RESUMEN
BACKGROUND: Magnesium sulfate (MgSO4) is utilized for fetal neuroprotection in preterm birth but its mechanism of action is still poorly understood. P2X7 receptor (P2X7R) is required for secretion of IL-1ß, and can be blocked by divalent cations such as magnesium (Mg) and its own antagonist, Brilliant Blue G (BBG). We sought to determine whether during inflammation MgSO4 can block endothelial IL-1ß secretion, using an in-vitro model. METHODS: Human umbilical vein endothelial cell (HUVEC) cultures were treated with varying doses of LPS, 2'(3)-Ο-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP), BBG and MgSO4 for 3- or 24 h. We determined cell cytotoxicity, apoptosis, IL-1ß mRNA expression, IL-1ß production and secretion and P2X7R expression on HUVECs. RESULTS: We demonstrated that MgSO4 is efficacious in blocking IL-1ß-mediated-inflammation in HUVECs, at both the initiation and propagation phases of inflammation. MgSO4 exerts these anti-inflammatory effects via downregulation of P2X7Rs on HUVECs. CONCLUSION: LPS-exposure increases IL-1ß production and secretion in HUVECs, which is further intensified by P2X7R agonist, BzATP while MgSO4 inhibits IL-1ß in both presence and absence of BzATP. This effect is similar to the results of P2X7R antagonist, BBG, suggesting that the anti-inflammatory effects of MgSO4 is through P2X7R.
Asunto(s)
Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inflamación/prevención & control , Sulfato de Magnesio/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/efectos de los fármacos , Apoptosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Vías Secretoras , Transducción de SeñalRESUMEN
Maternal inflammation (MI) is associated with many adverse perinatal outcomes. The placenta plays a vital role in mediating maternal-fetal resource allocation. Studies have shown that MI contributes to placental dysfunction, which then leads to adverse birth outcomes and high health risks throughout childhood. Placental mammalian target of rapamycin complex 1 (mTORC1) signaling pathway links maternal nutrient availability to fetal growth; however, the impact of MI on mTORC1 signaling in the placenta remains unclear. In this study, we sought to explore the changes of mTORC1 signaling in the mouse placenta at late gestation by using two models of MI employing lipopolysaccharide (LPS) and interleukin-1ß (IL-1ß) to mimic acute (aMI) and sub-chronic (cMI) inflammatory states, respectively. We determined placental mTORC1 activity by measuring the activity of mTORC1 downstream molecules, including S6k, 4Ebp1, and rpS6. In the aMI model, we found that mTORC1 activity was significantly decreased in the placental decidual and junctional zone at 2 and 6 h after LPS surgery, respectively; however, mTORC1 activity was significantly increased in the placental labyrinth zone at 2, 6, and 24 h after LPS treatment, respectively. In the cMI model, we observed that mTORC1 activity was increased only in the placental labyrinth zone after consecutive IL-1ß exposure. Our study reveals that different parts of the mouse placenta react differently to MI, leading to variable mTORC1 activity throughout the placenta. This suggests that different downstream molecules of mTORC1 from different parts of the mouse placenta may be used in clinical research to monitor the fetal well-being during MI.
Asunto(s)
Inflamación/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Placenta/metabolismo , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Interleucina-1beta/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Fosforilación , Embarazo , Distribución Aleatoria , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismoRESUMEN
Interleukin-1 beta (IL-1ß) is a cytokine mediator of perinatal brain injury. The effect of sub-chronic systemic IL-1ß exposure in perinatal and offspring outcomes is unclear. The aim of this study was to examine the effects of maternal IL-1ß exposure on pregnancy and offspring outcomes. At E15, CD1 dams were allocated to receive intraperitoneal injection of phosphate buffered saline or mouse recombinant IL-1ß (1â¯mcg) for four consecutive days. We analyzed pup survivaland neurobehavioral status. At E18, placental H&E staining and fetal brain Nissl staining was performed. Placental gene expression was analyzed by qPCR and T cell infiltration was analyzed by flow cytometry. Effects of inflammation on feto-placental blood flow were analyzed by Doppler ultrasonography. IL-1ß decreased pup survival (Pâ¯<â¯.0001) and adversely affected offspring performance on neurodevelopmental tests (Pâ¯<â¯.05). Placentas of exposed dams exhibited significant thinning of maternal and fetal sides, and fetal brain exhibited cortical thinning. Placental qPCR analysis revealed significant upregulation of NFκB2 (Pâ¯=â¯.0021) and CXCL11 (Pâ¯=â¯.0401). While maternal IL-1ß exposure did not affect feto-placental blood flow, placental flow cytometry showed an increase in placental infiltration of CD4+ T cells at 24â¯h post-injection (hpi, Pâ¯<â¯.0001) and CD8+ T cells at 72â¯hpi (Pâ¯=â¯.0217). Maternal sub-chronic, systemic inflammation with IL-1ß decreased pup survival and played a key role in perinatal brain injury. The mechanisms behind these outcomes may involve immune system activation and alterations in placental T cell trafficking.
Asunto(s)
Interleucina-1beta/efectos adversos , Placenta/inmunología , Efectos Tardíos de la Exposición Prenatal/inmunología , Animales , Lesiones Encefálicas/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Femenino , Feto/metabolismo , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/fisiología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos , EmbarazoRESUMEN
Melatonin has been shown to reduce oxidative stress and mitigate hypercoagulability. We hypothesized that maternally administered melatonin may reduce placental oxidative stress and hypercoagulability associated with exposure to intrauterine inflammation (IUI) and consequently improve fetoplacental blood flow and fetal sequelae. Mice were randomized to the following groups: control (C), melatonin (M), lipopolysaccharide (LPS; a model of IUI) (L), and LPS with melatonin (ML). The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. The systolic/diastolic ratio, resistance index, and pulsatility index in uterine artery (UtA) and umbilical artery (UA) were significantly increased in L compared with other groups when analyzed by Doppler ultrasonography. The expression of antioxidant mediators in the placenta was significantly decreased, while that of pro-inflammatory mediators was significantly increased in L compared to C and ML. Vascular endothelial damage and thrombi formation, as evidenced by fibrin deposits, were similarly increased in L compared to other groups. Maternal pretreatment with melatonin appears to modulate maternal placental malperfusion, fetal cardiovascular compromise, and fetal neuroinflammation induced by IUI through its antioxidant properties.
Asunto(s)
Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Melatonina/uso terapéutico , Placenta/efectos de los fármacos , Placenta/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Hemodinámica/efectos de los fármacos , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Ratones , Insuficiencia Placentaria/tratamiento farmacológico , Insuficiencia Placentaria/metabolismo , Embarazo , Ultrasonografía DopplerRESUMEN
A number of published studies have questioned the serotonin neurotoxic potential of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") and related drugs (fenfluramine, p-chloroamphetamine) based upon results from Western blot studies using a custom synthesized serotonin transporter (SERT) antibody that found no reduction in the abundance of a 50kDa protein after substituted amphetamine treatment. The purpose of this study was to collect Western blot data using the same SERT antibody used in those studies, but with positive and negative controls to identify the SERT protein signal. A 63-68 kDa band that had the regional distribution expected of rat brain SERT, was decreased by 5,7-DHT, and was absent in SERT KO animals was identified as the SERT protein. Significant, lasting decreases in the abundance of the 63-68 kDa band were evident in the rat brain after treatment with MDMA and related drugs (FEN, PCA). Thus, when the band corresponding to the SERT protein is identified in Western blots through the use of positive and negative controls, reduced abundance of the SERT protein can be readily demonstrated after substituted amphetamine treatment. These data provide further evidence of lasting loss of the SERT protein after exposure to MDMA and other substituted amphetamines.
Asunto(s)
Western Blotting/métodos , Western Blotting/normas , N-Metil-3,4-metilenodioxianfetamina/toxicidad , Proteínas de Transporte de Serotonina en la Membrana Plasmática/fisiología , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Técnicas de Inactivación de Genes , Valor Predictivo de las Pruebas , Ratas , Serotonina/deficiencia , Serotoninérgicos/toxicidad , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genéticaRESUMEN
The objective of this study was to determine if mouse bone marrow-derived mesenchymal stem cells (BMMSCs) ameliorate preterm birth and perinatal brain injury induced by intrauterine inflammation (IUI). A mouse model of IUI-induced perinatal brain injury at embryonic (E) day 17 was utilized. BMMSCs were derived from GFP-transgenic mice and phenotypically confirmed to be CD44+, Sca-1+, CD45-, CD34-, CD11b-, and CD11c- by flow cytometry and sorted by fluorescence-activated cell sorting (FACS). Dams were assigned to four groups: phosphate-buffered saline (PBS) + PBS, PBS + BMMSCs, lipopolysaccharide (LPS) + PBS, and LPS + BMMSCs. Following maternal IUI, there was a significant increase in CD8+ T cells in the placentas. Maternally administered BMMSCs trafficked to the fetal side of the placenta and resulted in significantly decreased placental CD8+ T cells. Furthermore, fetal trafficking of maternally administered BMMSCs correlated with an improved performance on offspring neurobehavioral testing in LPS + BMMSC group compared with LPS + PBS group. Our data support that maternal administration of BMMSCs can alleviate perinatal inflammation-induced brain injury and improve neurobehavioral outcomes in the offspring via CD8+ T cell immunomodulation at the feto-placental interface.
Asunto(s)
Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/prevención & control , Linfocitos T CD8-positivos/metabolismo , Modelos Animales de Enfermedad , Trasplante de Células Madre Mesenquimatosas/métodos , Útero/metabolismo , Animales , Animales Recién Nacidos , Médula Ósea/fisiología , Lesiones Encefálicas/etiología , Células Cultivadas , Femenino , Inflamación/complicaciones , Inflamación/metabolismo , Inflamación/patología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Transgénicos , Embarazo , Nacimiento Prematuro/etiología , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/prevención & controlRESUMEN
Zika virus (ZIKV) infection during pregnancy causes significant adverse sequelae in the developing fetus, and results in long-term structural and neurologic defects. Most preventive and therapeutic efforts have focused on the development of vaccines, antivirals, and antibodies. The placental immunologic response to ZIKV, however, has been largely overlooked as a target for therapeutic intervention. The placental inflammatory response, specifically IL-1ß secretion and signaling, is induced by ZIKV infection and represents an environmental factor that is known to increase the risk of perinatal developmental abnormalities. We show in a mouse model that maternally administrated IL-1 receptor antagonist (IRA; Kineret, or anakinra), following ZIKV exposure, can preserve placental function (by improving trophoblast invasion and placental vasculature), increase fetal viability, and reduce neurobehavioral deficits in the offspring. We further demonstrate that while ZIKV RNA is highly detectable in placentas, it is not correlated with fetal viability. Beyond its effects in the placenta, we show that IL-1 blockade may also directly decrease fetal neuroinflammation by mitigating fetal microglial activation in a dose-dependent manner. Our studies distinguish the role of placental inflammation during ZIKV-infected pregnancies, and demonstrate that maternal IRA may attenuate fetal neuroinflammation and improve perinatal outcomes.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Placentación/efectos de los fármacos , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Efectos Tardíos de la Exposición Prenatal/prevención & control , Infección por el Virus Zika/complicaciones , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Encéfalo/inmunología , Modelos Animales de Enfermedad , Femenino , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Ratones , Microglía/efectos de los fármacos , Microglía/inmunología , Placenta/irrigación sanguínea , Placenta/efectos de los fármacos , Placenta/inmunología , Placenta/patología , Placentación/inmunología , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/patología , Complicaciones Infecciosas del Embarazo/virología , Efectos Tardíos de la Exposición Prenatal/inmunología , Efectos Tardíos de la Exposición Prenatal/virología , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/inmunología , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
Maternal periodontal disease has been linked to adverse pregnancy sequelae, including preterm birth (PTB); yet, root planing and scaling in pregnancy has not been associated with improved perinatal outcomes. Fluoride, a cariostatic agent, has been added to drinking water and dental products to prevent caries and improve dental health. The objective of this study was to explore the effects of fluoride supplementation using a mouse model of preterm birth and perinatal sequalae. Pregnant mice were fed low dose fluoride (LF-) or high dose fluoride (HF-) and given intrauterine injections of lipopolysaccharide (LPS) or phosphate-buffered saline (PBS). We found that LPS + LF- significantly increased livebirths, pup survival, and litter size compared to LPS alone. Moreover, offspring from the LPS + LF- group exhibited significantly improved neuromotor performance and more neurons compared to those from the LPS group. Additionally, LF- treatment on human umbilical vein endothelial cells (HUVECs) increased cell viability and decreased oxidative stress after treatment with LPS. Collectively, our data demonstrates that maternal LF- supplementation during pregnancy postpones the onset of PTB, acts to increase the liveborn rate and survival time of newborns, and reduces perinatal brain injury in cases of intrauterine inflammation.
Asunto(s)
Fluoruros/farmacología , Enfermedades Periodontales , Nacimiento Prematuro , Animales , Modelos Animales de Enfermedad , Femenino , Fluoruros/efectos adversos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos/toxicidad , Ratones , Enfermedades Periodontales/tratamiento farmacológico , Enfermedades Periodontales/metabolismo , Enfermedades Periodontales/patología , Embarazo , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/patologíaRESUMEN
PROBLEM: Exposure to systemic maternal inflammation (i.e., maternal sepsis, influenza, human immunodeficiency virus, or pyelonephritis) and intrauterine (IU) inflammation (i.e., chorioamnionitis or preterm labor) have been associated with adverse perinatal sequelae. Whether systemic and localized inflammation leading to adverse outcomes have similar placental mechanisms remain unclear. METHOD OF STUDY: We conducted a study by murine modeling systemic and localized IU inflammation with injections of either intraperitoneal (IP) or IU interleukin-1ß (IL-1ß) and compared fetoplacental hemodynamic changes, cytokine/chemokine expression, and fetal loss. RESULTS: IU IL-1ß exposure reduced offspring survival by 31.1% and IP IL-1ß exposure by 34.5% when compared with control pups. Despite this similar outcome in offspring survival, Doppler analysis revealed a stark difference: IU group displayed worsened fetoplacental hemodynamic changes while no differences were found between IP and control groups. While both IU and IP groups had increases in pro-inflammatory cytokines and chemokines, specific gene expression trends differed between the two groups, once again highlighting their mechanistic differences. CONCLUSION: While both IP and IU IL-1ß exposure similarly affected pup survival, only IU inflammation resulted in fetoplacental hemodynamic changes. We speculate that exposure to maternal systemic and IU inflammation plays a key role in fetal injury by utilizing different placental inflammatory pathways and mechanisms.
Asunto(s)
Corioamnionitis/inmunología , Intercambio Materno-Fetal/inmunología , Placenta/inmunología , Nacimiento Prematuro/inmunología , Animales , Corioamnionitis/diagnóstico por imagen , Corioamnionitis/mortalidad , Corioamnionitis/patología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Feto/diagnóstico por imagen , Feto/inmunología , Humanos , Interleucina-1beta/administración & dosificación , Interleucina-1beta/inmunología , Ratones , Placenta/patología , Circulación Placentaria/inmunología , Embarazo , Nacimiento Prematuro/mortalidad , Nacimiento Prematuro/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Tasa de Supervivencia , Ultrasonografía DopplerRESUMEN
PROBLEM: Maternal inflammation leads to preterm birth and perinatal brain injury. Melatonin, through its anti-inflammatory effects, has been shown to be protective against inflammation-induced perinatal adverse effects. However, the immunomodulatory effects of melatonin on preterm birth and prematurity-related morbidity remain unknown. We wanted to investigate the effects of maternally administered melatonin on preterm birth and perinatal brain injury in a mouse model of maternal inflammation. METHOD OF STUDY: A model of maternal inflammation employing lipopolysaccharide (LPS) was used to mimic the most common clinical scenario of preterm birth, that of maternal inflammation. Mice were randomly divided into the following groups: control, LPS, and LPS with melatonin pre-treatment. Doppler ultrasonography was used to obtain fetal and maternal hemodynamic measurements in utero. Placenta and fetal brains were harvested and analyzed for proinflammatory markers and signs of perinatal brain injury, respectively. Surviving offspring were assessed for neuromotor outcomes. RESULTS: Melatonin pre-treatment lowered the level of proinflammatory cytokines in the uterus and the placenta, significantly improved LPS-induced acute fetal neuroinflammation and perinatal brain injury, as well as significantly upregulated the SIRT1/Nrf2 signaling pathway to reduce LPS-induced inflammation. Melatonin also prevented adverse neuromotor outcomes in offspring exposed to maternal inflammation. CONCLUSION: Maternally administered melatonin modulated immune responses to maternal inflammation and decreased preterm birth and perinatal brain injury. These results suggest that melatonin, a safe treatment during pregnancy, may be used as an experimental therapeutic in clinical trials.
Asunto(s)
Lesiones Encefálicas/terapia , Enfermedades Fetales/terapia , Inflamación/terapia , Exposición Materna/efectos adversos , Melatonina/uso terapéutico , Nacimiento Prematuro/terapia , Animales , Modelos Animales de Enfermedad , Femenino , Hemodinámica , Humanos , Inmunomodulación , Inyecciones Intraperitoneales , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos ICR , Embarazo , Resultado del EmbarazoRESUMEN
Exposure to intrauterine inflammation (IUI) is associated with short- and long-term adverse perinatal outcomes. However, little data exist on utilizing placenta to prognosticate fetal injury in this scenario. Our study aimed to utilize imaging modalities to evaluate mechanisms contributing to placental injury following IUI exposure and correlated it to concomitant fetal brain injury. CD1 pregnant dams underwent laparotomies and received intrauterine injections of either lipopolysaccharide (LPS; a model of IUI) or phosphate-buffered saline (PBS). In utero ultrasound Doppler velocimetry of uterine and umbilical arteries and magnetic resonance imaging (MRI) of placental volumes with confirmatory immunohistochemical (vimentin) and histochemistry (fibrin) analyses were performed. ELISA for thrombosis markers, fibrinogen and fibrin was performed to analyze thrombi in placenta. Fetal brain immunohistochemistry was performed to detect microglial activation (ionized calcium-binding adaptor molecule 1, Iba1). On ultrasound, LPS group demonstrated elevated resistance indices, pulsatility indices and a greater occurrence of absent end-diastolic flow in the umbilical and uterine arteries. In the fetus, there was an increased cardiac Tei indices in the LPS group. MRI revealed decreased volume of placenta in the LPS group associated with placental thinning and placental endothelial damage on immunohistochemistry. Decreased fibrinogen content and more thrombi staining in placenta exposed to maternal LPS indicated the hypercoagulability. Furthermore, the expression of Iba1was significantly associated with placental thickness (r = -0.7890, Pearson correlation coefficient). Our data indicate that IUI can trigger events leading to maternal placental malperfusion and fetal vessel resistance, as well as predispose the developing fetus to cardiac dysfunction and brain damage. Furthermore, our data suggest that prenatal ultrasound can be a real-time clinical tool for assessing fetal risk for adverse neurologic outcomes following the potential IUI exposure.
Asunto(s)
Lesiones Encefálicas , Enfermedades Fetales , Inflamación , Lipopolisacáridos/toxicidad , Enfermedades Placentarias , Placenta , Animales , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Femenino , Enfermedades Fetales/inducido químicamente , Enfermedades Fetales/metabolismo , Enfermedades Fetales/patología , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Ratones , Placenta/lesiones , Placenta/metabolismo , Placenta/fisiología , Enfermedades Placentarias/inducido químicamente , Enfermedades Placentarias/metabolismo , Enfermedades Placentarias/patología , EmbarazoRESUMEN
We investigated the mechanisms by which CD8+ T-cell trafficking in placenta contributes to perinatal brain injury by studying effects of maternal CD8+ T-cell depletion (DEP) in a mouse model of intrauterine inflammation (IUI). Maternal CD8+ T cells were depleted with anti-CD8+ antibodies. IUI was induced with lipopolysaccharide (LPS). DEP was confirmed using flow cytometry. Preterm birth rate was evaluated. Offspring neurologic sequelae were assessed by Nissl staining, immune arrays, confirmatory individual TaqMan® gene assays, and neurobehavioral tests. DEP did not significantly prevent LPS-induced preterm birth but improved neurobehavioral performance (P < .001) and increased cortical neuronal density (P < .05) in LPS-exposed pups compared to controls. These changes were associated with decreased CCL3 and CXCL10 and increased CCL5 in DEP LPS-exposed mice. We demonstrate that DEP reduces perinatal brain injury following IUI. This supports a role for maternal CD8+ T-cell trafficking in placenta in mediating perinatal brain injury separate from preterm birth mechanisms.
Asunto(s)
Lesiones Encefálicas/inmunología , Linfocitos T CD8-positivos/inmunología , Inflamación/inmunología , Placenta/inmunología , Animales , Quimiocina CCL3/inmunología , Quimiocina CCL5/inmunología , Quimiocina CXCL10/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Lipopolisacáridos/inmunología , Depleción Linfocítica/métodos , Ratones , Neuronas/inmunología , Embarazo , Nacimiento Prematuro/inmunologíaRESUMEN
We studied in vivo expression of the serotonin transporter (SERT) protein after 3,4-methylenedioxymethamphetamine (MDMA), p-chloroamphetamine (PCA), or fenfluramine (FEN) treatments, and compared the effects of substituted amphetamines to those of 5,7-dihydroxytryptamine (5,7-DHT), an established serotonin (5-HT) neurotoxin. All drug treatments produced lasting reductions in 5-HT, 5-HIAA, and [(3)H]paroxetine binding, but no significant change in the density of a 70 kDa band initially thought to correspond to the SERT protein. Additional Western blot studies, however, showed that the 70 kDa band did not correspond to the SERT protein, and that a diffuse band at 63-68 kDa, one that had the anticipated regional brain distribution of SERT protein (midbrain>striatum>neocortex>cerebellum), was reduced after 5,7-DHT and was absent in SERT-null animals, was decreased after MDMA, PCA, or FEN treatments. In situ immunocytochemical (ICC) studies with the same two SERT antisera used in Western blot studies showed loss of SERT-immunoreactive (IR) axons after 5,7-DHT and MDMA treatments. In the same animals, tryptophan hydroxylase (TPH)-IR axon density was comparably reduced, indicating that serotonergic deficits after substituted amphetamines differ from those in SERT-null animals, which have normal TPH levels but, in the absence of SERT, develop apparent neuroadaptive changes in 5-HT metabolism. Together, these results suggest that lasting serotonergic deficits after MDMA and related drugs are unlikely to represent neuroadaptive metabolic responses to changes in SERT trafficking, and favor the view that substituted amphetamines have the potential to produce a distal axotomy of brain 5-HT neurons.
Asunto(s)
Encéfalo/efectos de los fármacos , N-Metil-3,4-metilenodioxianfetamina/efectos adversos , Neurotoxinas/efectos adversos , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Serotonina/biosíntesis , Degeneración Walleriana/inducido químicamente , 5,7-Dihidroxitriptamina/toxicidad , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Fenfluramina/toxicidad , Inmunohistoquímica , Masculino , N-Metil-3,4-metilenodioxianfetamina/química , Neurotoxinas/química , Ratas , Ratas Sprague-Dawley , Serotoninérgicos/efectos adversos , Serotoninérgicos/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Triptófano Hidroxilasa/metabolismo , Degeneración Walleriana/metabolismo , Degeneración Walleriana/fisiopatología , p-Cloroanfetamina/toxicidadRESUMEN
PURPOSE: To investigate the impact of somatic mutations in homologous recombination (HR) genes on the chemotherapeutic response and survival of patients with epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: We performed targeted massively parallel sequencing of tumor DNA from 158 patients with EOC. We associated adjuvant chemotherapy and clinical outcome with mutations in selected genes, focusing on those encoding HR proteins. RESULTS: HR mutations were found in 47 (30%) tumors. We did not detect an overall survival (OS) difference in advanced stage patients whose tumors had HR mutations compared to those without (median OS of 49.6 months (95% CI 29.9-57.7) vs. 43.3 months (95% CI 31.9-75.47), p = 0.87). However, when stratified by chemotherapy regimen, patients whose tumors had TP53 and HR mutations demonstrated a marked survival advantage when treated with platinum and paclitaxel vs. platinum +/- cyclophosphamide (median OS of 90 months (95% CI 50-NA) vs. 29.5 months (95% CI 17.7-50.5), p = 0.0005). CONCLUSIONS: Previous studies demonstrating a survival advantage for EOC patients with somatic HR mutations have been conducted with almost universal use of both platinum and paclitaxel. Our study is the first to our knowledge to compare cohorts with somatic HR gene mutations treated with and without paclitaxel containing platinum regimens. The survival benefit attributed to the platinum sensitivity of HR deficient ovarian cancers may depend upon the combined use of paclitaxel.
Asunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Recombinación Homóloga/genética , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/mortalidad , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Paclitaxel/uso terapéutico , Carcinoma Epitelial de Ovario , Quimioterapia Adyuvante , Estudios de Cohortes , Ciclofosfamida/uso terapéutico , Variaciones en el Número de Copia de ADN , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Compuestos de Platino/uso terapéutico , Análisis de Secuencia de ADN , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In recent years, there has been growing interest in the use of Western blot analysis to monitor changes in the abundance of the serotonin transporter (SERT) protein. In the Western blot procedure, heat denaturation is a common, early step. We now report that heating samples to 90 degrees C decreases the abundance of the SERT protein band and causes dispersion of a majority of the SERT signal to a high molecular weight smear. These observations are in keeping with the fact that heating can influence the electrophoretic behavior of some proteins. By omitting the heat denaturation step in the Western blot procedure, better detection of the SERT protein is achieved.