Expression patterns of cytokines, p53 and nitric oxide synthase enzymes in corpora lutea of pseudopregnant rabbits during spontaneous luteolysis

The gene expressions for macrophage chemoattractant protein-1 (MCP-1), interleukin (IL)-1 beta, IL-2 and p53 were examined by semi-quantitative RT-PCR in corpora lutea (CL) of rabbits during spontaneous luteolysis at days 13, 15, 18 and 22 of pseudopregnancy. In the same luteal tissue, total activity of nitric oxide (NO) synthase (NOS) and genes for both endothelial (eNOS) and inducible (iNOS) isoforms were also analysed. From day 13 to 15, MCP-1 and IL-1 beta mRNA levels rose (P < or = 0.01) almost 2-fold, and the transcript for p53 almost 8-fold, but then all dropped (P < or = 0.05) from day 18 onward. IL-2 mRNA abundance was higher (P < or = 0.01) on day 13 and then gradually declined. During luteolysis, eNOS mRNA decreased 40% (P < or = 0.05) by day 15, but thereafter remained unchanged, while iNOS mRNA was barely detectable and did not show any clear age-related pattern throughout the late luteal stages. Total NOS activity progressively increased (P < or = 0.01) from day 13 to 18 of pseudopregnancy and then dropped to the lowest (P < or = 0.01) levels on day 22. Luteal progesterone content also declined during CL regression from 411 to 17 pg/mg found on days 13 and 22 respectively, in parallel with the decrease in blood progesterone concentrations. These data further support a physiological role of NO as modulator of luteal demise in rabbits. Locally, luteal cytokines may be involved in the up-regulation of NOS activity, while downstream NO may inhibit steroroidogenesis and induce expression of p53 gene after removal of the protective action of progesterone

The physiological dilemma of the high progesterone syndrome in rabbit does

This work focused on the mechanisms that may cause multiple asynchronous ovulations and alter normal ovarian function in order to characterize the high progesterone (P+) syndrome in rabbit does, that, having abnormally high plasma progesterone concentration at the time of insemination, fail to become pregnant. At different luteal stages, at either days 4, 9, or 13 of pseudopregnancy, induced by GnRH injection (d-0), two groups of rabbits (n=5/group) were treated with saline or 0.8 \ub5g GnRH. Blood samples were collected from d-0 to d-26 of pseudopregnancy. At d-4, GnRH injection prolonged (P<0.05) the functional CL life span by 3 to 4 d over that of controls. At d-9, GnRH caused a transient decline (P<0.01) of progesterone for the following 3 d but, thereafter, increased again and remained higher (P<0.01) than controls up to d-26. At d-13, progesterone fell to 1 ng/ml within one day following GnRH, but then gradually increased. Based of these progesterone profiles, it can be argued that, at both mid- and late-luteal phase, GnRH triggered luteolysis and induced ovulation followed by the formation of a new generation of CL. For the in vitro study, CL, collected at days 4, 9, and 13 of pseudopregnancy, were incubated with GnRH, GnRH-antagonist, PLA2 inhibitor, and PLC inhibitor. GnRH decreased (P<0.01) progesterone secretion by d-9 and d-13 CL cultured in vitro; by converse, GnRH antagonist, increased (P<0.01) progesterone release from d-4 CL. Co-incubation of GnRH with GnRH antagonist increased (P<0.01) progesterone release in d-4 CL, but had an opposite effect (P<0.01) on d-9 and d-13 CL. PLC inhibitor reversed the GnRH effects in both d-9 and d-13 CL, while PLA2 inhibitor did not change progesterone release. These data suggest that rabbit CL express a functional receptor for GnRH, likely of type II, that utilizes the PLC post transductional cascade. Luteal FSH-R and LH-R mRNA relative abundances did not differ between d-4 and d-9 CL, but were two- to three-fold (P 640.01) higher, respectively, at d-13. StAR mRNA was highly expressed at d-4 of pseudopregnancy, but then markedly declined (P 640.01) at d-9 and d-13. Taken together, our results show that GnRH triggers i) functional regression when CL acquire luteolytic capacity from d 9 of pseudopregnancy onward, and ii) multiple asynchronous ovulations, thus partly explaining the P+ syndrome associated with the simultaneous coexistence of two population of \u201cfresh\u201d and \u201cold\u201d CL, although not yet the underlying causes

Gluten-free diet and gut microbiome

As the only effective therapy against diagnosed celiac disease (CD), the gluten-free diet (GFD) has inevitable repercussion on the gut microbiome composition and functionality. Being the cause or the consequence of the disease, an altered homeostasis of the gut microbiome usually affects CD patients at diagnosis. After describing the main features of this altered physiological condition, this review defines the main nutritional aspects of the GFD and elucidates how this diet regimen does not fully restore the optimal gut microbiome composition and functionality. Unbalanced ratios between beneficial and potentially harmful bacteria are frequently present in fecal materials, biopsy specimens and saliva, used as ecological model systems to observe CD. Metabolome analyses also show how an altered microbiome synthesize different metabolite with respect to healthy conditions. The review concludes illustrating the current supplementations (biotics family), which fortify the GFD with the aim of restoring the homeostasis of the gut microbiome

Feeding with sustainably sourdough bread has the potential to promote the healthy microbiota metabolism at the colon level

The contribution of sustainably food processing to healthy intestinal microbial functions is of recent acquisition. The sourdough fermentation fits well with the most sustainable bread making. We manufactured baker's yeast (BYB) and sourdough (t-SB30) breads, which first underwent to an in-depth characterization. According to nutritional questionnaires, we selected 40 volunteers adhering to the Mediterranean diet. Data on their fecal microbiota and metabolome allowed the selection of two highly representative fecal donors to separately run the Twin Mucosal-SHIME (Twin M-SHIME) under 2-week feeding with BYB and t-SB30. Bread feeding did not affect the microbial composition at phylum and family levels of both donors, in all Twin M-SHIME colon tracts, and lumen and mucosal compartments. The genus core microbiota showed few significant fluctuations, which regarded the relative abundances of Lactobacillus and Leuconostoc according to feeding with BYB and t-SB30, respectively. Compared with BYB, the content of all short chain fatty acids (SCFA), and isovaleric and 2-methylbutyric acids significantly increased with t-SB30 feeding. This was evident for all Twin M-SHIME colon tracts and both donors. The same was found for the content of Asp, Thr, Glu, GABA, and Orn. The bread characterization made possible to identify the main features responsible for this metabolic response. Compared with BYB, t-SB30 had much higher contents of resistant starch, peptides, and free amino acids, and an inhomogeneous microstructure. We used the most efficient approach to investigate a staple food component, excluding interferences from other dietary factors and attenuating human physiology overlaps. The daily consumption of sourdough bread may promote the healthy microbiota metabolism at colon level. IMPORTANCE Knowledge on environmental factors, which may compose the gut microbiota, and drive the host physiology and health is of paramount importance. Human dietary habits and food compositions are pivotal drivers to assemble the human gut microbiota, but, inevitably, unmapped for many diet components, which are poorly investigated individually. Approximately 30% of the human diet consists of fermented foods and beverages. Bread, a fermented/leavened food, is a basic component of the human diet. Its potential effect on gut microbiota composition and functionality is challenging. In this study, we industrially made baker's yeast and sourdough breads, which were used to feed the Twin Mucosal-SHIME, a worldwide scientifically validated gastrointestinal simulator. Only the consumption of sourdough bread has the potential to enhance the synthesis of short chain fatty acids and free amino acids at the colon level

A survey of the main technology, biochemical and microbiological features influencing the concentration of biogenic amines of twenty Apulian and Sicilian (Southern Italy) cheeses

Abstract Twenty Apulian and Sicilian cheeses were analysed for their concentrations of eight biogenic amines (BAs), free amino acids, pH, water activity, and subjected to microbiological characterisation. In addition, lactic acid bacteria isolated from cheeses were assayed for their capacity to generate BAs. Principal component analysis was performed to find the effect of different parameters on the distribution of the cheeses. Although short-ripened (≤30 d) cheeses did not show significant BA concentrations, the only BA showing high positive correlation with time of ripening was histamine. Concentration of histidine and, especially, percentage of histidine-decarboxylase bacteria presumably affected histamine concentration. High pH values were negatively correlated to the concentration of tyramine, putrescine, and cadaverine. Fifty percent of the cheeses contained at least one BA at potentially toxic concentrations. Unambiguous and ever-valid relations among parameters and BAs are difficult to determine, because BAs are the result of combined and varied factors

Vasoactive peptides in the luteolytic process activated by PGF2alpha in pseudopregnant rabbits at different luteal stages

To study the role of endothelial factors in luteal function, the dynamic profiles of genes for endothelin 1 (EDN1), its receptor subtypes, EDNRA and EDNRB, and angiotensin converting enzyme (ACE) were examined in corpora lutea (CL) obtained from rabbits on Days 4 and 9 of pseudopregnancy after prostaglandin (PG) 172a analogue (alfaprostol) treatment. The cell type distribution of EDN1 in the ovaries and its mechanisms of actions in vitro and in vivo were also studied. Positive immunostaining for EDN1 was localized in the luteal and endothelial cells, in granulosa cells of the follicles, and in the ovarian epithelium. The basal mRNA levels for EDNRA, EDNRB, and ACE were lower (P <= 0.01) in Day-4 CL than in Day-9 CL, whereas those for EDNi did not differ between these two time-points. On Day 4, the luteal EDN1, EDNRA, EDNRB, and ACE mRNA levels were similarly increased two-fold (P <= 0.01) 1.5 h after alfaprostol injection, and did not show further changes in the subsequent 24 h. On Day 9, alfaprostol challenge transiently up-regulated (P < 0.01) the luteal ACE transcripts at 1.5 h, and those of EDN1 at 1.5 h and 3 h, whereas the EDNRA and EDNR8 transcript levels remained unchanged during the course of luteal regression. EDN1 decreased (P < 0.01) progesterone release and increased (P <= 0.01) PGF2 alpha secretion and NOS activity via the PLC/PKC pathway in Day-9 CL, but not in Day-4 CL, cultured in vitro. EDN1-induced, but not alfaprostol-induced luteolysis, was blocked by cotreatment in vivo with the ACE antagonist captopril. These findings support the hypothesis that PGF2 alpha regulates luteolysis through intraluteal activation of the reninangiotensin/EDN1 systems in CL that have acquired luteolytic competence

Effects of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 in IBS patients

Background: Irritable bowel syndrome (IBS) is a common gastrointestinal disorder, which still lacks effective therapy. We aimed to investigate the effects of a novel formulation of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001 with vitamin B6 (LBB) on symptoms, intestinal permeability, cultivable bacteria and metabolome in IBS subjects. Materials and methods: Twenty-five IBS patients (Rome IV criteria) (M:F = 8:17; age 48 years ± 11 SD) were randomized to treatment (LBB) or placebo (one month each) in a crossover randomized double-blind controlled trial. Symptoms, intestinal habits, disease severity, intestinal permeability and intestinal microbiota were analysed at 0, 30, 45 and 60 days. Results: Percentage decrease from baseline of abdominal pain (−48.8% vs −3.5%), bloating (−36.35% vs +7.35%) and severity of disease (−30.1% vs −0.4%) was significantly (P <.0001) greater with LBB than placebo, respectively. In IBS-D patients, the improvement from baseline of Bristol score was more consistent with LBB (from 6 ± 0.4 to 4.3 ± 1.1, P <.00001) than placebo (from 6.2 ± 0.7 to 5.3 ± 1.1, P =.04). In IBS-C patients, Bristol score tended to improve from baseline after LBB (2.6 ± 1.1 vs 3.2 ± 0.5, P =.06). LBB significantly improved the percentage of sucralose recovery (colonic permeability) (1.86 ± 0.1 vs 1.1 ± 0.2, P =.01). During treatment, presumptive lactic acid bacteria and bifidobacteria, relative abundance of propanoic, butanoic, pentanoic acids and hydrocarbons increased, while phenol decreased. Conclusions: The novel formulation of B. longum BB536 and L. rhamnosus HN001 with B6 vitamin improves symptoms and severity of disease, restores intestinal permeability and gut microbiota in IBS patients

Role of endothelin-1 system in the luteolytic process of pseudopregnant rabbits

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 mug iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ETA/ETB receptor antagonist, or cyclooxygenase ( COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F-2alpha (PGF(2alpha)). In CL cultured in vitro, ET-1 increased (P less than or equal to 0.01) both PGF(2alpha) production and luteal nitric oxide synthase activity but decreased (P less than or equal to 0.01) progesterone release. Addition of ETA receptor antagonist BQ123 or COX inhibitor blocked the ET-1 luteolytic effects. Positive staining for ET-1 receptors was localized in ovarian blood vessels, granulosa cells of large follicles, and luteal cells. Immunoblot analysis of ET-1 receptor protein revealed a strong band of approximately 48 kDa in d-9 CL. Up to d 6 of pseudopregnancy, ET-1 mRNA abundance in CL was poorly expressed but then increased (P less than or equal to 0.01) at d 9 and 13. ETA-receptor transcript increased (P less than or equal to 0.01) at d 6, remained at the same level up to d 13, and then declined to the lowest (P less than or equal to 0.01) levels at d 22. ETB-receptor mRNA increased (P less than or equal to 0.01) throughout the late-luteal stage from d 13 up to d 18. Our data suggest that the luteolytic action of ET-1 may be a result of PGF(2alpha) synthesis from both luteal and accessory cells, via the COX pathways

Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2alpha and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2alpha, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2alpha release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2alpha production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system

Prostaglandin receptors and role of G protein-activated pathways on corpora lutea of pseudopregnant rabbit in vitro

Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions