Placental alkaline phosphatase (PLAP) enzyme activity and binding to IgG in Chagas' disease.

Placentas and plasma from women with and without Chagas' disease and cultures of human placental villi with Trypanosoma cruzi, neuraminidase, phospholipase A2 and phospholipase C were analyzed in order to verify if the alterations in placental alkaline phosphatase (PLAP) enzyme activity are caused by T. cruzi as observed in previous works. As IgG receptivity happens to be one of the proposed functions of PLAP, general IgG binding ability of the placentas treated with the mentioned enzymes, which are present on the parasite's surface, were also tested. The phospholipases caused an increase of PLAP's enzyme activity in the supernatant of infected placentas and a decrease of enzyme activity in the membrane of cultured placentas, therefore suggesting the cleavage of PLAP by parasitic enzymes. Desialylation could also partially inhibit PLAP's enzyme activity in supernatant and membrane of placenta culture. Placentas from healthy patients presented higher IgG receptivity than those from patients with Chagas' disease. In vitro infection of healthy placentas with T. cruzi caused no difference in IgG receptivity in placental sections with respect to controls but the phospholipases and neuraminidase increased the IgG receptivity of cultured placentas. The IgG transference index was higher for patients with Chagas' disease than for those without it. Although binding to IgG does not completely inhibit the enzyme activity of PLAP, it interferes with the enzyme activity of PLAP. We concluded that the enzymes on the surface of T. cruzi trypomastigotes can not only affect PLAP's enzyme activity but also increase the IgG binding ability of the placenta and this can be related to the actions of neuraminidase-transsialidase, phospholipase A2 and phospholipase C on the parasite surface. The modification of PLAP from women with Chagas' disease should be considered as a result of multiple factors.

Placental alkaline phosphatase (PLAP) study in diabetic human placental villi infected with Trypanosoma cruzi.

Previous work has demonstrated that PLAP activity decreases in serum and placental villi from term chagasic and diabetic pregnant women. In vitro, T. cruzi induces changes in human syncytiotrophoblast's PLAP. Our aim was to determine if infection with T. cruzi induces changes in PLAP activity in diabetic and chagasic women's placenta, in order to elucidate if PLAP plays a role in the mechanisms of interaction between placenta and T. cruzi, and whether hyperglycemic conditions could worsen the placental infection. Using zymogrammes, Western blot, biochemical and immunohistological techniques, PLAP activity was determined in placental villi from diabetic and chagasic women, and in normal placentas cultured under hyperglycemic conditions with or without trypomastigotes. A significant reduction of PLAP expression was immunologically detected in infected diabetic and normal placental villi cultured under hyperglycemic conditions of 71 and 81%, respectively, compared with controls. A significant decrease of PLAP specific activity was registered in homogenates and in the culture media from both infected diabetic and normal placentas under hyperglycemic conditions (of about 50-70%), and in chagasic ones (of about 87%), when compared with controls. Thus, PLAP might be involved in parasite invasion and diabetic and hyperglycemic placentas could be more susceptible to T. cruzi infection.

Epidermal growth factor (EGF) in the human placental infection with Trypanosoma cruzi.

Maternal infection of Trypanosoma cruzi is associated with premature births, abortions and placentitis. A decrease in EGF levels has been suggested to occur in animals infected by T. cruzi, but there is no research about the levels of EGF in human patients with Chagas' disease. We evaluated serum EGF levels in pregnant women with and without the disease, and with immunological methods detected EGF receptors and EGF in both groups of placentae and in cultures of normal placental villi with and without parasites. PLAP in placentae from those women was also immunologically detected, since EGF can induce the release of PLAP from the trophoblast surface and PLAP is suggested to be a receptor allowing parasite invasion of the placenta. Plasma from women with Chagas' disease contained lower level of EGF when compared to plasma of healthy women. Placentae from women with Chagas' disease showed lower PLAP expression but same level of detectable EGF receptors and EGF when compared with placentae from women without the disease. Culture with parasites did not reduce EGFr level. Results suggest a lower availability of EGF in women with Chagas' disease, which could explain several malfunctions of the placenta associated with maternal Chagas' disease.

Role of placental alkaline phosphatase in the internalization of trypomatigotes of Trypanosoma cruzi into HEp2 cells.

BACKGROUND: In vitro, Trypanosoma cruzi invades a wide variety of mammalian cells by an unique process that is still poorly understood. Trypomastigotes adhere to specific receptors on the outer membrane of host cells before intracellular invasion, causing calcium ion mobilization and rearrangement of host cell microfilaments. OBJECTIVE: To test if placental alkaline phosphatase (PLAP), a trophoblast plasma membrane protein anchored by a glycosylphosphatidylinositol molecule, is involved in the transplacental transmission of this parasite. METHOD: We cultured HEp2 cells with the parasite and studied PLAP and actin microfilaments. The results were correlated with invasion rate. RESULTS: Human HEp2 tumour cells express PLAP. HEp2 cells infected with trypomastigotes showed alteration in their alkaline phosphatase activity and a different pattern of actin organization, compared to control cells. Perturbation of PLAP from HEp2 cells before infection with T. cruzi trypomastigotes decreased the invasion rate. CONCLUSION: Placental alkaline phosphatase could be involved in the internalization of T. cruzi into HEp2 cells, via activation of tyrosine kinase and rearrangement of actin microfilaments.

Trypanosoma cruzi infection induces microfilament depletion in human placenta syncytiotrophoblast.

Congenital Chagas disease, endemic in Latin America, is associated with premature labour, miscarriage, and placentitis. Metacyclic trypomastigotes adhere to specific receptors on the outer membrane of host cells as a prelude to intracellular invasion, causing calcium ion mobilization, rearrangement of host cell microfilaments, recruitment of lysosomes and parasite internalization. The actin cytoskeleton plays an important role in many cellular processes including the parasite invasion into mammalian cells. In order to observe if placental cytoskeleton is altered in the process of parasite invasion into placental villi, actin microfilaments were studied. Using immunohistochemical techniques, it was observed that the presence of actin in the syncytiotrophoblast was intense throughout the brush border in control placentae belonging to non-chagasic women. But after culture with the trypomastigote, this labelling disappeared, indicating that the parasite induced disassembly of the cortical actin cytoskeleton when the placenta was infected. As a control, placentae from chagasic women were studied, and no actin was found. The same results were obtained by the electron microscope. We confirmed that cortical actin rearrangements may be an early step in the Trypanosoma cruzi invasion mechanism into placental cells, in order to allow lysosomes access to the plasma membrane, and formation of the parasitophorous vacuole. The recruitment of lysosomes occurs directly beneath the invasion site, and this process is required for parasite internalization.

Role of placental alkaline phosphatase in the interaction between human placental trophoblast and Trypanosoma cruzi.

Congenital Chagas disease, due to the intracellular parasite Trypanosoma cruzi, is associated with premature labor, miscarriage, and placentitis. Human enzyme placental alkaline phosphatase (PLAP) (EC 3.1.3.1.) is membrane-anchored through glycosylphosphatidylinositol (GPI). PLAP is present in plasma in late pregnancy, 36 to 40 weeks; there are lower levels in maternal Chagas disease. Infants born to such mothers may have congenital Chagas disease. Human placental villi (PV) were treated with phospholipase-C (PL-C) and then cultured with T. cruzi to determine the effect of the parasites on PLAP activity as an in vitro model. There is less PLAP activity after treatment by PL-C and during culture with T. cruzi. Pretreatment of PV with PL-C before culture with T. cruzi yielded essentially normal specific activity of PLAP and prevented or greatly reduced infective penetration of villi by parasites. The results are consistent with a pathogenetic role for placental alkaline phosphatase in congenital Chagas disease. Receptor activation of membrane attachment to PLAP may be a device used by T. cruzi to enable parasite invasion of human trophoblast.

The effect of placental subfractions on Trypanosoma cruzi.

Five subfractions were collected from six term placentas by mincing and differential centrifugation: homogenate, nuclear, mitochondrial, lysosomal, and supernatant. The effect of each subfraction on Trypanosoma cruzi was assessed by trypan blue exclusion, relative infectivity of mice, and penetration of susceptible cultured VERO cells. Ultrastructural changes in trypomastigotes were identified after high cell mortality was shown by dye exclusion following treatment with lysosomal and supernatant fractions. Trypomastigotes treated with other subfractions or preheated subfractions, those recovered from infected VERO cells, and controls remained unaffected. This was confirmed by the ability of treated trypomastigotes to infect mice or to penetrate susceptible cultured VERO cells. There were a 48% decrease in parasitemia and fewer myocardial lesions in Balb/c mice following treatment with the lysosomal subfraction compared to homogenate and controls. VERO cells were invaded about half as often after lysosomal treatment compared to controls (P < 0. 05); an 11% decrease in cell invasion following homogenate treatment was not significant. Placental lysosomal enzyme activity was unaffected by trypomastigotes. Human placentas contain one or more heat-labile substances in lysosomal and supernatant subfractions which inhibit or injure trypomastigotes of T. cruzi in cell-free systems.

Trypanosoma cruzi: increased 5'-nucleotidase activity associated with dysfunction of adrenergic receptors in acutely infected albino Swiss mice.

Adenosine, derived from hydrolysis of 5'-AMP by 5'-nucleotidase activity, may be involved in coupling coronary blood flow to cardiac function and metabolism; it has been postulated as a cardioprotective substance in ischemic myocardium. The stimulation of beta-adrenergic receptors produces an increase in adenosine by 5'-AMP hydrolysis. In addition, it has been demonstrated that in Chagas' disease there is decreased cardiac perfusion. We show in this paper by histochemical and densitometric procedures that ecto-5'-nucleotidase activity increases in ventricles of acutely Trypanosoma cruzi-infected mice and that the density of beta-adrenergic receptors is significantly diminished with affinity similar to controls, showing that a compensatory mechanism was absent. The increase of ecto-5'-nucleotidase in heart myocytes from infected mice may produce cardioprotective adenosine that may be independent of beta-adrenergic function, based on the hypoperfusion conditions of acute chagasic cardiomyopathy.

Histochemical study of magellanic penguin (Spheniscus magellanicus) minor salivary glands during postnatal growth.

The histological and histochemical features of the minor salivary glands during postnatal development have been generally associated with the type of food ingested. However, recent studies support the fact that these salivary glands develop independently of the diet; in fact, minor salivary glands have similar morphological and histochemical characteristics in adult individuals of species with different diet regimens. Thus, the aim of this study was to characterize the developmental morphology of the penguin minor salivary glands and to contrast them with minor salivary glands of other species. The tongue, palatine, and mouth cavity (bottom) minor salivary glands of newborn, 1- to 20-day-old, and adult magellanic penguins were studied with hematoxylin-eosin, periodic acid-Schiff, alcian blue, toluidine blue, and lectin histochemistry. Minor salivary glands were present at all ages, although they were only moderately developed in animals less than 15 days old. After this age, glands were abundant in all age groups; in addition, cells from the glandular epithelium were functionally mature and secreted mucins. Nevertheless, in newborn to 15-day-old penguins, mucins were located only at the apical cytoplasm of mucous cells. In all ages, mucous cells displayed periodic acid-Schiff-positive, alcianophilic, and metachromatic reactions; among mucous cells, other orthochromatic cells appeared interspersed. From 15 days on, histochemical reactions became more intense until adulthood, and the cytoplasm of secretory cells was filled with glycoproteins and sulfomucins. Moreover, lectins bound to different oligosaccharides in mucous cells, depending on the stage of maturation of the glands. In conclusion, penguin minor salivary glands are already present at birth, and show progressive and quantitative increases in mucous secretion during postnatal development. These changes are necessary not only for nutrient ingestion, but also for nonimmune protection of the buccal cavity.

Structural and cytochemical modifications in the lingual glands of the newborn chicken irradiated with He-Ne laser.

Despite the increasing and successful use of laser in Medicine and Odontology, the possible iatrogenic and otherwise deleterious side effects of this radiation remain mostly unknown. In previous studies, it was shown that both the embryonic and the post-hatched chicken constitute reliable experimental models for this type of studies. Hence, the purpose of the present work was to analyze the structural and cytochemical alterations of the lingual glands of the newborn chicken irradiated with low energy He-Ne laser. This laser produced regressive structural changes of the glands towards the embryonic stage as well as hyperplasia of the reserve glandular basal cells. Furthermore, a decrease in the glycoprotein content and a rise in the sulphated glycosaminoglycans were also found. These results corroborate the pathogenic effects of the He-Ne- laser on the experimental model employed and, at the same time, emphasize the importance of considering, regarding clinical applications, possible previous neoplastic alterations as well as adverse reactions which might appear once laser therapy has been installed.

Alkaline phosphatase activity in plasma of pregnant chagasic patients.

The kinetic properties of plasma placental alkaline phosphatase patients with Chagas' disease were studied. When Cl2Mg was used as activator the same increase of activity (17-20%) was found in the chagasic and non chagasic groups. The enzyme was not inhibited by F-ion in any of the groups. No significant differences were detected between the two groups (chagasic and non chagasic) when the enzyme was treated with inhibitors such as EDTA and L-phenylalanine. However, when the CN-ion was used, the enzyme of the normal pregnant women followed a Michaelian curve, whereas in the chagasic group a sigmoideal plot was observed. Thus, the Hill coefficient was 1.1 for the normal group and over 1.5 for the chagasic.

Structural and biochemical modifications in parotid gland induced by fasting.

Structural, ultrastructural and biochemical modifications produced by fasting in the parotid gland of guinea pig, were studied. The highest storage of secretory granules was found in the apical cytoplasm after a 12 hour fasting period. The curve of soluble proteins showed that the highest storage of proteins in the parenchyma took place after a 10/12 hour fasting period. Amylase activity reached its highest point after a 10 hour fasting period. We suggest that granules stored in cellular cytoplasm after a 12 hour fasting period would have completed their maturation cycle.

[Primary malignant esophageal melanoma: report of a case]. / Melanoma maligno primario de esófago: presentación de un caso.

Primary malignant melanoma of the esophagus is rare. We identified one patient over a period of 15 years. This patient was a 80 years-old caucasian man. No association was found with tobacco or ethanol use, nor was there a personal or family history of malignant melanoma. Symptoms were related to obstruction. This tumor was polypoid in its upper part and ulcero-infiltrant in its lower part. Histologically the melanoma had epithelioid spindle cells. The neoplasm was immunoreactive for S-100 protein and non reactive with anti-cytokeratins. This patient was treated by Garlock type, esophagectomy with excision of 13 cm of esophagus and 2 cm of stomach. The survival was of only 3 days, because he developed acute respiratory and cardiac disease syndrome and died.

In vivo and in vitro analysis of lysosomes and acid phosphatase activity in human chagasic placentas.

A structural, cytochemical, stereological, and biochemical study of lysosomes and a lysosome marker, the enzyme acid phosphatase, was performed, both in placentas at term from chagasic pregnant women without fetal infection and in normal placentas at term cocultured in vitro with Trypanosoma cruzi. It was found that in placentas from chagasic women lysosomes were normally distributed in the trophoblast. Stereological analysis showed that lysosomes and cytochemical acid phosphatase (AcP) activity were increased in the trophoblast of chagasic placentas. AcP activity increased in subcellular fractions of the isolated trophoblast from chagasic placentas, and the lysosomal fraction of those placentas exhibited the highest value of enzymatic activity in comparison to controls (P < 0.05). No differences in AcP activity were observed between homogenates of normal placentas cocultured with T. cruzi and controls. These data suggest that the placental lysosome population might be involved in the process of placental infection by T. cruzi.

[Human chagasic placenta: structural and cytochemical changes of blood vessels]. / Placenta humana chagásica: alteración estructural y citoquímica de vasos sanguíneos.

Various authors have demonstrated that coronary blood vessels could have some participation in the pathogenesis of the cardiac alterations of Chagas' disease. The purpose of this work was to detect structural and cytochemical modifications of blood vessels in human chagasic placentas at term with optical and electron microscopy due their possible participation in the pathogenesis of the congenital transmission of the disease. In two of the six chagasic placentas at term from pregnant women with positive serology, there was diminution and occlusion of the lumen of the chorionic villi blood vessels, with hialine aspect of their walls. An increase of acid phosphatase activity in the endothelium was also observed with electron microscopy. The diminished blood vessel lumen could be due to smooth muscle and endothelium participation.

[Morphological evaluation of intercellular junctions in the ovary of chick embryos: action of steroid and gonadotropic hormones in vitro]. / Evaluación morfológica de las uniones intercelulares en el ovario del embrión de pollo: acción de hormonas esteroideas y gonadotróficas in vitro.

Variations of the intercellular junctions of the germ and epithelial cells of the ovarian epithelium produced by gonadotropic and esteroid hormones were determined on ovaries of the chick embryo at 7 days of development. Explants of right and left ovaries were cultured without (control) or with hormones eddition (experimental) for 4 days. Cultures were processed for their ultrastructural study. In both control ovaries the union complexes were similar to those identified in ovo. Under the action of 17 beta-estradiol, an increase and a greater development of adherens junctions was observed in the left ovary; in the right ovary, adherens junctions diminished by action of 17 beta-estradiol. The response of the left ovary to progesterone and testosterone was similar to that seen with the estrogen. No changes were observed in the right gonad. A disminution of intercellular junctions was produced in both ovaries under the action of FSH. The changes produced by LH and hCG were similar to those find in the left ovary with the estrogen, consisting of an increase of the union complexes, mainly of the adherens type. These results indicate that steroid and gonadotropic hormones act by modifying intercellular junctions and would participate in the processes of growth and atrophy that occur in the ovaries of the chick embryo.

Evaluación morfológica de las uniones intercelulares en el ovario del embrión de pollo: acción de hormonas esteroideas y gonadotróficas in vitro. / [Morphological evaluation of intercellular junctions in the ovary of chick embryos: action of steroid and gonadotropic hormones in vitro]

Variations of the intercellular junctions of the germ and epithelial cells of the ovarian epithelium produced by gonadotropic and esteroid hormones were determined on ovaries of the chick embryo at 7 days of development. Explants of right and left ovaries were cultured without (control) or with hormones eddition (experimental) for 4 days. Cultures were processed for their ultrastructural study. In both control ovaries the union complexes were similar to those identified in ovo. Under the action of 17 beta-estradiol, an increase and a greater development of adherens junctions was observed in the left ovary; in the right ovary, adherens junctions diminished by action of 17 beta-estradiol. The response of the left ovary to progesterone and testosterone was similar to that seen with the estrogen. No changes were observed in the right gonad. A disminution of intercellular junctions was produced in both ovaries under the action of FSH. The changes produced by LH and hCG were similar to those find in the left ovary with the estrogen, consisting of an increase of the union complexes, mainly of the adherens type. These results indicate that steroid and gonadotropic hormones act by modifying intercellular junctions and would participate in the processes of growth and atrophy that occur in the ovaries of the chick embryo.

Placenta humana chagásica: alteración estructural y citoquímica de vasos sanguíneos. / [Human chagasic placenta: structural and cytochemical changes of blood vessels]

Various authors have demonstrated that coronary blood vessels could have some participation in the pathogenesis of the cardiac alterations of Chagas disease. The purpose of this work was to detect structural and cytochemical modifications of blood vessels in human chagasic placentas at term with optical and electron microscopy due their possible participation in the pathogenesis of the congenital transmission of the disease. In two of the six chagasic placentas at term from pregnant women with positive serology, there was diminution and occlusion of the lumen of the chorionic villi blood vessels, with hialine aspect of their walls. An increase of acid phosphatase activity in the endothelium was also observed with electron microscopy. The diminished blood vessel lumen could be due to smooth muscle and endothelium participation.

[Increase of Hofbauer cells in human placentas cocultured in vitro with Trypanosoma cruzi]. / Aumento de células de Hofbauer en placentas humanas cocultivadas in vitro con Trypanosoma cruzi.

To analyze the interaction between normal human placentas with Trypanosoma cruzi, optical and electron microscopy of chorionic villi stroma cocultured in vitro with 1.5 x 106 Tulahuen strain Trypomastigotes of the T. cruzi for 1 h, 3 hs and 12 hs in Eagle minimal essential medium were done. An agglutination of chorionic villi in experimental cultures (with T. cruzi) from 1 h cultures was observed that was not present in control ones. this phenomenon resisted soft mechanical agitation to separate the isolated villi. Microscopical observations of stromal villi showed edema, separated structures and increment of Hofbauer cells as found by qualitative analysis. The chorionic villi agglutination could be caused by glycoproteins' modifications of the trophoblast, which in turn could be caused by secreted products of T. cruzi, as other authors have postulated in various cells' types. The increment of Hofbauer cells could represent a regulator mechanism of the placenta to equilibrate the intracellular water of the villi stroma.