healthy early post partum Holstein cows.¶Treatment: Mastitis was induced by intramammary infusion of 104 cfu E. coli P4:O32. Six cows were treated twice according to the usual enrofloxacin therapy: 5 mg/kg enrofloxacin 1) intravenously at 10 h and 2) subcutaneously at 30 h after challenge. The other 6 cows served as non-treated controls.¶Methods: Blood and milk samples were collected at several time points after challenge. LPS in plasma was quantified using the limulus amœbocyte lysate (LAL) assay. The somatic cell count (SCC) and cfu of milk samples were also analysed.¶Results: Occasional LPS peaks were detected in the plasma of 2 control cows at 6 h post-challenge and of 1 enrofloxacin-treated cow at 10 h post-challenge (P < 0.01 and P < 0.05, respectively, in comparison with time 0), just before enrofloxacin treatment. After enrofloxacin treatment, no significant LPS amounts were detected in the plasma of treated cows, but neither in the control cows.¶Conclusion: During induced coliform mastitis, LPS resorption in plasma occured only sporadically and within 10 h post-challenge. Whereas enrofloxacin treatment clearly limited bacterial growth in milk, significant effects on LPS resorption could not be detected. This suggests that enrofloxacin treatment of E. coli mastitis is predominantly beneficial by its bactericidal activity and is not associated with enhanced resorption of endotoxins.

Short- and long-term follow-up of glomerular and tubular renal markers of kidney function in hyperthyroid cats after treatment with radioiodine ☆

Hyperthyroidism can mask co-existing chronic kidney disease (CKD). Previous studies showed that post-treatment renal azotemia can be predicted by pre-treatment assessment of glomerular filtration rate (GFR). We hypothesized that treatment of hyperthyroidism may have different effects on glomerular and tubular function and these changes might be predicted by additional pre-treatment variables than GFR. Serum total T4 (TT4), creatinine and blood urea nitrogen (BUN), blood pressure (BP), body weight (BW), GFR, urine specific gravity (USG), urinary protein/creatinine ratio (UPC) and retinol binding protein/creatinine ratio (uRBP/c) were evaluated before and 1, 4, 12 and 24 weeks post-treatment with radioiodine ((131)I) in 21 non-azotemic hyperthyroid cats. Cats were divided 24 weeks post-treatment into group A (normal kidney function, n=16) and group B (impaired kidney function, n=5). Serum TT4, GFR, UPC and uRBP/c decreased significantly after treatment for the complete group and group A (P<0.05), although GFR and uRBP/c did not change in group B. Serum creatinine and BW increased significantly from 1 week after treatment (P<0.05). There was no change in BUN, USG or BP. Pre-treatment serum TT4, GFR and USG differed significantly between group A and B (P<0.05). GFR at 4 weeks after treatment and maximum decrease in GFR could be partially predicted by a formula using pre-treatment GFR, serum TT4, serum creatinine, BUN and/or USG. Significant changes in kidney function occur within 4 weeks post-treatment and none thereafter. Pre-treatment measurement of GFR, USG and serum TT4 can have possible predictive value regarding the development of post-treatment renal azotemia.

The Quorum Sensing Inhibitor Hamamelitannin Increases Antibiotic Susceptibility of Staphylococcus aureus Biofilms by Affecting Peptidoglycan Biosynthesis and eDNA Release.

Treatment of Staphylococcus aureus infections has become increasingly challenging due to the rapid emergence and dissemination of methicillin-resistant strains. In addition, S. aureus reside within biofilms at the site of infection. Few novel antibacterial agents have been developed in recent years and their bacteriostatic or bactericidal activity results in selective pressure, inevitably inducing antimicrobial resistance. Consequently, innovative antimicrobials with other modes of action are urgently needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. Hamamelitannin (2′,5-di-O-galloyl-d-hamamelose; HAM) was previously suggested to block QS through the TraP QS system and was shown to increase S. aureus biofilm susceptibility towards vancomycin (VAN) although mechanistic insights are still lacking. In the present study we provide evidence that HAM specifically affects S. aureus biofilm susceptibility through the TraP receptor by affecting cell wall synthesis and extracellular DNA release of S. aureus. We further provide evidence that HAM can increase the susceptibility of S. aureus biofilms towards different classes of antibiotics in vitro. Finally, we show that HAM increases the susceptibility of S. aureus to antibiotic treatment in in vivo Caenorhabditis elegans and mouse mammary gland infection models.