Cord blood mesenchymal stem cells suppress DC-T Cell proliferation via prostaglandin B2.

Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases.

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes.

Osteoblast-derived oxysterol is a migration-inducing factor for human breast cancer cells.

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.

Structural insights into human serum albumin-mediated prostaglandin catalysis.

Previous studies have shown that many arachidonic acid metabolites bind to human serum albumin (HSA) and that the metabolism of these molecules is altered as a result of binding. The present study attempted to gain insights into the mechanisms by which prostaglandins bound to subdomain 2A of HSA are metabolized by catalytic processes. The breakdown of the prostaglandin 15-keto-PGE(2) to 15-keto-PGA(2) and 15-keto-PGB(2) in the presence of wild-type HSA and a number of subdomain 2A mutants was examined using a previously validated spectroscopic method which monitors absorbance at 505 nm. The species examined using this method were wild-type HSA, K195M, K199M, F211V, W214L, R218M, R218P, R218H, R222M, H242V, R257M, and bovine serum albumin. Previous studies of HSA-mediated catalysis indicated that the breakdown of HSA-bound prostaglandins results from an alkaline microenvironment in the binding site. Our results show that the catalytic breakdown of HSA-bound 15-keto-PGE(2) to 15-keto-PGB(2) results from two specific processes which are modulated by specific amino acid residues. Specifically, some amino acid residues modulate the rate of step 1, the conversion of 15-keto-PGE(2) to 15-keto-PGA(2), while other residues modulate the rate of step 2, the conversion of 15-keto-PGA(2) to 15-keto-PGB(2). Some residues modulate the rate of steps 1 and 2. In total, while our results support the involvement of certain basic amino acid residues in the catabolism of HSA-bound 15-keto-PGE(2), our data suggest that metabolism of HSA-bound prostaglandins may be a more complex and specific process than previously thought.

The wide binding properties of a wheat nonspecific lipid transfer protein. Solution structure of a complex with prostaglandin B2.

The 3D solution structure of wheat nonspecific lipid transfer protein (ns-LTP) complexed with prostaglandin B2, a lipid with both vinyl and hydroxylated groups, has been determined by 1H 2D NMR. The global fold of the protein is close to the previously published structures of wheat, maize, barley and rice ns-LTPs. The ligand is almost completely embedded in the hydrophobic core of the protein. Structure comparisons of free and bound wheat ns-LTP reveal that the binding of prostaglandin B2 hardly affects the global fold of the protein. The structural data on this unusual complex are discussed and compared with other known ns-LTP lipid-complexes.

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60 percent, at the same dose PGA1 suppressed virus replication by more than 90 percent. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells.

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 micrograms/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

Arachidonic acid metabolism during early development of ovine embryos: a possible relationship to shedding of the zona pellucida.

Two experiments were conducted to determine if early ovine embryos produce prostaglandins and if prostaglandins may have a role in the shedding of the zona pellucida, i.e., embryo hatching. For Experiment 1, embryos were collected on day (d) 4, 8, 10, 12 or 14 of pregnancy and incubated with 1 microCi of [14C] arachidonic acid (AA) for 24 h. Based upon high-performance liquid chromatography, embryos from all days converted AA to a number of compounds; the amounts produced differed with day. Primarily, embryos produced an unidentified polar compound, 6-keto-PGF1 alpha, PGF2 alpha, PGE2, 13,14-dihydro-15-keto-PGF2 alpha and PGB2. For Experiment 2, embryos collected on d 7 of pregnancy were incubated for a maximum of 6 d in 500 microL of medium containing either ethanol (control; 20 microL), indomethacin (INDO; 10(-4) M), PGE2 (2 ng) or INDO (10(-4) M) + PGE2 (2 ng). Embryos were evaluated daily for hatching from the zona pellucida. The hatching rates (percentage) for control, INDO, PGE2 and INDO + PGE2 were 46.4, 34.5, 60.0, and 30.0, respectively. There was a main effect (P < .09) of treatment, and the hatching rate for embryos treated with PGE2 alone was greater (P < .05) than that for embryos in any group with INDO. The results indicate that early ovine embryos can convert AA to various compounds in vitro, and prostaglandins may have a role in the hatching of sheep embryos from the zona pellucida.

Prostaglandin E2 binding sites in human renal tissue: characterization and localization by radioligand binding and autoradiography.

The prostaglandin E2 (PGE2) binding site in human kidney was characterized in membrane preparations from cortex, outer medulla and inner medulla using radioligand binding techniques. The localization of the binding sites for [3H]PGE2 was visualized autoradiographically. In the membrane suspensions, the highest level of specific [3H]PGE2 binding was detected in the outer medulla (Bmax = 335 +/- 28 fmol mg-1 protein) followed by the inner medulla (Bmax = 258 +/- 21 fmol mg-1 protein) and the cortex (Bmax = 143 +/- 22 fmol mg-1 protein). The binding was of high affinity with KD values between 3.7 and 6.2 nM in the various regions. Unlabelled prostaglandins competed for the [3H]PGE2 binding sites in the following rank order of potency: PGE2 approximately PGE1 greater than PGF2 alpha approximately PGA2 greater than PGB2 greater than PGI2 approximately PGD2. Autoradiographs revealed that a high density of [3H]PGE2 (2 nM) binding sites were located on the distal tubule, particularly on the thick ascending limbs of Henle. Lower densities of [3H]PGE2 binding sites were found on the medullary collecting ducts and possibly on the thin loops of Henle. In contrast, no specific [3H]PGE2 binding could be found on the proximal tubule, glomeruli or on blood vessels. This distribution is in accordance with the assumed site of action for the salt and water regulatory function of PGE2.

Oxidative metabolism of dihomogammalinolenic acid by guinea pig epidermis: evidence of generation of anti-inflammatory products.

Reports that vegetable oils which contain gamma-linolenic acid (18:3n-6) may exert beneficial effects on cutaneous disorders prompted us to investigate whether epidermis possesses the ability to transform dihomogammalinolenic acid (20:3n-6), the epidermal elongase product of 18:3n-6, into oxidative metabolites with anti-inflammatory potential. Incubations of [1-14C]20:3n-6 with the 105,000 g particulate (microsomal) fraction from guinea pig epidermal homogenate resulted in the formation of the 1-series prostaglandin PGE1. The identity of this product was confirmed by argentation thin-layer chromatography (TLC), reverse phase-HPLC, and conversion with alkali treatment to PGB1. Incubations of [1-14C]20:3n-6 with the 105,000 g supernatant (cytosolic) fraction from guinea pig epidermal homogenate resulted in the formation of the 15-lipoxygenase product 15-hydroxy-8, 11, 13-eicosatrienoic acid (15-OH-20:3n6). The identity of this product was confirmed by normal phase-HPLC and gas chromatography/mass spectrometry (GC/MS). Thus, data from these studies indicate the capacity of enzymes in the microsomal and cytosolic fractions of guinea pig epidermal homogenates to transform 20:3n-6 to the eicosanoids PGE1 and 15-OH 20:3n-6, products which reportedly have anti-inflammatory properties. The in vivo significance of these findings remains to be explored.

Isolation of 19,20-dehydroprostaglandins E1 and E2 in human seminal fluid and further studies on 18,19-dehydroprostaglandins E1 and E2.

Human seminal fluid was recently found to contain 18,19-dehydroprostaglandins E1 and E2 (E. H. Oliw, H. Sprecher, and M. Hamberg, (1986) J. Biol. Chem. 261, 2675-2683). In the present study, the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2 were prepared by incubation of microsomes of ram vesicular glands and glutathione with the precursor fatty acids, 8(Z),11(Z),14(Z),18(E/Z)-eicosatetraenoic acids, and 5(Z),8(Z),11(Z),14(Z),18(E/Z)-eicosapentaenoic acids, and used as references to characterize the 18,19-dehydroprostaglandins of human seminal fluid. Based on separation by reversed-phase high-performance liquid chromatography, capillary gas chromatography-mass spectrometry, and ozonolysis of the (-)-menthoxycarbonyl derivatives and on comparison with the authentic compounds, human seminal fluid was found to contain both the cis and trans isomers of 18,19-dehydroprostaglandins E1 and E2. Furthermore, human seminal fluid contained two related compounds, viz. 19,20-dehydroprostaglandins E1 and E2. The structures of these compounds were established by conversion into the corresponding prostaglandin B compounds, by mass spectrometric analysis and by chemical degradation by oxidative ozonolysis, which afforded, inter alia, 2(S)-hydroxy-adipic acid.

Role of endogenous gastric mucosal prostaglandins in the formation of acute gastric mucosal lesions induced by aspirin, ethanol, HCl and CH3COOH.

The role of endogenous mucosal prostaglandins (PGs) in the production of acute gastric mucosal lesions (AGML) was examined in rats. Aspirin, ethanol or 0.6 N-HCl was given intragastrically and 20% acetic acid was injected into the gastric wall. Endogenous gastric mucosal PG (A + B), PGE and PGF were determined by radioimmunoassay. Their gastric contents were markedly reduced by aspirin administration (p less than 0.001). The level of gastric mucosal PGs still remained low (p less than 0.001) after the aspirin-induced AGML began to heal. Furthermore, rats with AGML induced by ethanol, HCl or acetic acid, showed no decrease in endogenous gastric mucosal PGs compared with the controls. These findings indicated that endogenous PGs are not necessary for either the induction or healing of experimental AGML.

The effect of NaCl intake on 9-ketoprostaglandin reductase activity in the rabbit kidney.

Renal 9-ketoprostaglandin reductase activity from rabbits fed 0.3 g or 2.5 g NaCl per 100 g chow was measured in both centrifuged homogenates and in purified enzyme fractions. There was no salt related increase in 9-ketoprostaglandin reductase activity. PGA1-glutathione, 9, 10-phenanthrenequinone, and 4-nitrobenzaldehyde were better substrates for the purified 9-ketoprostaglandin reductases than was PGE2. Several carbonyl reductases were isolated which used PGA1-glutathione, 9, 10-phenanthrenequinone, and 4-nitrobenzaldehyde, but not PGE2, as substrates. Although PGA1-glutathione was a more faithful indicator of PGE2-related 9-ketoprostaglandin reductase activity than either 9, 10-phenanthrenequione or 4-nitrobenzaldehyde, it did not always provide an accurate estimate of that activity.

Presence of prostaglandins (PGs) in Tetrahymena pyriformis, GL and the effect of aspirin.

Tetrahymena pyriformis GL appears to require prostaglandins, either B, E or F series for growth, as demonstrated by the deleterious effect of aspirin. The latter inhibits prostaglandin synthetase (cyclooxygenase). Aspirin inhibited 50% growth of a 24 hr culture of T. pyriformis at a dose of approximately 200 micrograms/ml and completely inhibited at 600 micrograms/ml. Extraction with acid ethyl acetate: isopropanol solvent of 2.8 x 10(8) cells yielded 41.3 mg of lipid of which 62.8% and 34.5% were PGE2 and PGB, respectively. It is suggested that PGs are important for the growth of T. pyriformis and that the organism may be a useful source of natural PGs. Additionally, T. pyriformis may be useful in studies of the PGs pathway.

Studies on the mechanism of mitochondrial protection by polymeric prostaglandin PGBx.

On the basis of the interaction between tritiated PGBx and rat liver mitochondria, it appears that PGBx interacts with rat liver mitochondria to form a complex. At low PGBx-mitochondrial ratios, one effect of this complex formation is to stabilize the phosphorylation activity of rat liver mitochondria when exposed for short times to hypotonic solutions. At higher PGBx-mitochondrial ratios, PGBx fails to show this effect. At low PGBx concentrations, PGBx is also shown to inhibit release of amino acids and proteins as well as glutamic acid dehydrogenase and monoamine oxidase from the mitochondria and to inhibit mitochondrial swelling.

[Prostaglandin levels in amniotic fluid during normal advanced pregnancy (author's transl)]. / Prostaglandinspiegel im Fruchtwasser während der normalen Spätschwangerschaft.

Radio-immuno assay was used, following extraction and fractionation, to measure primary prostaglandins in the amniotic fluid of 88 women in advanced pregnancy under normal conditions. No change was recordable from prostaglandin fraction B + A, whereas exponential rises were determined over the last four to six weeks of pregnancy for prostaglandins F and E. Prostaglandin E levels went up from 100 pg/ml amniotic fluid, between the 29th and 34th weeks of pregnancy, to 380 pg/ml, between the 39th and 40th weeks, whereafter they remained constant. Prostaglandin F levels were between 70 pg/ml and 100 pg/ml and went up to 422 pg/ml, with a mean value as high as 494 pg/ml being measured in the 41st and 42nd weeks of pregnancy. The prostaglandin E/F quotient decreased close to full term, between the 39th and 42nd weeks of pregnancy, on account of steeper rise of prostaglandin F. Prostaglandin approached nanogram values, more pronouncedly characteristic of childbirth, few hours before onset of regular pains.