Peroxisome proliferator-activated receptor-gamma ligand, 15-deoxy-Delta A(12,14)-prostaglandin J(2), reduces neutrophil migration via a nitric oxide pathway

Ligands for peroxisome proliferator-activated receptor gamma (PPAR-gamma), such as 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) have been implicated as a new class of anti-inflammatory compounds with possible clinical applications. Based on this concept, this investigation was designed to determine the effect of 15d-PGJ(2)-mediated activation of PPAR-gamma ligand on neutrophil migration after an inflammatory stimulus and clarify the underlying molecular mechanisms using a mouse model of peritonitis. Our results demonstrated that 15d-PGJ(2) administration decreases leukocyte rolling and adhesion to the inflammated mesenteric tissues by a mechanism dependent on NO. Specifically, pharmacological inhibitors of NO synthase remarkably abrogated the 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory site. Moreover, inducible NOS(-/-) mice were not susceptible to 15d-PGJ(2)-mediated suppression of neutrophil migration to the inflammatory sites when compared with their wild type. In addition, 15d-PGJ(2)-mediated suppression of neutrophil migration appeared to be independent of the production of cytokines and chemokines, since their production were not significantly affected in the carrageenan-injected peritoneal cavities. Finally, up-regulation of carrageenan-triggered ICAM-I expression in the mesenteric microcirculation vessels was abrogated by pretreatment of wild-type mice with 15d-PGJ(2), whereas 15d-PGJ(2) inhibited F-actin rearrangement process in neutrophils. Taken together these findings demonstrated that 15d-PGJ(2) suppresses inflammation-initiated neutrophil migration in a mechanism dependent on NO production in mesenteric tissues.

Antihypertensive effects exerted by enalapril in mild to moderate hypertension are not associated with changes in the circulating levels of nitric oxide-related markers

The antihypertensive effects of angiotensin-converting enzyme inhibitors (ACEi) are explained, at least in part, by enhanced bradykinin-dependent nitric oxide (NO) formation and decreased angiotensin II-induced oxidative stress and vasoconstriction. We examined for the first time whether treatment with enalapril increases the plasma levels of markers of NO formation and decreases oxidative stress in mild to moderate hypertensive patients. Eighteen untreated hypertensive patients were treated with enalapril 10 mg/day (n = 10) or 20 mg/day (n = 8) for 60 days. Eighteen normotensive healthy controls were followed for the same period. Venous blood samples were collected at baseline and after 30/60 days of treatment with enalapril. Plasma NOx (nitrites + nitrates) concentrations were determined by using the Griess reaction. Plasma nitrite and whole blood nitrite concentrations were determined by using an ozone-based chemiluminescence assay. Plasma thiobarbituric acid-reactive species (TBARS) and 8-isoprostane concentrations were determined by a fluorimetric method and by ELISA, respectively. Treatment with enalapril decreased blood pressure in hypertensive patients. However, we found no significant changes in plasma NOx, nitrite, whole blood nitrite, and in the levels of markers of oxidative stress in both normotensive controls and hypertensive patients treated with enalapril. Our data show that enalapril 10-20 mg/day does not affect the concentrations of relevant markers of NO formation or markers of oxidative stress in mild to moderately hypertensive subjects, despite satisfactory blood pressure control. Our findings do not rule out the possibility that ACEi may produce such effects in more severely hypertensive patients treated with higher doses of ACEi.

Nitric oxide inhibition in paraventricular nucleus on cardiovascular and autonomic modulation after exercise training in unanesthetized rats

It is well known that regular physical exercise alter cardiac function and autonomic modulation of heart rate variability (HRV). The paraventricular nucleus of hypothalamus (PVN) is an important site of integration for autonomic and cardiovascular responses, where nitric oxide (NO) plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after nitric oxide synthase (NOS) inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After swimming training protocol, adult male Wistar rats, instrumented with guide cannulas to PVN and femoral artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, the physical training induced a resting bradycardia (S: 374 +/- 5, ST: 346 +/- 1 bpm) and promoted adaptations in HRV characterized by an increase in high-frequency oscillations (HF; 26.43 +/- 6.91 to 88.96 +/- 244) and a decrease in low-frequency oscillations (LF; 73.57 +/- 6.91 to 11.04 +/- 2.44) in normalized units. The microinjection of N(omega)-nitro-L-arginine methyl ester (L-NAME) in the PVN of sedentary and trained rats promoted increase in MAP and HR. L-NAME in the PVN did not significantly alter the spectral parameters of HRV of sedentary animals, however in the trained rats increased LF oscillations (11.04 +/- 2.44 to 27.62 +/- 6.97) and decreased HF oscillations (88.96 +/- 2.44 to 72.38 +/- 6.97) in normalized units compared with baseline. Our results suggest that NO in the PVN may collaborate to cardiac autonomic modulation after exercise training. (c) 2010 Elsevier B.V. All rights reserved.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: Involvement of nitric oxide

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1,3, 5 and 7 h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production. (C) 2010 Elsevier Inc. All rights reserved.

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: Involvement of cytokines and nitric oxide

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 mu g/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 mu g/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1 beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 mu g/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1 beta, TNF-alpha and CINC-1. (C) 2009 Elsevier Ltd. All rights reserved.

NOC-9, a selective nitric oxide donor, induces flight reactions in the dorsolateral periaqueductal gray of rats by activating soluble guanylate cyclase

Previous studies have showed that SIN-1, a nitric oxide (NO) donor, injected into the dorsolateral column of the periaqueductal gray (dlPAG) induces flight reactions. This drug, however, can also produce peroxynitrite, which may interfere in this effect. In addition, it is also unknown if this effect is mediated by local activation of soluble guanylate cyclase (sGC). The aims of this study, therefore, were (1) to investigate if NOC-9 (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), a NO donor that does not produce peroxynitrite, would produce flight reactions after intra-dlPAG administration similar to those induced by SIN-1; (2) to verify if these responses could be prevented by local injection of a selective guanylate cyclase inhibitor (ODQ). Male Wistar rats (n = 5-12) with cannulae aimed at the dlPAG received injections of TRIS (pH 10.0, 0.5 mu l), NOC-9 (75 and 150 nmol), saline or SIN-1 (200 nmol) and were placed in an open arena for 10 min. In a subsequent experiment animals (n = 7-8) were pretreated with ODQ (1 nmol/0.5 mu l) before receiving NOC-9 150 nmol. NOC-9 induced a significant dose-dependent increase in flight reactions in the first minute after injection (% of animals displaying flight: vehicle = 0%, NOC 75 = 67%. NOC 150 = 75%). SIN-1 had a similar effect (100% of animals showing flight) but the effects lasted longer (10 min) than those of NOC-9. The effect of NOC-9 (150 nmol) was prevented by pretreatment with ODQ (% of animals displaying flight: vehicle + NOC 150 = 71 %, ODQ + NOC 150 = 37%). The results suggest that NO donors injected into the dlPAG induce defensive responses that are not mediated by secondary peroxynitrite production. Moreover, they also indicate that these defensive responses depend on activation of local sGC. The data strengthen the proposal that NO can modulate defensive reactions in the dlPAG. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Central but not systemic inhibition of inducible nitric oxide synthase modulates oxytocin release during endotoxemic shock

Previous studies have shown that immunological challenges as lipopolysaccharide (LPS) administration increases plasma oxytocin (OT) concentration. Nitric oxide (NO), a free radical gas directly related to the immune system has been implicated in the central modulation of neuroendocrine adaptive responses to immunological stress. This study aimed to test the hypothesis that the NO pathway participates in the control of OT release induced by LPS injection. For this purpose, adult male Wistar rats received bolus intravenous (i.v.) injection of LPS, preceded or not by iv. or intracerebroventricular (i.c.v.) injections of aminoguanidine (AG), a selective inducible nitric oxide synthase (iNOS) inhibitor. Rats were decapitated after 2, 4 and 6 h of treatment, for measurement of OT by radioimmunoassay. In a separate set of experiments, mean arterial pressure (MAP) and heart rate (HR) were measured every 15 min over 6 h, using a polygraph. These studies revealed that LPS reduced MAP and increased HR at 4 and 6 h post-injection. LPS significantly increased plasma OT concentration at 2 and 4 h post-injection. Pre-treatment with i.c.v. AG further increased plasma OT concentration and attenuated the LPS-induced decrease in MAP, however, i.v. AG failed to show similar effects. Thus, iNOS pathway may activate a central inhibitory control mechanism that attenuates OT secretion during endotoxemic shock. (C) 2009 Elsevier Inc. All rights reserved.

GABA and nitric oxide in the PVN are involved in arterial pressure control but not in the chemoreflex responses in rats

GABAergic, nitrergic and glutamatergic mechanisms in the PVN on the baseline mean arterial pressure (MAP), heart rate (HR) and on the cardiovascular responses to chemoreflex activation in awake rat were evaluated. Chemoreflex was activated with KCN before and after microinjections into the PVN. Bicuculline into the PVN increased baseline MAP (94+/-3 vs 113+/-5 mmHg) and HR (350+/-9 vs 439+/-18 bpm) but had no effect on the pressor (49+/-5 vs 47+/-6 mmHg) or bradicardic (-213+/-23 vs -256+/-42 bpm) responses (n=7). Kynurenic acid into the PVN (n=6) produced no significant changes in the MAP (98+/-3 vs 100+/-3 mmHg), HR (330+/-5 vs 339+/-12 mmHg) or in the pressor (50+/-4 vs 42+/-4 mmHg) and bradicardic (-252+/-4 vs -285+/-16 bpm) responses to chemoreflex. L-NAME into the PVN (n=8) produced increase in the MAP (94+/-3 vs 113+/-5 mmHg) and HR (350+/-9 vs 439+/-18 bpm) but had no effect on the pressor (52+/-5 vs 47+/-6 mmHg) or bradicardic (-253+/-19 vs -320+/-25 bpm) responses to chemoreflex. We conclude that GABA(A) and nitric oxide in the PVN are involved in the maintenance of the baseline MAP but not in the modulation of the responses to chemoreflex. The results also show that Glutamate receptors in the PVN are not involved in maintenance of the baseline MAP, HR or in the cardiovascular responses to chemoreflex in awake rats. (C) 2008 Elsevier B.V. All rights reserved.

Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect Of L-arginine, a precursor for NO synthesis, and N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 mu M) and DL-2-amino-5-phosphonovaleric acid (DL-AP5) (30 mu M) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 mu M) and saclofen (30 mu M) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 mu M L-arginine decreased the firing rate from 3.7 +/- 0.6 Hz to 1.3 +/- 0.3 Hz. Conversely, 100 mu M L-NAME increased the firing rate from 3.0 +/- 0.3 Hz to 5.8 +/- 0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1 +/- 0.8 mV to -62.2 +/- 1.1 mV in the presence of L-arginine and from -59.8 +/- 0.7 mV to -56.9 +/- 0.8 mV by L-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4 +/- 1.2 mV to 16.8 +/- 0.7 mV by L-arginine, but without significant changes by L-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Interethnic differences in ADMA concentrations and negative association with nitric oxide formation in preeclampsia

Background: Recent studies have suggested that impaired nitric oxide (NO) formation in preeclampsia may result from increased concentrations of an endogenous NO synthase inhibitor, the asymmetric dimethylarginine (ADMA). However, no previous study has examined whether a negative association exists between ADMA and nitrite concentrations in preeclampsia. Moreover, no previous study has compared ADMA and nitrite levels in black and white preeclamptic pregnant women. Methods: We measured plasma nitrite concentrations using an ozone-based chemiluminescence assay, and plasma ADMA levels using enzyme immunoassays in 94 pregnant (47 healthy pregnant: 16 blacks and 31 whites; and 47 preeclamptic: 14 blacks and 33 whites). Results: We found higher ADMA (2.199 +/- 0.016 mu mol/l vs. 2.112 +/- 0.012 mu mol/l; P < 0.0001) and lower plasma nitrite levels (102 +/- 7.1 nmol/l vs. 214.8 +/- 26.1 nmol/l; P<0.0001) in preeclamptic compared with healthy pregnant women. Black pregnant had higher ADMA levels than white pregnant women (P<0.05), both in preeclamptic (2.239 +/- 0.020 mu mol/l vs. 2.144 +/- 0.019 mu mol/l) and in healthy pregnant (2.172 +/- 0.025 mu mol/l vs. 2.077 +/- 0.018 mu mol/l). Conversely, we found no significant effects of ethnicity on the plasma nitrite levels, both in healthy pregnant and in preeclamptic women (P>0.05). We found a significant negative correlation (P<0.05) between these markers (r = 0.28; P<0.05). Conclusions: Our findings show higher ADMA and lower nitrite levels in preeclamptic compared with healthy pregnant, and the concentrations of these biomarkers are inversely associated. While ethnicity affected ADMA concentrations, no such effect was found with respect to nitrite levels. These results may have important implications for studies on NO biology and therapeutic approaches of preeclampsia. (C) 2010 Elsevier B.V. All rights reserved.

Ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures

Study objective: To compare the effects of ethinylestradiol (EE) and 17 beta-estradiol (E(2)) on nitric oxide (NO) production and protection against oxidative stress in human endothelial cell cultures. Design: Experimental study. Settings: Research laboratory. Material: Human ECV304 endothelial cell cultures. Intervention(s): The NO synthesis was determined by flow cytometry, and oxidative stress was determined by a cell viability assay, after exposure to hydrogen peroxide (H(2)O(2)) and stimulation of endothelial cells with EE at concentrations similar to those of a contraceptive containing 30 mu g EE. Main Outcome Measure(s): The effects of EE were compared with those of E(2) at concentrations similar to those occurring during the follicular phase. Result(s): Ethinylestradiol did not increase NO synthesis and did not protect cells against oxidative stress. The viability of the cells incubated with E(2) in combination with H(2)O(2) was greater than the viability obtained with H(2)O(2) only or with H(2)O(2) in combination with EE. The cells stimulated with E(2) presented a significant increase in NO production compared with control. Conclusion(s): In contrast to the effects of E(2), EE did not protect human ECV304 endothelial cells against oxidative stress and did not increase their production of NO. (Fertil Steril (R) 2010; 94: 1578-82. (C) 2010 by American Society for Reproductive Medicine.)

Alterations in cyclic GMP levels in preeclampsia may reflect increased B-type natriuretic peptide levels and not impaired nitric oxide activity

Objectives: We compared nitrite, B-type natriuretic peptide (BNP), and cGMP levels in preeclamptic with those found in healthy pregnant. Methods: We studied 21 healthy pregnant and 27 preeclamptic. Plasma cGMP and BNP levels were determined by ELISA. Nitrite levels were determined by chemiluminescence. Results: Higher cGMP and BNP, and lower nitrite levels were found in preeclamptic versus healthy pregnant Conclusions: Altered cGMP levels reflect increased BNP levels and not impaired nitric oxide activity in preeclampsia. (C) 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Increased circulating cell-free hemoglobin levels reduce nitric oxide bioavailability in preeclampsia

Contrasting with increased nitric oxide (NO) formation during healthy pregnancy, reduced NO bioavailability plays a role in preeclampsia. However, no study has examined whether increased NO consumption by enhanced circulating levels of cell-free hemoglobin plays a role in preeclampsia. We studied 82 pregnant women (38 healthy pregnant and 44 with preeclampsia). To assess NO bioavailability, we measured plasma and whole blood nitrite concentrations using an ozone-based chemiluminescence assay. Plasma ceruloplasmin concentrations and plasma NO consumption (pNOc) were assessed and plasma hemoglobin (pHb) concentrations were measured with a commercial immunoassay. We found lower whole blood and plasma nitrite concentrations in preeclamptic patients (-48 and -39%, respectively; both P<0.05) compared with healthy pregnant women. Plasma samples from preeclamptic women consumed 63% more NO (P=0.003) and had 53% higher pHb and 10% higher ceruloplasmin levels than those found in healthy pregnant women (P<0.01). We found significant positive correlations between pHb and pNOc (r=0.61; P<0.0001), negative correlations between pNOc and whole blood or plasma nitrite concentrations (P=0.02; r=-0.32 and P=0.01: r=-0.34, respectively), and negative correlations between pHb and whole blood or plasma nitrite concentrations (P=0.03; r=-0.36 and P=0.01: r=-0.38, respectively). These findings suggest that increased pHb levels lead to increased NO consumption and lower NO bioavailability in preeclamptic compared with healthy pregnant women. (C) 2010 Elsevier Inc. All rights reserved.