Sonia Gagioti

Participation of the Mouse Implanting Trophoblast in Nitric Oxide Production During Pregnancy

Abstract While considerable progress has been made in elucidating nitric oxide (NO) regulatory mechanisms in the later stages of gestation, much less is known about its synthesis and role during embryo implantation. Thus, to evaluate the participation of the trophoblast in the production of NO during this phase, this study focused on NADPH-diaphorase activity and the distribution of NO synthase isoforms (NOS) using immunohistochemistry in pre- and postimplantation mouse embryos in situ and in vitro, as well as on NO production itself, measured as total nitrite, in trophoblast culture supernatants (Griess reaction). No NADPH-diaphorase activity was found in preimplanting embryos except after culturing for at least 48 h, when a few trophoblastic giant cells were positive. Conversely, postimplantation trophoblast cells either lodged into the implantation chamber (in situ) or after culturing (in vitro) showed intense NADPH-diaphorase activity. Also in the postimplantation trophoblast, the endothelial and inducible NOS (eNOS and iNOS) isoforms were immunodetected, under both in situ and in vitro conditions, although in different patterns. Extracts of ectoplacental cone also revealed bands of 135 and 130 kDa on SDS-PAGE that reacted with anti-eNOS and anti-iNOS, respectively, on Western blot. Analysis of the culture supernatant demonstrated that the nitrite concentration was 1) proportional to the number of cultured trophoblast cells, 2) almost completely abolished in the presence of Nω-nitro-l-arginine methyl ester, and 3) increased 2-fold in cultures stimulated with γ-interferon. These results strongly suggest the production of NO from constitutive and inducible isoforms of NOS by the implanting mouse trophoblast. They also emphasize the possibility of the participation of these cells in vasodilatation and angiogenesis, and in cytotoxic mechanisms involved in the intense phagocytosis of injured maternal cells, which occur during the implantation process.

Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Placental morphology of rats prenatally exposed to methyl parathion

Although prenatal exposure to pesticides has been associated with numerous adverse reproductive effects, data on the effects of such toxics in the placenta is limited. Thus, the present study was carried out to determine the morphology of the rat placenta exposed to the organophosphate pesticide methyl parathion (MP) in ad libitum fed and restricted diet animals. The pregnant females were randomly divided into control groups and experimental groups, the latter of which received MP at the doses of 0.0, 1.0, 1.5 and 2.0 mg/kg. Most cells in the maternal-fetal interface showed significant alterations in the presence of MP. Trophoblast giant cells exhibited either prominent characteristics of degeneration or normal morphology with many phagosome vacuoles, apparently containing cell debris. Vascular congestion was also more frequent in the labyrinth of the treated animals. Areas of fibrosis and hemorrhage were found in the decidua, as well as decidual cells presenting pyknotic nuclei and acidophilic cytoplasm. In the placentas of females treated with both restricted diet and MP, such changes were much more severe. Together, these alterations suggest a direct, toxic effect of MP on the placental cells. The phagocytic activity exhibited by trophoblast cells, may be playing a role in the removal of death cells from the maternal-placental interface and/or in a compensatory mechanism to maintain the uptake of maternal nutrients, following decreased metabolic exchange functions of the labyrinth due to the toxic effect of the MP.

Interferon-gamma alters the phagocytic activity of the mouse trophoblast

Interferon-gamma (IFN-gamma) mediates diverse functions in bone marrow-derived phagocytes, including phagocytosis and microbe destruction. This cytokine has also been detected at implantation sites under both physiological and pathological conditions in many different species. At these particular sites, the outermost embryonic cell layer in close contact with the maternal tissues, the trophoblast exhibits intense phagocytic activity. To determine whether IFN-gamma affects phagocytosis of mouse-trophoblast cells, ectoplacental cone-derived trophoblast was cultured and evaluated for erythrophagocytosis. Phagocytic activity was monitored ultrastructurally and expressed as percentage of phagocytic trophoblast in total trophoblast cells. Conditioned medium from concanavalin-A-stimulated spleen cells significantly enhanced trophoblast phagocytosis. This effect was blocked by pre-incubation with an anti-IFN-gamma neutralizing antibody. Introduction of mouse recombinant IFN-gamma (mrIFN-gamma) to cultures did not increase cell death, but augmented the percentage of phagocytic cells in a dose-dependent manner. Ectoplacental cones from mice deficient for IFN-gamma receptor alpha-chain showed a significant decrease of the phagocytosis, even under mrIFN-gamma stimulation, suggesting that IFN-gamma-induced phagocytosis are receptor-mediated. Reverse transcriptase-PCR analyses confirmed the presence of mRNA for IFN-gamma receptor alpha and beta-chains in trophoblast cells and detected a significant increase in the mRNA levels of IFN-gamma receptor beta-chain, mainly, when cultured cells were exposed to IFN-gamma. Immunohistochemistry and Western blot analyses also revealed protein expression of the IFN-gamma receptor alpha-chain. These results suggest that IFN-gamma may participate in the phagocytic activation of the mouse trophoblast, albeit the exact mechanism was not hereby elucidated. Protective and/or nutritional fetal benefit may result from this physiological response. In addition, our data also shed some light on the understanding of trophoblast tolerance to inflammatory/immune cytokines during normal gestation.

During stationary phase, Beijerinckia derxii shows nitrogenase activity concomitant with the release and accumulation of nitrogenated substances

Beijerinckia derxii, a free-living nitrogen-fixing bacterium, maintained an increasing nitrogenase specific activity during the stationary growth phase. To verify the destination of the nitrogen fixed during this phase, intra and extracellular nitrogenated contents were analyzed. Organic nitrogen and amino acids were detected in the supernatant of the cultures. An increase in intracellular content of both nitrogen and protein occurred. Cytoplasmic granules indicated the presence of arginine. The ability of a non-diazotrophic bacterium (E. coli) to use B. derxii proteins as a source of nitrogen was observed concomitantly with E. coli growth. There is a suggestion that B. derxii contributes to the environment by both releasing nitrogenated substances and accumulating substances capable of being consumed after its death.

NADPH‐diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal‐maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH‐diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH‐diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre‐ and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH‐diaphorase activity nor inducible or endothelial NOS isoforms were found in pre‐implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal‐maternal interface.

Polineuropatia por Parathion: estudo clinico, eletrofisiológico e histológico de um caso

A case of 38 year old man who worked with organochlorinated and Parathion during 5 years is reported. His follow-up was up to 2 years. The onset of the disease was characterized by cholinergic signs, headache, loss of weight, trembling, miokimias, fasciculations, ataxia, myotonic phenomena (in hands only) and motor sensitive peripheral polyneuropathy (affecting the lower limbs symmetrically). Low concentrations of blood cholinesterases confirmed the etiology. Myotonic phenomena disappeared spontaneously 6 months after the initial observation. One year later, the concentration of erythrocyte acetylcholinesterase was found to be low and plasma cholinesterase was normal, suggesting that the patient was carrier of a congenital deficiency of acetylcholinesterase. In literature relationship between myotonia and intoxication due to organophosphorus was not found. The whole clinical picture, cholinergic symptoms, transitory phenomena and spontaneous motor activity could be explained by an excess of acetylcholine. Electromyography (EMG) in the first observation showed neuromuscular transmission blocking characterized by deficiency or absence of voluntary activity, unexcitability of fibular nerves, with fibrillations and positive peaks as described previously with Mipafox (another organophosphorus agent). During 2 years of observation numerous end-plates potentials of muscular fibres persisted in the EMG. A progressive increase in voluntary activity showed by unit motor potential of almost normal amplitude and very increased duration was observed. No potentials of reinnervation were noted. The results of EMG were explained as disturbances of neuromuscular transmission associated with moderate signs of denervation. The Eaton-Lamberts test and the stimulation of a single unit motor potential confirmed disorder of neuromuscular synapses. The histochemistry of brachial biceps showed scattered atrophic and angulated type I and II fibres. Teased-fibres preparations showed nerve fibres with B, C, and G alterations as defined by Dyck et al. indicating axonal degeneration. These results were according to velocity of sensitive conduction. The conduction velocity of fibular nerves was strongly delayed during all the evolution indicating serious disorders of motor nerves myelin.(ABSTRACT TRUNCATED AT 400 WORDS)

Miopatia miotubular: estudo clinico, eletrofisiologico e histologico de um caso

A cases of myotubular myopathy in a 10 years old girl is reported. Clinically, palpebral ptosis, ocular movements limitation, facial diplegia, positivity of Gowers test, muscular hypotrophy distal, foot drop and deep absent reflexes were found. These signals were described by most of authors, besides symptoms referred, like partial urinary incontinency and frequent vomits. Reflexes H absents and teary in our patient were observed but were not described in the other cases of the literature. Routine laboratory tests were within normal limits; only aldolase was lightly elevated. In all muscles examined it was noted a spontaneous electromyographic activity with +- of 2,64 +/- 1,33 ms, 36,87 +/- 30,87 microV and 88,13 +/- 24,82 /s of frequency without characteristics of desenervation potentials of myoneural plates. The voluntary electromyographic activity was of myopathic pattern. A curve I/D made in the motor point of braquial biceps muscle was normal. The biopsy was made in the motor point of this muscle for histochemical, electron immunofluorescence and vital stain microscopy. The biopsy showed 35% of fibras with central nuclei, predominance and hypotrophy of type I fibres, in some of them there were not myofibrils in the central zone, and poor differentiation between the fibre types in oxidative enzymes reactions. The electron microscopy confirmed the histochemical studies. The direct immunofluorescence was positive in some fibres. The vital stain showed beaded subterminal motor fibres. The clinical, electromyographic and principally histological findings suggest a innervation congenital disorder of muscle fibres.

McArdle's disease: a case report

This disease was described by McArdle as an inherited autosomal recessive affection characterized by glicogen storage with normal chain in the skeletal muscles, due to absence of myophosphorylase activity. Under a clinical aspect, excessive fatigability, cramps and myoglobinuria appear following physical exercise. A case of this disease in a 36-year-old male patient is reported. Failure of elevation of venous blood lactate after physical effort under anaerobic conditions, as well as muscle histochemistry, made diagnosis confirmation possible. The authors comment on the differential diagnosis betwen McArdles disease and the other causes of myoglobinuria, specially phosphofructokinase and carnitine-palmityl-transferase deficiency.

Liver Damage Induced by Succinylacetone: A Shared Redox Imbalance Mechanism between Tyrosinemia and Hepatic Porphyrias

To show liver failure mediated by 5-aminolevulinic acid (ALA), a heme precursor accumulated in inborn and acquired porphyrias, rats were treated with succinylacetone methyl ester (SAME). Treated rats underwent the expected ALA accumulation in plasma, liver and urine as a result from inhibition of ALA dehydratase (ALAD) activity. The enzyme activity decreased concomitantly with diminished urinary coproporphyrin levels. Additionally, liver protein carbonyls, iron and ferritin were higher in groups treated with a lower concentration of SAME whereas malondialdehyde was higher in the group treated with a higher ester dose. Consistent with these biochemical data, chronic treatment SAME was associated with induced oxidative subcellular and tissue damage revealed by cytosolic and mitochondrial changes within the liver cells. Altogether, these data expand the understanding of the direct biochemical mechanism for liver cell death in hepatic inborn disorders by generating excess ALA and may foster future therapeutic-driven strategies to preserve liver function.