Sindrome de Marshall- Smith en mujer adulta: nuevos retos en anticoncepción / Marshall-Smith syndrome in an adult woman: new challenges in contraception

Se presenta el caso de una mujer de 38 años con Síndrome de Marshall-Smith. Se trata de una enfermedad rara de etiología desconocida, cuyas características incluyen anomalías craneofaciales, maduración ósea acelerada, alteraciones en el desarrollo neurológico y en las vías respiratorias con compromiso de la vía aérea y escasa supervivencia a largo plazo debido a problemas respiratorios. Sin embargo, los avances en el soporte respiratorio han permitido que algunos pacientes lleguen a la etapa adulta. Nuestra paciente, sin retraso intelectual ni psicomotor, solicita método anticonceptivo. Los pacientes con enfermedades raras necesitan ser atendidos con especial dedicación intentando reducir al mínimo la transmisión genética de dichas entidades, y mejorando al máximo su calidad de vida. Se ofrece un método anticonceptivo reversible de larga duración, sin riesgos para la evolución de su patología respiratoria, y atendiendo a los criterios medicos de elegibilidad de método anticonceptivo de la OMS, se indica la utilización de un implante subdérmico de etonogestrel. Con un perfil de seguridad y farmacocinética equivalente a los métodos de solo gestágeno y mayor comodidad.

A 38-year-old female patient with a history of Marshall-Smith syndrome is reported. It is a rare congenital disorder of unknown aetiology, which features include craneo facial dysmorphism, accelerated bone maturation, neurodevelopmental abnormalities, and upper and lower airways compromise. Long term survival is a problem due to respiratory complications, but it has decreased since airway support has improved, and that allows survival into adulthood. Our patient has neither intelectual nor psychomotor delay, so she asks for contraception method. As a rare genetic condition it needs to be attended with special consideration in order to reduce the disorder's transmission and to increase the life's quality of patients. A secure contraception method should be offered with no risk at all, attending to medical elegibility criteria for contraception use. We considered progestogen-only options and the patient's choice was etonogestrel subcutaneous implant.

TH17- and IL-17- mediated autoantibodies and placental oxidative stress play a role in the pathophysiology of pre-eclampsia.

Pre-eclampsia is a multisystem disorder of pregnancy that affects 5-8% of pregnancies. The pathophysiologic mechanisms that lead to the development of pre-eclampsia are poorly understood. Higher than normal levels of circulating TH17 is observed in preeclamptic women compared to women with normal pregnancy. TH17 cells are a subset of CD4+ T helper cells that are characterized by their secretion of IL-17. Recent studies suggest a role for TH17 cells and IL-17 in the pathophysiology of pre-eclampsia. In this review, we will discuss the known function of TH17 cells and IL-17 in immunity and vascular function. We will then review the role of IL-17 and TH17 cells in normal pregnancy and their association with pre-eclampsia, followed by a discussion of the literature to examine a potential role for IL-17 and TH17 cells in mediating pathophysiology in pre-eclampsia.

Randomized, placebo controlled, double blind trial evaluating early pregnancy phytonutrient supplementation in the prevention of preeclampsia.

OBJECTIVE: Daily provision of pregnant patients with dietary supplements containing antioxidants and phytonutrients, if initiated in the first trimester of pregnancy and continued throughout the gestation, may significantly decrease the incidence of preeclampsia. STUDY DESIGN: We conducted a single center, randomized, placebo-controlled investigation in which women were randomized by their risk status and assigned to daily ingestion of a supplement consisting primarily of a blended fruit and vegetable juice powder concentrate or placebo. RESULT: Of the 684 patients randomized to the trial, 267 (39.0%) completed it. The final analysis is based on those participants who completed the study. For the primary outcome of preeclampsia, there was no difference observed between the phytonutrient supplement group and the placebo group: 15.9% vs 16.3%, respectively, (R.R. 0.97 (0.56-1.69)). Non-significant trends toward lower placenta-related obstetrical complications were observed in the supplement group compared with the placebo cohort (8.3% vs 15.5%, respectively, (R.R. 0.57 (0.29-1.14). Those infants born to mothers taking the supplement in the high-risk stratified group demonstrated non-significant trends toward lower rates of respiratory distress syndrome (RDS); 5.3% in the supplement group vs 15.4% in the placebo group: R.R. 0.34 (0.12-1.01). CONCLUSION: Initiation of antioxidant/phytonutrient supplementation in the first trimester did not decrease rates of preeclampsia. Non-significant trends toward lower incidences of placental derived morbidity in those mothers taking the supplement in addition to decreased rates of RDS in infants born to supplemented mothers considered to be high-risk for preeclampsia, warrant further investigation.

Control of soluble fms-like tyrosine-1 (sFlt-1) production response to placental ischemia/hypoxia: role of tumor necrosis factor-α.

Although abnormal soluble fms-like tyrosine kinase-1 (sFlt-1) production is thought to be an important factor in the pathogenesis of preeclampsia (PE), the mechanisms that regulate the production of sFlt-1 during PE are unclear. While our laboratory has shown tumor necrosis factor-α (TNF-α) and sFlt-1 to be elevated in pregnant rats in response to placental ischemia, the importance of TNF-α in the regulation of sFlt-1 production is unknown. Therefore, the purpose of this study was to determine the role of TNF-α in mediating the increase in sFlt-1 in response to placental ischemia or hypoxia. Reductions in uterine perfusion pressure in pregnant rats significantly increased plasma levels of sFlt-1 and tended to increase TNF-α, an effect markedly attenuated by pretreatment with a TNF-α inhibitor etanercept (0.4 mg/kg). To further assess chronic interactions between TNF-α and sFlt-1, we examined a chronic effect of TNF-α infusion (50 ng/day) into normal pregnant rats to increase plasma sFlt-1 levels, as well as the effects of acute hypoxia on placental sFlt-1 production in the absence and presence of TNF-α blockade. Placental explants exposed to hypoxic conditions had enhanced TNF-α levels versus normoxic conditions, as well as increased sFlt-1 production. Pretreatment of placental explants with etanercept (15 µM) significantly reduced TNF-α levels in response to hypoxia but did not attenuate sFlt-1 production. These data suggest that while TNF-α may not play an important role in stimulating sFlt-1 production in response to acute hypoxia, a more chronic hypoxia, or placental ischemia may be an important stimulus for enhanced sFlt-l production.

Vitamin D Supplementation Suppresses Hypoxia-Stimulated Placental Cytokine Secretion, Hypertension and CD4+ T Cell Stimulation in Response to Placental Ischemia.

OBJECTIVE: To investigate a role of Vitamin D in the pathogenesis of preeclampsia (PE), and to discern any potential benefits of Vitamin D supplementation on hypertension in the RUPP rat model of PE. STUDY DESIGN: Blood and placentas from normal pregnancies (NP) and PE were collected following elective cesarean delivery without evidence of infection. Circulating Vitamin D was extracted by HPLC and measured via mass spectrometry. Media for placenta explants was supplemented with Vitamin D and exposed to hypoxic (1% O2) or normoxic (6% O2) conditions for 24 hours. ELISAs were performed on media and normalized to total protein to determine cytokine secretion. RUPP rats were supplemented with vitamin D by oral gavage, and blood pressure (MAP) and pup weights were measured in NP and RUPP rats with or without Vitamin D supplementation. Flow cytometry was used to evaluate CD4+ Tcells in control RUPP rats and RUPP rats treated with Vitamin D. RESULTS: Inflammatory cytokine secretion was higher (p<0.05) while the anti-inflammatory cytokine, IL-10, was significantly lower in the media of PE placentas compared to NP (p=0.005). Vitamin D supplementation decreased hypoxia stimulated pro-inflammatory cytokine secretion (p=0.003) in the media of PE placentas. Vitamin D decreased MAP and circulating CD4+ T cells in the RUPP rat model of PE (p<0.05). CONCLUSION: Vitamin D supplementation may be useful in the treatment or prevention of hypertensive disorders in pregnancy.

Neoplasia vaginal intraepitelial / Vaginal intraepithelial neoplasia

Objetivo: actualizar los conocimientos disponibles sobre la neoplasia vaginal intraepitelial (VAIN) especialmente en el diagnóstico y tratamiento. Métodos: revisión de la literatura en Pubmed de los últimos 20 años, especialmente de los publicados desde 2005 hasta la actualidad y considerando sobre todo los ensayos clínicos aleatorizados. Resultados: su prevalencia real es desconocida, aunque es una patología rara generalmente en mujeres posmenopaúsicas. Su fisiopatología es similar a la neoplasia cervical intraepitelial, con el HPV como principal factor de riesgo, sobre todo el serotipo 16, pero su progresión a cáncer es menor. Un grupo importante es el de VAIN tras histerectomía, que supone el 48-70 por ciento del total de las VAIN. La colposcopia para el diagnóstico no es sencilla y se aconseja preparación con estrógenos en las pacientes menopáusicas y utilización de ácido acético y lugol para identificar las zonas sospechosas. Las tres opciones terapéuticas son la cirugía (excisional, láser, ultrasonidos), braquiterapia y tratamiento médico (imiquimod, 5-fluorouracilo, ácido tricloroacético). Se expone la técnica y las ventajas e inconvenientes de cada uno de ellos. Conclusión: aunque la VAIN es una entidad rara, es preciso tenerla en cuenta ante un diagnóstico de lesión citológica. Su diagnóstico a veces no es sencillo y el tratamiento dependerá del grado, la localización y el tamaño de la lesión y las circunstancias personales de la paciente. Se necesitan más ensayos aleatorizados que comparen la eficacia entre las distintas opciones terapéuticas y su repercusión en la calidad de vida de las pacientes.

Aims: update the knowledge about vaginal intraepithelial neoplasia (VAIN) with special emphasis on diagnosis and therapeutic management. Method: electronic search of Pubmed of all kinds of articles about the VAIN, for the last 20 years with special attention to those published from 2005 to the present and considering especially randomized clinical trials. Results: its prevalence is unknown, although it is a rare condition that usually occurs in postmenopausal women. The physiopathology is similar to cervical intraepithelial neoplasia, being HPV the main risk factor, particularly serotype 16, but its progression to cancer is lower. An important group is VAIN after hysterectomy, 48-70 percent of total VaIN. Colposcopy for the diagnosis is not easy and in patients with postmenopausal the preparation with local estrogen is necessary and use both of acetic acid and lugol to identify suspicious areas. The three treatment options are surgery (excisional, laser, ultrasound), brachytherapy and medical management (imiquimod, 5-fluorouracil, tricholoroacetic acid). The technique and the advantages and disadvantages of each are explained. Conclusion: although VAIN is rare, it must take it into account before a cytological diagnosis of dysplasia. Its diagnosis is sometimes not easy and the treatment depends on the extent, location and size of the lesion and the individual preferences of the patient. We need more randomized trials comparing different treatment options and also their impact on quality of life of patients.

Endothelial dysfunction. An important mediator in the pathophysiology of hypertension during pre-eclampsia.

Pre-eclampsia is defined as new onset hypertension with proteinuria during pregnancy. It affects approximately 5% of pregnancies in the US with a subset of those progressing into more severe forms of the disease, known as HELLP or eclampsia. Pre-eclampsia is associated with intrauterine growth restriction, chronic immune activation and multi-organ endothelial dysfunction thus contributing to the clinically visible elevation in maternal blood pressure. The end result is increased infant and maternal morbidity and mortality thereby contributing to the gross health care expenditure nationwide. Although the underlying cause of this disease is still unknown, the most well accepted hypothesis is that placental ischemia/hypoxia results from inadequate uteroplacental vascular remodeling, which leads to a decrease in placental blood flow. The ischemic placenta releases factors such as the soluble VEGF receptor-1 (sFlt-1), the angiotensin II type-1 receptor autoantibody (AT1-AA), and cytokines such as TNF-α and Interleukin 6 which cause maternal endothelial dysfunction characterized by elevated circulating endothelin (ET-1), reactive oxygen species (ROS), and enhanced vascular sensitivity to angiotensinII. These factors act in concert to decrease renal function and cause hypertension during pregnancy. Understanding the link between placental ischemia, endothelial dysfunction and hypertension during pregnancy will lend to better prediction, prevention and treatment strategies for women and children stricken by this devastating disease.

OS066. Intrauterine CYP2J2 expression and circulatingepoxyeicosatrienoic acid levels in preeclampsia.

INTRODUCTION: The cytochrome P450 (CYP)-system regulates vascular functions, inflammation, and angiogenesis that are mechanistically important in preeclampsia. OBJECTIVES: The aim of this study was to analyze the dysregulation of the Cytochrome P450 in the pathogenesis of preeclampsia. METHODS: We performed microarray screening of placenta and decidua from 25 preeclamptic women and 23 controls. Results were confirmed by realtime RT-PCR, immunohistochemistry and Serum of patients were analyzed by HPLC tandem mass spectrometry. For functional testing we did cardiomyocyte contraction bioassay and myograph studies. The reduced uterine perfusion pressure (RUPP) rat model was proceed for interventional study. RESULTS: In microarray studies the CYP subfamily 2J polypeptide 2 (CYP2J2) was upregulated in preeclamptic decidual tissue (3.9 fold, p<0.0001) and in preeclamptic placenta (1.55 fold, p<0.001). RT-PCR confirmed the upregulation and immunohistochemistry, localized CYP2J2 in trophoblasts of villi and deciduas at week 12 and term. The CYP2J2 metabolites were analyzed by HPLC tandem mass spectrometry. 5,6- epoxyeicosatrienoic acids (EET), 14,15-EET, and the corresponding dihydroxyeicosatrienoic acids (DHET), were elevated in preeclamptic women compared to controls in the latter two-thirds of pregnancy and after delivery. Stimulation of the trophoblast-derived cell line SGHPL-4 with the preeclampsia-associated cytokine tumor necrosis factor-a enhanced CYP2J2 gene and protein expression. For functional testing, 5,6-EET increased the beating rate of neonatal cardiomyocytes in a bioassay and downregulated large-conductance calcium-activated potassium channel KCa 1.1 activity. In the RUPP rat model of preeclampsia, we observed elevated EET, DHET, and preeclamptic features that were ameliorated by the CYP inhibitor MsPPOH. Uterine arterial rings of rats also dilated in response to MsPPOH. CONCLUSION: Our data implicate CYP2J2 in the pathogenesis of preeclampsia and as a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

PP039. The importance of intentionally considering class 1 HELLP syndrome outcomes versus all other categories of HELLP syndrome or severe preeclampsia.

INTRODUCTION: Combining HELLP syndrome patient groups in publications and presentations may obfuscate any potential differences among patient groups with regard to maternal-perinatal outcomes and rendered therapies. OBJECTIVES: We explored the prevalence of major maternal morbidity (MMM) for patients with severe preeclampsia (SPRE) and each defined group of HELLP syndrome. METHODS: Retrospective cohort study 2000-2007 of patients categorized either as class 1 HELLP syndrome (HELLP1, platelets⩽50,000, AST⩽70,LDH⩽600), class 2 (HELLP2, platelets>50,000 to ⩽100,000), class 3 (HELLP3, platelets>100,000 to ⩽150,000), or partial/incomplete (HELLP4) with only 2 of 3 diagnostic parameters present. All SPRE patients (no HELLP) of 2005-2007 were also evaluated. Total MMM for each group was determined. MMM included cardiopulmonary [cardiogenic or noncardiogenic pulmonary edema, pleural or pericardial effusion, pulmonary embolus, indicated intubation, myocardial infarction or arrest], hematologic/coagulation [DIC, transfused blood products], central nervous system/visual [stroke, cerebral edema, hypertensive encephalopathy, vision loss], hepatic [subcapsular hematoma or rupture] or renal complications [acute tubular necrosis or renal failure]. All HELLP1 and HELLP2 patients received corticosteroids, magnesium sulfate and anti-hypertensives. Comparison among groups was done using Chi-square or Fisher exact test at 95% CI. RESULTS: Four hundred and twenty patients had a form of HELLP syndrome 2000-2007; 688 patients had SPRE 2005-2007.The prevalence of MMM for each patient group was determined: HELLP1=41.5%; HELLP2=10.3%; HELLP3=20.0%; HELLP4=21.0%; and SPRE=17.7%. MMM in HELLP1 was significantly increased over all other groups (P<0.001). Combining MMM for HELLP1+HELLP2 produced a prevalence of 22.1% MMM, insignificantly different from all others including HELLP3, HELLP4 and SPRE (p=0.19), thereby obscuring the significantly elevated MMM of HELLP1 patients. CONCLUSION: Only patients with HELLP1 have significantly increased MMM compared to other HELLP groups or SPRE. Failing to separately evaluate patients with HELLP1 in studies of HELLP syndrome could lead to mistaken conclusions about the effectiveness of a treatment to reduce MMM. All publications reviewing HELLP syndrome management should address how well it functions to reduce patient development of HELLP1 and thus minimize MMM.

PP085. Angiotensin II type 1 receptor antibodies increase angiotensin II sensitivity in pregnant rats.

INTRODUCTION: Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. OBJECTIVES: The aim of the study was to show if AT1-AB generated by immunisation alters Ang II sensitivity in pregnant rats. METHODS: We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. RESULTS: We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-alpha and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1alpha was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. CONCLUSION: Our mechanistic study supports the existence of an "autoimmune-activating receptor" that could contribute to Ang II sensitivity and possibly to preeclampsia.

PP137. Posterior reversible encephalopathy syndrome (PRES) is a constant component of eclampsia.

INTRODUCTION: Posterior reversible encephalopathy syndrome (PRES) has been reported to occur in patients with eclampsia. In both conditions there is evidence to suggest disordered cerebral autoregulation. OBJECTIVES: We sought to investigate the concurrence of PRES with eclampsia and to describe the associated obstetric, radiologic and critical care correlates. METHODS: Single center 2001-2010 retrospective cohort study of all patients with eclampsia who underwent neuroimaging via magnetic resonance imaging (MRI) or computerized tomography (CT) with or without contrast. The medical records of all patients with eclampsia during the study interval were identified, evaluated and extracted for pertinent data; a diagnosis of PRES was made by radiologists using standard criteria. RESULTS: Forty-six of forty-seven (97.9%) patients with eclampsia revealed PRES on neuroimaging using one or more modalities: MRI without contrast=41 (87.2%), MRI with contrast=27 (57.4%), CT without contrast=16 (34%), CT with contrast=7 (14.8%) and/or MRA/MRV=2 (4.3%). PRES was identified within the parietal (36, 78.3%), occipital (35, 76.1%), frontal (29, 63%), temporal (13, 28.3%) and basal ganglia/ brainstem/cerebellum (12, 26.1%). Eclampsia occurred antepartum in 23 patients, postpartum in 24 patients with 22 vaginal/25 cesarean deliveries at a mean maternal age of 21.8 years (range 15-39) and a mean gestational age of 33.9 weeks (range 22.4-41.7 weeks). Ethnicity was African-American in 38 patients. Headache was the most common presenting symptom (87.2%) followed by altered mental status (51.1%), visual disturbances (34%) and nausea/vomiting (19.1%). Severe systolic hypertension was present in 22 (47%) of patients.Use of antihypertensives (87%), magnesium sulfate (100%), diuretics (66%) and corticosteroids (50%) facilitated maternal recovery in all cases with usually a brief hospitalization (mean 3.9 days, range 1-20 days). CONCLUSION: The common finding of PRES in patients with eclampsia suggests that PRES may be part of the pathogenesis of eclampsia. We speculate that therapy targeted at prevention or reversal of PRES pathogenesis will prevent or facilitate recovery from eclampsia.

The role of immune activation in contributing to vascular dysfunction and the pathophysiology of hypertension during preeclampsia.

Preeclampsia remains a leading cause of maternal death and perinatal morbidity and still the pathophysiological mechanisms of the disease remain largely unknown. The most well accepted hypothesis for the genesis of the disease is that placental ischemia/hypoxia results from inadequate remodeling of the maternal uterine spiral arteries, which leads to a decrease in uteroplacental blood flow. Subsequently factors are released from the ischemic placenta showering the maternal vascular endothelium. These factors include a host of molecules such as the soluble VEGF receptor-1 (sFlt-1), the angiotensin II type-1 receptor autoantibody (AT1-AA), and cytokines such as TNF-a and Interleukin 6 which in turn generate widespread dysfunction of the maternal vascular endothelium. This dysfunction results in elevated circulating endothelin (ET-1), reactive oxygen species (ROS), and augmented vascular sensitivity to angiotensin II as well as decreased formation of vasodilators such as nitric oxide and prostacyclin. These alterations in vascular function lead to hypertension with multi-organ dysfunction, especially in cases of early onset preeclampsia. Therefore, identifying the connection between placental ischemia and maternal cardiovascular abnormalities is an important area of investigation.

Role of endothelin in mediating soluble fms-like tyrosine kinase 1-induced hypertension in pregnant rats.

Although soluble fms-like tyrosine kinase 1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor, has been implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms whereby enhanced sFlt-1 production leads to hypertension remain unclear. Both sFlt-1 and endothelin 1 productions are elevated in women with preeclampsia and in placental ischemic animal models of preeclampsia; however, the importance of endothelin 1 and sFlt-1 interactions in the control of blood pressure during pregnancy is unknown. The purpose of this study was to determine the role of endothelin 1 in mediating sFlt-1-induced hypertension in pregnant rats. To achieve this goal, sFlt-1 (3.7 microg/kg per day for 6 days) was infused into normal pregnant rats and pregnant rats treated with a selective endothelin type A receptor antagonist, ABT 627 (5 mg/kg per day for 6 days). Plasma concentration of sFlt-1 increased from 735+/-34 pg/mL in normal pregnant rats to 2498+/-645 pg/mL (P<0.05) with infusion of sFlt-1. Arterial pressure increased from 100+/-1 mm Hg in normal pregnant rats to 122+/-3 mm Hg (P<0.05) in sFlt-1-infused rats. Chronic increases in plasma sFlt-1 in normal pregnant rats increased preproendothelin mRNA expression in the renal cortices by approximately 3-fold. In addition, chronic endothelin type A receptor blockade completely abolished the blood pressure response to sFlt-1 in pregnant rats (104+/-3 versus 100+/-1 mm Hg; P<0.05), whereas the endothelin A receptor antagonist had no effect on arterial pressure in NP rats (105+/-2 versus 100+/-1 mm Hg). In conclusion, this study demonstrates that endothelin 1, via endothelin type A receptor activation, plays an important role in mediating the hypertension in response to excess sFlt-1 during pregnancy.

Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of interleukin 6.

The purpose of this study was to determine the role of interleukin (IL) 6 in mediating the increase in arterial pressure (AP) in response to chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats. AP was higher in RUPP rats (138+/-1 mm Hg) than in normal pregnant (NP) rats (104+/-1 mm Hg). Serum IL-6 levels in the RUPP rats were 104.5+/-28.6 pg/mL as compared with 36.6+/-7.4 pg/mL in NP rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma IL-6 on renal function and AP in pregnant rats, we infused IL-6 for 5 days (2.5 ng/day) in NP rats starting at day 14 of gestation. Five days later, serum IL-6 levels were 55.5+/-6.5 pg/mL in the control NP rats and 157.0+/-36.1 pg/mL in the IL-6-treated NP rats. AP was higher in the IL-6-treated NP rats (115+/-3 mm Hg) as compared with NP controls (101+/-1 mm Hg) at day 19 of gestation. Renal plasma flow and GFR were lower in the IL-6-treated NP rats than in the NP group. IL-6 increased plasma renin activity but did not affect endothelin in IL-6-treated NP rats. In contrast to the NP rats, IL-6 had no effect on AP or renal hemodynamics in virgin rats. In summary, these data indicate that plasma IL-6 is elevated in response to chronic reductions in uterine perfusion in pregnant rats and that a comparable elevation in plasma IL-6 increases AP and reduces renal function in pregnant rats.

Reduced uterine perfusion pressure (RUPP) model for studying cardiovascular-renal dysfunction in response to placental ischemia.

Despite being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are unknown. The initiating event in preeclampsia has been postulated to be reduced uteroplacental perfusion. Placental ischemia/hypoxia is thought to lead to widespread activation/dysfunction of the maternal vascular endothelium, vasoconstriction and hypertension. Experimental induction of chronic uteroplacental ischemia appears to be the most promising animal model to study potential mechanisms of preeclampsia since reductions in uteroplacental blood flow in a variety of animal models lead to a hypertensive state that closely resembles preeclampsia in women. This chapter details the methods we use in our laboratory to produce the reduced uterine perfusion pressure (RUPP) model in the pregnant rat.

Enhanced endothelin synthesis by endothelial cells exposed to sera from pregnant rats with decreased uterine perfusion.

The initiating event in preeclampsia is thought be to reduced uteroplacental perfusion. Although we have reported previously that chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats results in hypertension and enhanced endothelin production, the factors linking placental ischemia and endothelial cell activation remain unclear. The purpose of this study was to determine the role of angiotensin II type-1 (AT1) receptor activation on endothelin production induced by serum from pregnant rats exposed to reductions in uterine perfusion. To achieve this goal, human umbilical vein endothelial cells were exposed to sera collected from RUPP rats or normal pregnant rats. Arterial pressure was significantly higher in RUPP rats (135+/-2 mm Hg) than in pregnant rats (106+/-1 mm Hg). Six hours after exposure to RUPP serum (n=17), cell media endothelin concentration was 18.4+/-2.7 pg/mL as compared with 9.22+/-1.3 pg/mL from cells exposed to serum from normal pregnant rats (n=9). Eighteen hours after exposure to RUPP serum (n=7), endothelin concentration was 30.5+/-3.8 pg/mL as compared with 12.8+/-5.3 pg/mL from cells exposed to normal pregnant rat serum (n=6). In contrast, serum from RUPP rats did not increase endothelin production in human umbilical vein endothelial cells pretreated with an AT1 receptor antagonist, losartan (15 micromol/L). Eighteen hours after exposure to RUPP serum and losartan (n=14), endothelin concentration was 21.3+/-2.2 pg/mL as compared with 16.4+/-3.3 pg/mL from cells exposed to normal pregnant rat serum and losartan (n=10). These data indicate that serum from pregnant rats exposed to reductions in uterine perfusion enhances endothelin production by endothelial cells via by AT1 receptor activation.

Hypertension produced by reductions in uterine perfusion in the pregnant rat: role of tumor necrosis factor-alpha.

Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha are elevated in preeclamptic women and are thought to be an important link between placental ischemia and endothelial dysfunction. The purpose of this study was to determine the role of TNF in mediating hypertension in response to chronic reductions in uterine perfusion (RUPPs) in pregnant rats. Arterial pressure was significantly higher in RUPP rats (138+/-1 mm Hg) than in pregnant rats (107+/-1 mm Hg). Serum TNF-alpha levels in the RUPP rats were 17+/-4 pg/mL compared with 8+/-1 pg/mL in normal pregnant rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma TNF-alpha on renal and systemic hemodynamics in pregnant rats, we infused TNF-alpha for 5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats. Serum levels were 7+/-2 pg/mL in the control pregnant rats and 14+/-2 pg/mL in the TNF-alpha-treated pregnant rats. Mean arterial pressure was higher in the TNF-alpha-treated pregnant rats (123+/-3 mm Hg) compared with pregnant controls (96+/-3 mm Hg) at day 19 of gestation. TNF-alpha increased renal vascular resistance in pregnant rats by 182%. Renal plasma flow was 5.4+/-1.2 mL/min in the TNF-alpha-treated group and 9.2+/-1.6 mL/min in the control group. Glomerular filtration rate was 1.7+/-0.4 mL/min in the TNF-alpha-treated group and 2.6+/-0.4 mL/min in the control group. In summary, these data suggest that TNF-alpha may play an important role in mediating the increased arterial pressure in response to chronic RUPPs in pregnant rats.

Role of endothelin in mediating tumor necrosis factor-induced hypertension in pregnant rats.

Hypertension during preeclampsia is associated with an increase in plasma levels of tumor necrosis factor (TNF)-alpha, a cytokine known to contribute to endothelial dysfunction. Recently, our laboratory reported that a 2-fold increase in plasma TNF-alpha produces hypertension in pregnant rats. Endothelin is also elevated in preeclampsia and endothelin synthesis is enhanced by TNF-alpha. The purpose of this study was to determine the role of endothlelin in mediating TNF-alpha-induced hypertension in pregnant rats. To achieve this goal, TNF-alpha (50 ng/d for 5 days) was infused into control pregnant rats and pregnant rats treated with an endothelin receptor A antagonist, ABT 627 (5 mg/kg per day for 5 days). At day 19 of gestation, arterial pressure was measured and aorta, kidneys, and placentas were harvested. Infusion of TNF-alpha into pregnant rats increased plasma concentration of TNF-alpha (13.5+/-0.8 to 28.0+/-3.7 pg/mL) and arterial pressure (101+/-2 to 122+/-1 mm Hg). The increase in arterial pressure was associated with an increase in preproendothelin mRNA expression in placenta, aorta, and kidneys measured by real-time polymerase chain reaction (PCR). Pretreatment with the endothelin receptor A antagonist completely abolished the blood pressure response to TNF-alpha in pregnant rats (105+/-1 versus 97+/-2 mm Hg). In sharp contrast, the ETA receptor antagonist had no effect on arterial pressure in normal pregnant rats (97+/-2 versus 101+/-2 mm Hg). Moreover, chronic infusion of TNF-alpha had no significant effect on arterial pressure or renal preproendothelin levels in virgin rats. These results suggest an important role for endothelin in mediating TNF-alpha-induced hypertension in pregnant rats.

Participation of fad and mbt genes in synthesis of mycobactin in Mycobacterium smegmatis.

Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 micro M Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin. Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M. smegmatis. Ferrimycobactin prepared from iron-restricted cells of the wild type had an R(f) of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm. Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an R(f) of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin. Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain. Mutant strain LUN9 produced no detectable mycobactin. Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid). The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE. The mutated gene in LUN8 had homology with M. tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin.