BACKGROUND

Pyomyositis is typically caused by gram-positive bacteria, especially Staphylococcus aureus. Few cases of Escherichia coli pyomyositis have been reported, including only 1 involving a patient with a hematologic malignancy.

METHODS

The clinical microbiology database at The M. D. Anderson Cancer Center (Houston, TX) was reviewed for the period January 2003 through December 2007 to identify cases of E. coli pyomyositis. Clinical characteristics, laboratory and radiologic findings, treatment, and outcomes were recorded. Available isolates underwent phylogenetic group determination, virulence genotyping, multilocus sequence typing, repetitive-element polymerase chain reaction, and pulsed-field gel electrophoresis.

RESULTS

Six cases of E. coli pyomyositis were identified. All patients were receiving chemotherapy for a hematologic malignancy; 5 were severely neutropenic. Three patients became hypotensive, 2 required intensive care, and 2 (33%) died, despite receiving carbapenem therapy. All E. coli isolates were fluoroquinolone resistant; 55% produced an extended-spectrum beta-lactamase (ESBL). Five of 6 available isolates belonged to phylogenetic group B2, had similar virulence factor profiles, exhibited>> 95% similar repetitive-element polymerase chain reaction profiles, and represented sequence type ST131; however, all had unique pulsed-field gel electrophoresis profiles.

CONCLUSIONS

E. coli pyomyositis has emerged as a serious problem among our patients with hematologic malignancy. It usually is caused by members of E. coli ST131, a recently identified cause of fluoroquinolone-resistant, ESBL-positive E. coli infection worldwide. Awareness of this emerging syndrome and the usual causative agent is important to ensure appropriate management when febrile, neutropenic patients with hematologic malignancy exhibit signs of localized muscle infection.

Two different types of low-density detergent-insoluble glycosphingolipid-enriched membrane domain (DIG) fractions were isolated from myelin by extraction with Triton X-100 (TX-100) in 50 mM sodium phosphate buffer at room temperature (20 degrees C) (procedure 1), in contrast to a single low-density fraction obtained by extraction with TX-100 in Tris buffer containing 150 mM NaCl and 5 mM EDTA at 4 degrees C (procedure 2). Procedure 1 has been used in the past by others for myelin extraction to preserve the cytoskeleton and/or radial component of oligodendrocytes and myelin, whereas procedure 2 is now more commonly used to isolate myelin DIG fractions. The two DIG fractions obtained by procedure 1 gave opaque bands, B1 and B2, at somewhat lower and higher sucrose density respectively than myelin itself. The single DIG fraction obtained by procedure 2 gave a single opaque band at a similar sucrose density to B1. Both B1 and B2 had characteristics of lipid rafts, i.e. high galactosylceramide and cholesterol content and enrichment in GPI-linked 120-kDa neural cell adhesion molecule (NCAM)120, as found by others for the single low-density DIG fraction obtained by procedure 2. However, B2 had most of the myelin GM1 and more of the sulfatide than B1, and they differed significantly in their protein composition. B2 contained 41% of the actin, 100% of the tubulin, and most of the flotillin-1 and caveolin in myelin, whereas B1 contained more NCAM120 and other proteins than B2. The single low-density DIG fraction obtained by procedure 2 contained only low amounts of actin and tubulin. B1 and B2 also had size-isoform selectivity for some proteins, suggesting specific interactions and different functions of the two membrane domains. We propose that B1 may come from non-caveolar raft domains whereas B2 may derive from caveolin-containing raft domains associated with cytoskeletal proteins. Some kinases present were active on myelin basic protein suggesting that the DIGs may come from signaling domains.

BACKGROUND

Iron is an important modulator of lipid peroxidation, and its levels have been associated with the progression of atherosclerosis. Little is known about the possibility that this metal, when released from tissue stores, may modulate the reactivity of blood cell components, in particular platelets. Therefore, we investigated a possible link between iron, oxygen free radical formation, and platelet function.

RESULTS

Human whole blood was stimulated with collagen 2 micrograms/mL, and an irreversible aggregation with thromboxane (Tx)B2 formation was observed (15+/-4 versus 130+/-10 ng/mL). Deferoxamine (DSF), a specific iron chelator, and catalase, an H2O2 scavenger, inhibited collagen-induced whole-blood aggregation. The aggregation was accompanied by an increase in hydroxyl radical (OH.) levels (30+/-8 versus 205+/-20 nmol/L dihydroxybenzoates), which were reduced by DSF and by 2 specific OH. scavengers, mannitol and deoxyribose. Iron (Fe2+) dose-dependently induced platelet aggregation, TxB2 formation (6+/-2 versus 135+/-8 ng/mL), and protein kinase C (PKC) translocation from the cytosol to the cell membrane when added to platelets that have been primed with a low concentration of collagen (0.2 micrograms/mL). In the same system, an increase in OH. levels was observed (37+/-12 versus 230+/-20 nmol/L dihydroxybenzoates). Mannitol and deoxyribose, but not urea, were able to reduce OH. formation, PKC activation, and platelet aggregation. Selective inhibition of PKC activity by GF 109203X prevented iron-dependent platelet aggregation without influencing OH. production.

CONCLUSIONS

The present study shows that iron can directly interact with human platelets, resulting in their activation. Its action is mediated by OH. formation and involves PKC activity. Our findings provide an additional contribution to the understanding of the mechanism(s) by which iron overload might promote atherosclerosis and coronary artery disease.

Rat carrageenin-induced pleurisy was used to clarify the role of prostaglandin H synthase (PGHS)-2 in acute inflammation. Intrapleural injection of 0.2 ml of 2% lambda-carrageenin induced accumulation of exudate and infiltration of leukocytes into the pleural cavity. When PGHS-1 and -2 proteins in the pleural exudate cells were analyzed by Western blot analysis, PGHS-2 was detectable from 1 hr after carrageenin injection. Its level rose sharply, remained high from 3 to 7 hr after injection, and then fell to near the detection limit. PGHS-1 was also detected, but kept almost the same level throughout the course of the pleurisy. Levels of prostaglandin (PG) E2 and thromboxane (TX) B2 in the exudate increased from hour 3 to hour 7, and then declined. Thus, the changes of the level of PGE2 were closely paralleled those of PGHS-2. The selective PGHS-2 inhibitors NS-398, nimesulide and SC-58125 suppressed the inflammatory reaction and caused a marked decrease in the level of PGE2 but not in those of TXB2 and 6-keto-PGF 1 alpha. These results suggest that the PGHS-2 expressed in the pleural exudate cells may be involved in PGE2 formation at the site of inflammation.

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

The formation of arachidonic acid metabolites [prostaglandin (PG)D2, PGE2, PGF2 alpha, 6oxo-PGF1 alpha and thromboxane (TX)B2] has been examined in the frontal, temporal, and parieto-occipital regions of the cortex in the postmortem brains from five patients with a pathologically verified Alzheimer-type dementia (ATD) and in eight, age-matched controls. The study disclosed that the amount of PGD2 and TXB2 found in the ATD brains was greater than in controls, and that the formation of PGD2 was significantly increased in the frontal cortex of the ATD brains. Other PG metabolites, however, showed no significant changes in the ATD group. Since it is known that PGD2 is synthesized mainly in neurons, our results seem to suggest that the preserved cortical neurons in the ATD brain have an accelerated PGD2 production.

Hypertension is the main side effect developing in patients suffering from renal anemia who are treated with recombinant human erythropoietin (rHuEPO). We investigated the effect of rHuEPO on the vascular tone of isolated rabbit aorta and carotid artery under isometric conditions. The production of prostacyclin and the vasoconstrictor prostanoids PGF2 alpha and TXB2 was investigated in arterial rings incubated with rHuEPO. Endothelial cells from human umbilical veins (HUVECs) were isolated and cultured in flasks (37 degrees C, 5% CO2). After incubation with rHuEPO, the formations of prostacyclin (as its stable metabolite 6-keto-PGF1 alpha), PGF2 alpha, PGE2, thromboxane (TX) B2 and of ET-1 were measured by radioimmunoassays. rHuEPO had no direct vasoconstrictor effect, but it enhanced noradrenalin-induced contractions. This effect was more prominent in rings with intact endothelium than in rings from which the endothelium had been mechanically removed, indicating that endothelial vasoactive factors might be involved. Relaxations to acetylcholine (ACh, 1 microM) were unaltered in the presence or absence of rHuEPO, suggesting that the endothelial NO-cGMP pathway was not impaired by rHuEPO. Incubation with rHuEPO (20 to 200 U/ml) increased the release of the vasoconstrictor mediators ET-1, PGF2 alpha and TXB2, and decreased prostacyclin formation in isolated rabbit arterial rings and in HUVECs, respectively. The cyclooxygenase inhibitor indomethacin abolished the rHuEPO-induced increase in vasoconstrictor prostanoid production. ET-1 formation by HUVECs was also increased by rHuEPO in a dose-dependent manner (maximal effect +90% by rHuEPO 200 U/ml, P < 0.05). Indomethacin and the selective ETA receptor antagonist BQ123 each partly inhibited the enhancement of vascular responsiveness to noradrenalin induced by rHuEPO in rabbit carotid artery, but simultaneous administration of rHuEPO with both antagonists completely abolished the force increment. In conclusion, these studies show that a dose-dependent shift in the balance of constrictor and relaxing prostanoids as well as an increased synthesis of ET-1 induced by rHuEPO lead to the enhanced vascular responsiveness to noradrenalin in isolated rabbit arteries. The increased vascular responsiveness to noradrenalin, which is in line with clinical observations, may contribute to the hypertensive side effect associated with rHuEPO therapy in patients with chronic renal failure.

Isolated perfused sensitized guinea pig hearts release relatively large amounts of radioimmunologically measurable thromboxane B2 (TXB2) as well as smaller amounts of prostaglandin (PGs) after antigenic challenge. Using thin layer chromatography the major PG released was shown to cochromatograph with PGD2, while smaller amounts of immunoreactive PGF2alpha were found. The TX-synthetase inhibitor imidazole (100 microgram/ml) significantly decreased TXB2 release and simultaneously increased PG release during cardiac anaphylaxis. On the other hand, the beta-sympathomimetic drug isoproterenol decreased both TXB2 and PG release from the anaphylactic hearts. While isoproterenol significantly diminished anaphylactic coronary flow reduction, imidazole was without effect in this respect. PGD2 (0.5 microgram/min and 5.0 microgram/min) infused intraaortally into non-sensitized guinea pig hearts reduced coronary flow dose-dependently. These results are compatible with the view that release of TX and PGs might contribute to coronary flow reduction in cardiac anaphylaxis.

A detailed time course of changes in plasma renin activity (PRA), urinary prostaglandin (PG) E2, PGF2 alpha, thromboxane (TX) B2 and sodium excretion rates following furosemide was obtained in 7 women. PRA increased within the first 15 min and remained elevated all through the experiment. PGE2, PGF2 alpha, TXB2 and sodium increased simultaneously, reached a peak between 15 and 45 min after furosemide and declined thereafter. It is concluded that furosemide induces a generalized activation of the renal PG system temporally related to the increase of renin release and natriuresis.

In order to investigate whether bronchopulmonary hyperresponsiveness represents a unique property of sensitized lungs, we examined the responses of lungs from either actively sensitized, passively sensitized, or nonsensitized (control) guinea pigs to in vitro bronchoconstriction (BC) and release of thromboxane (TX) B2, 6-keto-PGF1 alpha, and histamine induced by platelet-activating factor (PAF-acether) or leukotriene (LT) D4. Guinea pigs were actively sensitized with 10 micrograms of either ovalbumin or Dermatophagoides farinae extract in AI(OH)3 injected intraperitoneally twice at a 2-wk interval. Seven days after the second injection (booster injection), the lungs were removed, ventilated, and perfused via the pulmonary artery with Krebs solution containing 2.5 g/L bovine serum albumin. In lungs from actively sensitized animals, BC was induced by significantly lower doses of PAF-acether and LTD4 than those required to elicit the same response in control preparations. In addition, sensitized lungs released more TxB2, 6-keto-PGF1 alpha, and histamine in response to PAF-acether and LTD4 than did control lungs. Increased mediator release was also observed upon challenge of lungs from actively sensitized animals with arachidonic acid and histamine. Lungs from guinea pigs passively sensitized with serum from actively sensitized animals did not exhibit increased responsiveness to PAF-acether as compared to control lungs. The hyperresponsiveness induced after booster injection of the antigen occurred concomitantly with an increase in the homocytotropic antibody titer (as measured by passive cutaneous anaphylaxis) and persisted for 3 months after sensitization, when the levels of circulating antibodies and lung response to antigen challenge returned to control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-vascular endothelial cell interactions are central to the maintenance of vascular homeostasis. Thromboxane A2 (TXA2) and prostacyclin (prostaglandin (PG)I2) are the major products of cyclooxygenase (COX) metabolism by platelets and the vascular endothelium, respectively. Here we report the effects of platelet-endothelial interactions on human umbilical vein endothelial cells (HUVECs) COX-2 expression and prostanoid synthesis. Co-incubation of platelets with HUVECs resulted in a dose-dependent induction in COX-2 expression. This was accompanied by a relatively small increase in thromboxane B2 synthesis (2 ng) by comparison to the production of 6-keto-PGF1alpha and PGE2, which increased by approximately 14 and 12 ng, respectively. Abrogation of platelet-HUVEC interactions excluded direct cell-cell contact as a required event. Preincubation of HUVECs with SQ29548, a TXA2 receptor antagonist, dose-dependently inhibited platelet-induced COX-2 expression and prostanoid synthesis. Similarly, if platelet TXA2 synthesis was inhibited no induction of COX-2 was observed. Furthermore, a TXA2 analog, carbocyclic TXA2, induced HUVEC COX-2 expression and the synthesis of 6-keto-PGF1alpha and PGE2. This was also associated with an increase in the expression and activity of PGI synthase and PGE synthase but not TX synthase. Platelet co-incubation (or TXA2) also selectively activated the p44/42 mitogen-activated protein kinase pathway to regulate HUVEC COX-2 expression. Thus it seems that platelet-derived TXA2 can act in a paracrine manner to up-regulate endothelial COX-2 expression and PGI2 synthesis. These observations are of particular importance given the recent observations regarding selective COX-2 inhibitors and the suppression of PGI2 synthesis.

Previous reports have given conflicting conclusions of the role platelets may play in initiating vaso-occlusive sickle cell crisis. Seven patients homozygous for sickle cell hemoglobin, and seven age, race and sex matched controls were each studied on at least two occasions in a six week period of normal health. The number of platelets circulating as aggregates, the plasma concentration of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) were significantly elevated compared with controls. These findings were confirmed with a second series of fourteen patients and nine controls. Patient's platelets in plasma adjusted for both platelet number and citrate concentration aggregated more in response to low concentrations (0.4 and 1 microM) but less to higher concentrations (4 and 20 microM) of ADP and needed significantly more prostacyclin (PGI2) to inhibit ADP induced aggregation than did platelets from control subjects. There was no significant difference in plasma concentration of fibrinopeptide A and thromboxane (Tx)B2, nor in the platelet generation of TxB2 and release of serotonin and beta TG induced by aggregating agents. Thus, the platelets of patients with sickle cell anemia in the steady state are readily activated and respond in vivo by increased formation of aggregates and release of beta TG and PF-4.

Lower torso ischemia leads on reperfusion to sequestration of polymorphonuclear leukocytes (PMN) in the lungs and increased permeability. This study tests the role of circulating leukocytes (WBC) in mediating this lung injury. Anesthetized sheep prepared with chronic lung lymph fistulae underwent 2 hours of bilateral hind limb tourniquet ischemia. In untreated controls (n = 7), 1 minute after reperfusion there were transient increases in mean pulmonary arterial pressure (MPAP) from 13 to 38 mmHg (p less than 0.05) and pulmonary microvascular pressure (Pmv) from 7 to 18 mmHg (p less than 0.05), changes temporally related to a rise in plasma thromboxane (Tx) B2 levels from 211 to 735 pg/ml (p less than 0.05). Lung lymph TxB2 levels rose from 400 to 1005 pg/ml at 30 minutes (p less than 0.05), and remained elevated longer than plasma levels. Lung lymph flow (QL) rose from 4.3 to 8.3 ml/30 minutes (p less than 0.05) after 30 minutes of reperfusion and remained elevated for 2 hours. The lymph/plasma (L/P) protein ratio was unchanged from 0.6, while the lymph protein clearance increased from 2.6 to 4.6 ml/30 minutes (p less than 0.05), suggesting increased microvascular permeability. WBC counts decreased within the first hour of reperfusion from 6853 to 3796/mm3 (p less than 0.05), and lung histology after 2 hours showed proteinaceous exudates and leukosequestration of 62 PMN/10 high-powered fields (HPF), higher than the 22 PMN/10 HPF (p less than 0.05) in sham animals (n = 3). Recruitment of the pulmonary vasculature by left atrial balloon inflation (n = 3) resulted in a rise in MPAP to 20 mmHg. After 3 hours of balloon inflation, QL stabilized at 9.8 ml/15 minutes, and a pressure-independent L/P protein ratio of 0.3 was achieved. During reperfusion, QL increased further to 11.2 ml/15 minutes, the L/P ratio rose to 0.56 and the calculated osmotic reflection coefficient decreased from 0.70 to 0.44, documenting an increase in lung microvascular permeability. In contrast to these untreated ischemic controls, sheep (n = 7) rendered leukopenic with hydroxyurea or nitrogen mustard and having a total WBC count of 760/mm3 and PMN count of 150/mm3 did not manifest reperfusion-induced increases in MPAP, Pmv, QL, lymph protein clearance, or lung lymph. TxB2 level (p less than 0.05). Plasma TxB2 levels rose slightly at 30 minutes from 199 to 288 pg/ml (p less than 0.05). Lung histology was normal. These data indicate that WBC mediate the ischemia-induced increase in pulmonary microvascular permeability.

Urinary excretion rates of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 were evaluated in three groups of cirrhotic patients [without ascites (group 1, 13 cases), with ascites and normal renal function (group 2, 15 cases), and with ascites and renal failure (group 3, 5 cases)] and in 14 healthy controls. All urinary arachidonate metabolites were significantly increased in group 2 patients. Patients with renal failure showed lower PGE2, PGF2 alpha, and TXB2 values than those from group 2; PGF2 alpha values were also lower than controls. Platelet TXA2 production during whole blood clotting was significantly reduced in all groups of patients. Administration of low-dose aspirin and sulindac, two cyclooxygenase inhibitors selectively sparing renal cyclooxygenase activity, effectively inhibited platelet TXA2 production without affecting urinary TXB2 excretion, thus ruling out platelets as a possible source of urinary TXB2. We conclude that patients with ascites and normal renal function show an overall activation of the renal PG system. Renal production of vasodilating PGE2 and PGI2 may be involved in supporting renal function in these patients. A reduced platelet synthesis of proaggregatory TXA2 also occurs in cirrhotic patients. This may play a role in the bleeding tendency of cirrhosis.

We assessed the role of bradykinin (BK) in allergen-induced early and late bronchial responses, airway inflammation, mediator release, and antigen-induced airway hyperresponsiveness in allergic sheep by studying the effects of the BK B2 receptor antagonist, NPC-567 (D-Arg-[Hyp3, D-Phe7]-BK), on these parameters. Antigen challenge was performed on two occasions greater than 3 wk apart, once with placebo (control) and once after high-dose (10 mg/ml) and low-dose (5 mg/ml) treatments with aerosol NPC-567. In the control trials (n = 14) antigen challenge resulted in an early and late increase in specific lung resistance (SRL). The early response was associated with increases (p less than 0.05) in prostaglandin (PG) D2, immunoreactive kinin, tosyl-L-arginine methyl ester (TAME)-esterase, and PGE2 in bronchoalveolar lavage (BAL) fluid. The late response was associated with increases (p less than 0.05) in leukotrienes (LT) B4 and C4, thromboxane (TX) B2, 6-keto-PGF10, and PGE2. There was a significant influx of neutrophils in the BAL fluid during the late response, and airway hyperresponsiveness to carbachol aerosol was apparent 4 h after challenge. In six sheep the high-dose NPC-567 treatment (given before, during, and 4 h after antigen challenge) did not attenuate the early bronchoconstrictor response or the early release of mediators but caused a significant reduction in the late response (p less than 0.05). This protective effect was accompanied by reductions (p less than 0.05) in both the concentrations of all the mediators associated with the late response and the severity of the BAL neutrophilia. High-dose NPC-567 did not attenuate the airway hyperresponsiveness or the cellular inflammatory response seen 24 h after challenge. In eight sheep treated with the low dose of NPC-567 (given before, during, and 4, 8, and 24 h after challenge) the early response was not blocked but the late response was again inhibited, as were the mediators associated with the late response. At the low dose the drug did not prevent the airway inflammation at 8 or 24 h. The additional treatments did, however, prevent the 24 h hyperresponsiveness. These data suggest that kinin generation during antigen-induced airway anaphylaxis may be important for controlling the release of arachidonic acid metabolites from airway inflammatory cells that contribute to the development of the late response in the allergic sheep model.

The effect of dazoxiben, a selective thromboxane (Tx) synthetase inhibitor, on systemic and pulmonary hemodynamics, eicosanoids, and lung permeability was assessed in awake goats with lung lymph fistulae following infusion of Escherichia coli endotoxin (1 microgram/kg). Animals received endotoxin either with no treatment or pretreatment with a bolus (25 mg/kg) followed by a maintenance infusion (10 mg/kg per h) of dazoxiben. In untreated animals, the peak rise of 26.8 cm H2O in pulmonary artery (Ppa) and of 13.5 cm H2O in wedge (Pw) pressures occurred at the same time as the peak elevations in plasma thromboxane B2 (T X B2). Maximum reduction in cardiac output (Qt) also occurred at the same time. Lung lymph flow (QL) increased during this period and remained elevated for at least 6 h after endotoxin. T X B2 levels had returned from a peak of 13.1 to 0.7 ng/ml by 2 h. In dazoxiben-treated animals, plasma concentrations of T X B2 were never significantly elevated. Increases in Ppa and Pw were markedly reduced and decreased Qt was transient. QL in treated animals began to increase by 30 min after endotoxin and reached a peak by 2 h. Increased QL in treated animals was not as great as in the untreated animals. Moreover, lymph-plasma protein ratios increased significantly in treated animals. Plasma prostaglandin (PG)F2 alpha and 6-keto-PGF1 alpha concentrations were elevated in both groups after endotoxin with values significantly greater in treated animals. We conclude that selective inhibition of Tx ameliorates many adverse hemodynamic consequences of endotoxemia but does not prevent lung permeability changes.

It has already been demonstrated in human and animal systems that PGE2 is a suppressor signal for many immune functions. These include T-lymphocyte blastogenesis, natural killer cell activity, and cytolytic T-lymphocyte activity. These functions are important for destruction of tumor cells. Conceivably, suppression of these functions by excessive PGE2 restricts tumor cell kill, and reversal of suppression by an inhibitor of prostaglandin synthesis such as indomethacin could increase tumor cell kill. The purpose of this study was to determine the kind of prostaglandins (PGs) produced by tissues with squamous cell carcinoma of head and neck and to measure the concentrations of PGE2, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in the tumor tissue and in the corresponding control tissue. Tumor and normal control tissues at the margin of the resection were obtained from surgical specimens. The production of PGs was determined by incubation of tissue homogenates with 14C-arachidonic acid, by thin layer chromatography, autoradiography, and scintillation counting. Concentrations of PGs were measured by radioimmunoassay. Tumor tissues produced PGD2, E2, TxB2, F2 alpha, and 6-keto-F1 alpha, and 15-, 12-, and 5-monohydroxyeicosatetraenoic acid (HETE). Concentrations of PGE2 were four times higher in the tumor tissues compared to those in control tissues. There was no difference between the levels of TxB2 and 6-keto-PGF1 alpha in the tumor tissues and those in control tissues. The results of this study will serve as basic information necessary for the potential use of inhibitors of PG-synthesis in the treatment of head and neck carcinoma.

Although long-chain n3 polyunsaturated fatty acids [n3 PUFAs; eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] have been reported to reduce platelet aggregation, the available evidence on this is equivocal. We previously demonstrated that the acute effects of n3 PUFA supplementation on platelet aggregation are sex specific. We aimed to determine if this gender bias is maintained during long-term n3 PUFA supplementation and whether this translates to other hemostatic markers. A double-blinded, randomized, placebo controlled trial was conducted in 94 healthy men and women. Platelet aggregation, thromboxane (TX) B2, P-selectin (P-sel), von Willebrand factor (vWF), and plasminogen activator inhibitor-1 were measured at baseline and 4 wk postsupplementation with EPA-rich (1000 mg EPA:200 mg DHA) or DHA-rich (200 mg EPA:1000 mg DHA) oil capsules daily. The effects of n3 PUFA on platelet activity were compared between men and women. In men and women combined, EPA and DHA reduced platelet aggregation following 4 wk of supplementation relative to placebo (-11.8%, P = 0.016; and -14.8%, P = 0.001, respectively). In subgroup analyses, in men, only the EPA treatment reduced platelet aggregation by -18.4% compared with placebo (P = 0.005) and women (P = 0.011). In contrast, in women, only the DHA treatment reduced platelet aggregation (-18.9%) compared with placebo (P = 0.001) and men (P = 0.017). Significant sex × treatment interactions were also observed on hemostatic markers and uptake of n3 PUFAs. The significant interactions between sex and specific, supplemental, long-chain n3 PUFAs result in platelet aggregation being differentially affected in men and women. With respect to thrombotic disease risk, men are more likely to benefit from supplementation with EPA, whereas women are more responsive to DHA.

OBJECTIVE

The role of prostacyclin in the development of venous thrombosis and vascular dysfunction in humans is unclear. In patients with deep vein thrombosis (DVT, n=34) and controls (matched for age, sex, indexes of systemic inflammation and metabolic status, n=20), we studied (i) differences on systemic markers of vascular disease and platelet activation and (ii) the influence of prostacyclin receptor gene (PTGIR) polymorphisms.

RESULTS

Enhanced levels of urinary 11-dehydro-thromboxane (TX)B2 and plasma [soluble(s)] P-selectin, mostly platelet derived, were detected in DVT patients, whereas plasma von Willebrand factor levels and intima-media thickness of the common carotid arteries were not significantly different. In all patients' cohorts, we identified five PTGIR polymorphisms (three nonsynonymous: P226T, R212C, V196L; two synonymous: V53V, S328S). In the four individuals carriers of R212C polymorphism (three in DVT, one in controls), intima-media thickness values were significantly (P=0.0043) higher than those detected in individuals of all cohorts [1.68+/-0.38, 1.55 (1.4-2.2) vs. 1.05+/-0.33, 1.08 (0.01-1.68) mm, respectively, mean+/-SD, median (range)]. Moreover, enhanced sP-selectin and 11-dehydro-TXB2, in DVT versus controls, were statistically significant only in carriers of both synonymous PTGIR polymorphisms V53V/S328S. Only the PTGIR mutant R212C was dysfunctional when examined in an in vitro overexpression system.

CONCLUSIONS

Our results suggest a propensity of enhanced platelet activation in DVT patients with PTGIR polymorphisms V53V/S328S. Moreover, we identified a dysfunctional PTGIR polymorphism (R212C) associated with intimal hyperplasia.

The effects of thromboxane (Tx) inhibition or arachidonic acid (AA) infusion were studied in anesthetized cats during acute myocardial ischemia (MI). AA (7.2 mg kg-1 h-1) or imidazole (25 mg kg-1 h-1) infusions were initiated 30 min after occlusion of the left anterior descending coronary artery. Assessment of the degree of protection of the ischemic myocardium was made by measurement of S-T segment elevation, plasma and myocardial creatine phosphokinase (CPK) activities, and myocardial amino-nitrogen content. Assessment of Tx inhibition was performed by radioimmunoassay. Administration of imidazole inhibited the sevenfold increase in plasma thromboxane B2 (TxB2) levels occurring in MI (p less than 0.001 at 2-5 h), markedly decreased S-T segment elevations at 2-k h (p less than 0.025), significantly prevented the elevation in plasma CPK (p less than 0.05, at 4 and 5 h), the increase in TxB2 post-MI, significantly decreased (p less than 0.025) S-T segment evaluations at 2-5 h, caused a decrease in plasmaCPK levels (p less than 0.05 at 5 h), but did not prevent loss of myocardial CPK or amino-nitrogen. In summary, the administration of imidazole resulted in significant protection of the myocardium in all indices of ischemic damage measured, while AA infusion resulted in only a partial protection. The mechanism of the imidazole protection of ischemic myocardial tissue appears to be via inhibition of Tx synthesis althoug we cannot exclude a hemodynamic or cytoprotective mechanism. These results suggest that specific inhibition of Tx formation is beneficial during acute MI.

To evaluate indicators of inflammatory changes in the airways of young smokers we have measured the levels of several eicosanoids in bronchoalveolar lavage (BAL) fluid of 18 female smokers (age 33 +/- 2 years) and 9 female non-smokers (age 29 +/- 2 years) who were hospitalized for treatment not related to any pulmonary disease. In each BAL specimen the following eicosanoids were determined by radioimmunoassay: prostaglandin (PG) E2; PGF2 alpha; 9 alpha, 11 beta-PGF2, a metabolite of PGD2; 6-keto PGF1 alpha, a metabolite of prostacyclin; thromboxane (Tx) B2, a metabolite of TxA2; the 5-lipoxygenase products 5-hydroxy-eicosa-tetraenoic acid (HETE), leukotriene (LT) B4 and LTC4; the 12-lipoxygenase product 12-HETE; and the 15-lipoxygenase product 15-HETE. The concentrations of the cyclooxygenase products (pg ml-1) in the BAL fluid of the non-smokers were: PGE2 15.4 +/- 1.9, PGF2 alpha 7.6 +/- 1.0, 9 alpha, 11 beta-PGF2 8.7 +/- 1.8, TxB2 8.8 +/- 1.3, and 6-keto PGF1 alpha only 1.5 +/- 0.8. The concentration of the lipoxygenase products were: 15-HETE 781 +/- 200, 12-HETE 193 +/- 33, 5-HETE 14.0 +/- 3.1, LTC4 9.5 +/- 3.1, LTB4 6.2 +/- 1.4. BAL fluid from smokers contained two- to three-fold higher levels of TxB2 and PGF2 alpha (P less than 0.05). The levels of TxB2 and PGF2 alpha were positively correlated to the number of package years (rs = 0.55 and rs = 0.65, P less than 0.02). The concentrations of 5-, 12- and 15-HETE tended to be higher in BAL fluid from smokers, but this was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Insufficient platelet function suppression by aspirin is a predictor of cardiovascular events in high-risk patients. The authors assessed the impact of obesity on platelet responsiveness before and after 2 weeks of aspirin 81 mg/d in 2014 people. Obese individuals had greater baseline platelet reactivity. Comparing obese and nonobese individuals after aspirin therapy, results for aggregometry to collagen were 6.7 vs 6.1 ohms, P=.008; aggregometry to adenosine diphosphate were 13.1 vs 11.8 ohms,P<.0001; aggregometry to arachidonic acid (AA) were 4.9% vs 8.3% nonzero aggregation, P=.002; urinary excretion of 11-dehydro-thromboxane B2 (Tx-M) were 4.9% vs 8.3% nonzero aggregation, P=.002; and aspirin resistance were 26.% vs 20.5%, P=.002; respectively. These remained significantly different for AA aggregation and Tx-M excretion after adjustment for covariates. Obese individuals have greater native platelet reactivity and retain greater reactivity after suppression by aspirin.

In an isotope dilution assay, prostaglandin (PG) E2, 6-keto-PGF1 alpha, thromboxane (Tx) B2 and their metabolites PGE-M (11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid), 2,3-dinor-6-keto-PGF1 alpha, 2,3-dinor-TxB2 and 11-dehydro-TxB2 were determined in urine by gas chromatography-triple stage quadrupole mass spectrometry (GC-MS-MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate-hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC-MS-MS. In each run, two or three prostanoids were determined.

Experimental data suggest that oxidative stress might be enhanced in hypertension and contribute to platelet activation. We hypothesized that both oxidative stress and platelet activation could be related to the clinical characteristics of hypertensive patients. The urinary excretion of 11-dehydrothromboxane (TX) B2, reflecting in vivo platelet activation, was measured in 75 patients with mild to severe essential hypertension and 75 pair-matched, healthy controls. The urinary excretion of 8-iso-prostaglandin (PG) F2alpha was determined as an index of in vivo lipid peroxidation. Urinary 11-dehydro-TXB2 was significantly higher in essential hypertensives compared with controls. Although no statistically significant difference in urinary 8-iso-PGF2alpha was observed between patients and controls, plasma vitamin C was lower and plasma homocysteine higher in hypertensive patients than in controls. Both urinary 11-dehydro-TXB2 and 8-iso-PGF2alpha were higher in patients with advanced hypertensive retinopathy compared with patients without retinopathy. Multivariate linear regression analysis identified urinary 8-iso-PGF2alpha, plasma fibrinogen, homocysteine, and vitamin E as the only variables independently correlated with urinary 11-dehydro-TXB2. Logistic regression analysis showed that high urinary 8-iso-PGF2alpha, plasma fibrinogen, and homocysteine, as well as low plasma vitamin E, advanced retinopathy, elevated diastolic blood pressure, and the absence of antihypertensive treatment, were predictors of high urinary 11-dehydro-TXB2. We demonstrated increased oxidative stress and persistent platelet activation in essential hypertensives with advanced vascular lesions. These findings might help identify hypertensive patients who are at increased risk of cardiovascular events and who might benefit from long-term antiplatelet therapy.