Plasma oxylipin profile of postpartum dairy cows categorized into different systemic inflammatory grades in the first week after parturition.

Oxygenated polyunsaturated fatty acids (oxylipins) are important mediators of inflammation ranging from pro- to anti-inflammatory actions. Research investigating differences in the oxylipin profile of dairy cows suffering from different degrees of systemic inflammation in the early postpartum period is lacking and can help advance knowledge on potential mechanisms leading to excessive inflammation. The objective of this preliminary study was to evaluate the plasma oxylipin profile of cows classified in 1 of 4 systemic inflammation categories based on plasma haptoglobin (Hp) concentrations assessed on days in milk (DIM) 1, 2, 3, 4, 5, and 7, in addition to the presence or absence of metritis within 10 DIM, and of cows without any clinical diseases within 21 DIM. Groups were classified as follows: (1) cows with a peak Hp concentration ≤3 DIM (EarlyHp) and diagnosed with metritis; (2) cows with a peak Hp concentration 3 < DIM ≤7 (LateHp) and diagnosed with metritis; (3) cows suffering from persistently elevated Hp concentrations assessed on DIM 4 and 7 while remaining apparently healthy during the first 21 DIM (PersistentHp); and (4) apparently healthy cows not suffering from persistently elevated Hp concentrations (LowHp). Six cows from each category were randomly selected from a plasma bank of a parent cohort study including 380 multiparous cows. Plasma samples on DIM 1 and 2, 3 and 4, and 5 and 7 were proportionally pooled to create 3 samples per cow for lipidomic analysis (i.e., pool 1 = DIM 1 and 2; pool 2 = DIM 3 and 4; pool 3: DIM 5 and 7). Statistical analyses were performed using SAS v9.4 (SAS Institute Inc.) and least squares means adjusted for multiple comparisons using the Tukey-Kramer test. Comparisons for EarlyHp and LateHp were only performed on pooled samples from DIM 1 and 2 (i.e., before metritis diagnosis). EarlyHp cows had decreased concentrations of 9(S)-HOTrE compared with LowHp cows of DIM 1-2 pooled samples. LateHp cows had decreased concentrations of 9(10)-DiHOME compared with LowHp cows. Next, we sought to investigate whether cows classified as PersistentHp had time-dependent differences in oxylipin profile versus LowHp cows. PersistentHp cows had decreased concentrations of 19(R)-HETE compared with LowHp cows in a time-dependent manner (only in pooled samples from DIM 5 and 7). Our results identified oxylipins of interest that warrant further investigation to elucidate their in vitro and in vivo functions in the postpartum inflammatory process of dairy cows.

Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system.

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300µM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4µl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300µM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Characterization of thoracal fat depots - expression of adipokines and remodeling factors and impact of adipocyte conditioned media in fibroblast scratch assays.

Adipose tissue is not only a connective tissue but also an endocrine organ secreting adipokines like Leptin and Adiponectin, lipokines such as palmitoileic acid and extracellular vesicles. These factors and the expression of matrix remodeling enzymes impact surrounding tissues via paracrine effects. The expression of selected secretion factors and the effect of adipocyte conditioned media from four thoracal adipose tissue origins - subcutaneous, perivascular, pericardial and epicardial adipose tissues - in a fibroblast proliferation/wound healing scratch assay model were investigated. Results were compared directly and according to the type 2 diabetic mellitus (T2DM) status of the patients the tissues are originated from. Adipocyte conditioned media from non-diabetic patients resulted in a significant higher scratch closure rate compared to the media with T2DM background. Linoleic acid incubation in scratch assay resulted in a reduced scratch closure rate. Leptin, Adiponectin and Visfatin/Nampt expression and MMP2, MMP9 and FSTL1 mRNA levels did not vary according to T2DM subgroups directly, leading to the assumption that these factors are not causal for scratch assay effects observed. In contrast significant mRNA expression differences were monitored between the thoracal tissue origins implying variations in the local effects of the different adipose tissue depots.

Antihypertensive and cardioprotective effects of the dipeptide isoleucine-tryptophan and whey protein hydrolysate.

AIMS: Angiotensin-converting enzyme inhibitors are treatment of choice in hypertensive patients. Clinically used inhibitors exhibit a structural similarity to naturally occurring peptides. This study evaluated antihypertensive and cardioprotective effects of ACE-inhibiting peptides derived from food proteins in spontaneously hypertensive rats. METHODS AND RESULTS: Isoleucine-tryptophan (in vitro IC50 for ACE = 0.7 µm), a whey protein hydrolysate containing an augmented fraction of isoleucine-tryptophan, or captopril was given to spontaneously hypertensive rats (n = 60) over 14 weeks. Two further groups, receiving either no supplement (Placebo) or intact whey protein, served as controls. Systolic blood pressure age-dependently increased in the Placebo group, whereas the blood pressure rise was effectively blunted by isoleucine-tryptophan, whey protein hydrolysate and captopril (-42 ± 3, -38 ± 5, -55 ± 4 mm Hg vs. Placebo). At study end, myocardial mass was lower in isoleucine-tryptophan and captopril groups but only partially in the hydrolysate group. Coronary flow reserve (1 µm adenosine) was improved in isoleucine-tryptophan and captopril groups. Plasma ACE activity was significantly decreased in isoleucine-tryptophan, hydrolysate and captopril groups, but in aortic tissue only after isoleucine-tryptophan or captopril treatment. This was associated with lowered expression and activity of matrix metalloproteinase-2. Following isoleucine-tryptophan and captopril treatments, gene expression of renin was significantly increased indicating an active feedback within renin-angiotensin system. CONCLUSION: Whey protein hydrolysate and isoleucine-tryptophan powerfully inhibit plasma ACE resulting in antihypertensive effects. Moreover, isoleucine-tryptophan blunts tissue ACE activity, reduces matrix metalloproteinase-2 activity and improves coronary flow reserve. Thus, whey protein hydrolysate and particularly isoleucine-tryptophan may serve as innovative food additives with the goal of attenuating hypertension.

Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR-S6K pathway.

Microbial metabolites, such as short-chain fatty acids (SCFAs), are highly produced in the intestine and potentially regulate the immune system. We studied the function of SCFAs in the regulation of T-cell differentiation into effector and regulatory T cells. We report that SCFAs can directly promote T-cell differentiation into T cells producing interleukin-17 (IL-17), interferon-γ, and/or IL-10 depending on cytokine milieu. This effect of SCFAs on T cells is independent of GPR41 or GPR43, but dependent on direct histone deacetylase (HDAC) inhibitor activity. Inhibition of HDACs in T cells by SCFAs increased the acetylation of p70 S6 kinase and phosphorylation rS6, regulating the mTOR pathway required for generation of Th17 (T helper type 17), Th1, and IL-10(+) T cells. Acetate (C2) administration enhanced the induction of Th1 and Th17 cells during Citrobacter rodentium infection, but decreased anti-CD3-induced inflammation in an IL-10-dependent manner. Our results indicate that SCFAs promote T-cell differentiation into both effector and regulatory T cells to promote either immunity or immune tolerance depending on immunological milieu.

Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster.

Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy-associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI-fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.