A study with pharyngeal and esophageal 24-hour pH-impedance monitoring in patients with laryngopharyngeal symptoms refractory to proton pump inhibitors.

BACKGROUND: The role of gastroesophageal reflux in chronic laryngeal symptoms is difficult to establish. The aim of this study was to characterize pharyngeal and esophageal pH-impedance reflux patterns in a group of patients with suspected laryngopharyngeal reflux and to determine predictive factors of response to proton pump inhibitors. METHODS: Patients with chronic pharyngolaryngeal symptoms were evaluated with a symptom score questionnaire, laryngoscopy, and 24-hour pharyngeal and esophageal pH-impedance monitoring at baseline and after 8-week treatment with esomeprazole 40 mg b.i.d. Response to treatment was defined by a diminution of more than 50% of the score for the primary symptom. Reflux patterns and baseline impedance values were compared to those obtained in 46 healthy subjects. KEY RESULTS: Twenty-four patients were included (17 women, median age 54 years), all previously refractory to antisecretory therapy. Symptom scores were 46 (32-62) and 40 (27-76) off and on therapy, respectively (P=.1). There was no significant difference between patients and controls for pH-impedance reflux parameters and baseline values off and on therapy in distal and proximal esophagus and in the pharynx. Median numbers of pharyngeal reflux were 0 and 0 off and on therapy, respectively. Only two patients were responders to treatment, both with excessive distal reflux but no pharyngeal reflux. Only one patient had abnormal pharyngeal reflux but did not respond to proton pump inhibitors. CONCLUSIONS: Patients with suspected laryngopharyngeal reflux refractory to therapy do not exhibit abnormal pharyngeal or esophageal pH-impedance reflux. In these patients, laryngopharyngeal reflux is unlikely.

Metabolism and effect of platelet-activating factor on the growth of human myeloma cell lines.

In this study we have investigated the presence of PAF receptor (PAF-R) on 5 myeloma cell lines (U266, L363, IM9, OPM2 and XG1), their metabolism of PAF and lyso PAF, and the effect of PAF on their growth. All myeloma cell lines express a PAF acetylhydrolase activity and metabolize [3H]PAF and [3H]lyso PAF in 1-alkyl-2-acyl analogue of phosphatidylcholine. Polymerase chain reaction on reverse transcript (RT-PCR) experiments indicate that OPM2, U266, IM9, XG1 and L363 cells express the PAF-R transcript 1 but not the PAF-R transcript 2. Flow cytometry experiments reveal that PAF-R are present on these myeloma cell lines. PAF and the non-metabolizable PAF agonist 1-O-hexadecyl-2-N-methycarbamyl-glycero-3-phosphocholine have no effect on the growth of OPM2, U266, IM9, XG1 and L363 assessed by [3H]thymidine incorporation into DNA. As a positive control of PAF effect on myeloma cells, PAF (1 microM) enhances by 100% the immunoglobulin synthesis by IM9 cells cultured for 48 h. In conclusion the five myeloma cell lines used in this study metabolize PAF through the deacetylation/reacylation pathway. They express membrane PAF-R through the PAF-R mRNA transcript 1 but PAF does not affect their growth.

Incorporation and effect of arachidonic acid on the growth of the human K562 cell line.

Lipoxygenase inhibitors reduce the growth of K562 cells (chronic myelogenous human leukaemia blasts) suggesting a role for endogenous lipoxygenase products of arachidonic acid (AA) in their proliferation. The objectives of this work are to investigate the incorporation of AA into K562 cells and to assess the effects of the exogenous addition of AA and lipoxygenase products on their growth. The mechanism of acylation of [3H]-AA indicates that K562 cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]-AA is incorporated unmodified into K562 phospholipids and is linked by an ester bond. Prelabelling-chase experiments indicate a transfer of labelled AA from phosphatidylcholine to phosphatidylethanolamine. The addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin B4, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE) has no effect on K562 cell proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while K562 cells readily incorporate AA into their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Arachidonic acid and freshly isolated human bone marrow mononuclear cells.

Arachidonic acid (AA), a fatty acid found in the human bone marrow plasma, is the precursor of eicosanoids that modulate bone marrow haematopoiesis. To further our understanding of the role of AA in the bone marrow physiology, we have assessed its incorporation in human bone marrow mononuclear cells. Gas chromatography analysis indicates the presence of AA in their fatty acid composition. In bone marrow mononuclear cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids, a result not observed with blood mononuclear cells. Prelabelling-chase experiments indicate a trafficking of labelled AA from phosphatidylcholine to phosphatidylethanolamine. Stimulation of prelabelled bone marrow mononuclear cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the release of a part of the incorporated labelled AA. Finally, exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow mononuclear cells participate to the control of marrow AA concentrations by incorporating AA into phospholipids and triglycerides. In turn, bone marrow mononuclear cells can release AA in response to the potent haematopoietic growth factor GM-CSF.

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines.

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.

Effect of platelet-activating factor on the growth of human erythroid and myeloid CD34+ progenitors.

We have assessed the effect of platelet-activating factor (PAF), a biologically active phospholipid present in the human marrow, on the growth of human marrow and blood CD34+ progenitors. While the metabolization rate of PAF by CD34+ cells is low (weak acetylhydrolase and acylation processes) it is readily catabolized by the acetylhydrolase activity present in the growth medium (10% fetal calf serum + 10% 5637-conditioned medium). Treatment of marrow CD34+ cells with the non-metabolizable PAF agonist C-PAF (1 nM to 100 nM) immediately before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E but not of CFU-GM colonies. Treatment of marrow or blood CD34+ cells with C-PAF (10-100 nM) for 3 days in liquid medium before semi-solid culture significantly (P < 0.01) decreased the number of BFU-E and CFU-GM colonies. Treatment of blood CD34+ cells with the two PAF receptor antagonists CV 3988 and BN 52021 (1 microM) had no significant effect on the number of BFU-E and CFU-GM colonies suggesting no role of endogenous PAF in these processes. These results show that exogenous PAF downregulates human erythropoiesis and myelopoiesis, a result that might be of importance during inflammatory states.

Arachidonic acid and human bone marrow stromal cells.

Human bone marrow stromal cells regulate the growth of marrow hematopoietic progenitors by secreting cytokines. Arachidonic acid (AA) is the fatty acid precursor of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that modulate the growth of human bone marrow progenitors. We have investigated the incorporation of AA in human bone marrow stromal cell cultures, their production of PGE2 and LTB4 and the effect of AA on their growth. Gas chromatography analysis reveals the presence of AA in the human bone marrow plasma and in bone marrow stromal cell cultures. In stromal cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids. Prelabeling-chase experiments indicate a preferential incorporation of AA into phosphatidylethanolamine and no trafficking of labeled AA between phospholipid species. Bone marrow stromal cells release PGE2 and LTB4 in response to phorbol myristic acetate (PMA) (1 microM) and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml). Exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow stromal cells capt exogenous AA and, thus, may participate to the control of marrow AA concentrations. They may also regulate human marrow hematopoiesis by secreting AA metabolites such as PGE2 and LTB4.

Platelet-activating factor and antagonists modulate DNA synthesis in human bone marrow stromal cell cultures.

Platelet-activating factor (PAF) is present in the human bone marrow. We have investigated the effect of PAF and antagonists (BN 52,021 and CV 3988) on the growth of human marrow stromal cells. PAF (1 microM) stimulates and PAF antagonists (0.1-1 microM) inhibit [3H]thymidine incorporation in cells grown in 5% serum. The catabolism of PAF by stromal cells was inhibited by CV 3988 suggesting the presence of specific PAF receptor on cells. PAF and antagonists (0.1 nM-10 microM) had no effect on cells cultured in high serum concentration (20%) or in low serum concentration (1%) with 0.5 ng/ml of basic fibroblast growth factor (bFGF). This study indicates for the first time that PAF modulates the serum-induced but not the bFGF-induced growth of marrow stromal cells. The interactions between PAF and stromal cells during inflammatory marrow events such as myelofibrosis deserve to be assessed.

Presence and metabolism of lyso platelet-activating factor in human bone marrow.

Lyso platelet-activating factor (PAF) is the precursor of PAF, an inflammatory phospholipid molecule present in human bone marrow. The present study shows that in healthy volunteers lyso PAF concentrations are significantly lower (P = 0.0001, Mann-Whitney U-test) in bone marrow plasma (594 +/- 67 ng/ml, n = 47) than in blood plasma (1448 +/- 99 ng/ml, n = 31). Marrow plasma lyso PAF concentrations are similar in patients with lymphoid and nonlymphoid malignancies as compared with controls. Freshly isolated mononuclear marrow cells and cultures of marrow stromal cells contain lyso PAF. Experiments with [3H]lyso PAF indicate that human mononuclear bone marrow cells and marrow stromal cells actively acylate lyso PAF into a 1-alkyl analogue of phosphatidylcholine. Results of this investigation indicate: (1) that lyso PAF is present in human marrow cells and plasma; and (2) that marrow cells and stromal cells metabolize it, thus suggesting their role in the regulation of lyso PAF amounts in human bone marrow.

Production and metabolism of platelet-activating factor by human bone marrow cells.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation present in the human bone marrow. Freshly isolated human mononuclear bone marrow cells and marrow stromal cell cultures produced PAF under calcium ionophore (2 microM) and LPS (10 micrograms/ml) stimulation. By contrast, M-CSF (1000 U/ml), GM-CSF (100 ng/ml), IL1, IL3, IL6 and stem cell factor (10 ng/ml) did not stimulate PAF production. Marrow stromal cells produced 50-fold more PAF than freshly isolated mononuclear marrow cells, suggesting that stromal cells might be the major source of the human marrow-derived PAF. Mononuclear marrow cells and stromal cell cultures metabolized PAF with 1-alkyl-2-acyl-glycerophosphocholine as the major metabolic product. PMSF and p-BPB decreased the catabolism of PAF by freshly isolated marrow cells, but not by stromal cell cultures. While stromal cells rather than haematopoietic progenitors might be a major source of the human bone-marrow-derived PAF, both cell types metabolize it, suggesting their putative role in the regulation of PAF concentration in the human bone marrow.

Production, metabolism and effect of platelet-activating factor on the growth of the human K562 erythroid cell line.

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [3H]PAF and [3H]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1-100 nM) stimulated [3H]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.

PAF and haematopoiesis XII: macrophage colony-stimulating factor (M-CSF) decreases platelet-activating factor (PAF) acetylhydrolase production in macrophagic J774 cells.

Platelet-activating factor (PAF) is a phospholipid mediator with major immunoregulatory activities. Macrophages produce PAF acetylhydrolase activity, which regulates blood PAF concentrations. Macrophage colony-stimulating factor (M-CSF) is involved in the differentiation and functions of cells from the monocytic/macrophagic lineage. We found that murine macrophagic J774 cells metabolized PAF with lyso PAF as the major metabolite product. As in mouse plasma, the metabolism of PAF by J774 cells was not inhibited by PMSF, p-BPB, DTNB and quinacrine, M-CSF (100-5000 U/ml) significantly decreased PAF acetylhydrolase activity of the J774 cell without exhibiting a significant effect on cell growth. Elevated concentrations of M-CSF are found in blood and tissues during inflammatory states. It could be suggested that a decreased PAF catabolism by tissue macrophages in response to M-CSF may induce local elevated PAF concentrations, thus amplifying the inflammatory response.

PAF and hematopoiesis. VIII. Biosynthesis and metabolism of PAF by human bone marrow stromal cells.

Human bone marrow stromal cells were studied for their ability to synthesize and to metabolize platelet-activating factor (PAF), a lipidic compound with potent immunoregulatory properties. When stimulated with 2 microM calcium ionophore for 60 minutes, cultures of stromal cells increased their PAF production (3.52 +/- 0.91 ng/1 x 10(6) cells) compared with controls (0.82 +/- 0.13 ng/1 x 10(6) cells). Addition of exogenous lyso PAF (100 nM) and acetyl-CoA (100 microM) during calcium ionophore stimulation did not change the PAF production. The synthesis of PAF was not influenced by the concentration of albumin in the incubation buffer. The PAF from stromal cells exhibited a hexadecyl chain at the sn-1 position of the molecule, as determined by reverse-phase HPLC. While stromal cells contained low amounts of PAF acetylhydrolase activity and did not secrete it in the culture medium, they metabolized exogenous PAF with 1-alkyl-2-acyl-glycero-phosphocholine and neutral lipids as the major metabolic products. The present results are the first to demonstrate the synthesis and metabolism of PAF by human bone marrow stromal cells. These data suggest that they might be a source of the PAF found in the human bone marrow and/or might be important in the regulation of its levels. The role of PAF on the proliferation and functions of human hematopoietic cells deserves investigation.

PAF and haematopoiesis. X. Macrophage colony-stimulating factor and granulocyte macrophage colony-stimulating factor enhance platelet-activating factor acetylhydrolase production by human blood-derived macrophages.

Platelet-activating factor (PAF), a phospholipid autacoid with potent regulatory functions, is synthesized by stimulated monocytes. Macrophages are a source of the plasma acetylhydrolase activity (AHA) which regulates PAF concentrations. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are involved in the differentiation and functions of cells from the monocytic/macrophagic lineage. This work reports that M-CSF and GM-CSF stimulated AHA production by human blood monocyte-derived macrophages in a time- and dose-dependent manner. After 7 days of culture without serum, a 6- and 4-fold increase was found in cells treated with M-CSF (1000 U/ml) and GM-CSF (50 ng/ml), respectively. M-CSF (up to 1000 U/ml) and GM-CSF (up to 10 ng/ml) did not induce PAF production by human blood monocytes. While GM-CSF (10 ng/ml) and interleukin-1 (10 U/ml) stimulated M-CSF production from monocyte-derived macrophages, PAF did not. These results indicate that M-CSF and GM-CSF enhance AHA production by human blood-derived macrophages cultured in low serum concentrations. Clearly the effects of growth factors on AHA production in vivo deserve to be assessed.

PAF and haematopoiesis. IV. Modifications of spleen and thymus PAF contents after a single dose of the chemotherapeutic drug 5-fluorouracil in mice.

The spleen and thymus of mice were examined for the presence of PAF after injection of 5-fluorouracil (5-FU) (200 mg/kg). A significant increase of the spleen (P = 0.005) and thymus (P < 0.05) PAF concentrations was noted 48 h after 5-FU infusion. PAF levels in thymus are similar to those of controls from days 4 to 14. By contrast, spleen PAF significantly decreased (0.005 < P < 0.03) from days 7 to 14. Conversely, the 5-FU administration did not modify the spleen and plasma acetylhydrolase activity, suggesting that the variations of PAF levels in thymus and spleen were mainly due to differences of local PAF production. Thus, the chemotherapeutic drug 5-FU modulates in vivo PAF production in haematopoietic organs of mice. Considering the effects of PAF in the processes of B- and T-cell proliferation and functions, these results could be of importance for the role of PAF during human cancer therapy and haematopoiesis in vivo.

Intestinal absorption of biliary and exogenous cholesterol in the rat.

Non-starved rats (fed a cholesterol-free diet prior to the experiments) with common bile fistula were infused intraduodenally with rat bile labelled with [1,2-3H]cholesterol at a constant rate (0.6 ml/h) and a nutritive mixture containing, in particular, olive oil and 1 mumol [4-14C]cholesterol per ml at rates of 1 ml/h (group B) or 2.3 ml/h (group A) for 5 h. Control rats (group C) were prepared as group B rats but the nutritive mixture was free of cholesterol. 1 h after the end of infusions, the animals were killed. Biliary and exogenous cholesterol were absorbed in the upper two-thirds of the small intestine; a large proportion of 3H and 14C radioactivity was present in the mucosa, but cholesterol from exogenous origin went across the mucosa more rapidly than cholesterol from biliary source. These observations suggest the existence of a non-homogeneous luminal mixture of molecules of cholesterol from different sources. The luminal dilution of [3H]- and [14C]sterols by non-labelled sterols increased from the proximal to the distal part of the small intestine. Precursor sterols and coprosterol were present in the stomach contents and in the lumen of caecum, colon and feces.