Trypanosoma cruzi: participation of cholesterol and placental alkaline phosphatase in the host cell invasion.

One of the most important parasitic endemic diseases in Latin America is Chagas disease, with almost 20 million people being infected and 60 million others at risk of infection. In the cell infection by Trypanosoma cruzi, the first step is contact with the host cell by receptors and ligands on the membrane. It is known that lipids play an important role in the interaction process between pathogens and host cells with lipid rafts being highly specialized regions of the plasma membrane that are enriched in cholesterol and sphingolipids. We explored whether the treatment with methyl-beta-cyclodextrin alone or by adding Mevinolin, an inhibitor of cholesterol synthesis could deplete membrane cholesterol of the HEp2 cell and if this treatment could affect the trypomastigote infection into the host cell. These treatments led to a leakage of cholesterol, and concomitantly, PLAP enzyme and unidentified proteins resulting in a decrease of the invasion process. However, the GGTP enzyme was not liberated from the host cell membranes.

Elicitation of specific, Th1-biased immune response precludes skeletal muscle damage in cruzipain-vaccinated mice.

Cruzipain (Cz), the major cystein proteinase of Trypanosoma cruzi, is able to induce protective immunity against parasite challenge. However, some concern has arisen regarding its potential to elicit pathogenic autoimmune reactivity. To determine whether the adverse myopathic effects of Cz-based immunization could be prevented, we evaluated the co-administration of Cz with different adjuvants. Mice were immunized with Cz adjuvantized by alum (Cz+alum), oligodeoxynucleotides containing CpG motifs (Cz+ODN-CpG) or Freund's preparation (Cz+CFA). Cz triggered a vigorous specific humoral response, irrespective of the adjuvant used. Alum mainly drove response towards Th2 phenotype, characterized by specific IgG1 antibodies and IL-10 induction, whereas Cz+ODN-CpG mice exhibited Th1-dominant immunity, with antibodies of the IgG2a isotype and enhanced IFN-gamma production. Histological examination of cardiac tissue demonstrated lesions in Cz+CFA but not in Cz+alum nor Cz+ODN-CpG immunized animals, suggesting that CFA is critical for Cz-mediated injury. Analysis of skeletal muscle revealed that mice receiving Cz+CFA exhibited disrupted and hyalinized myofibers, whereas [Cz+alum]-immunized animals showed hyalinization, architecture modifications and small inflammatory foci. Conversely, no abnormalities were observed in the striated muscle from the Cz+ODN-CpG group. Hence, generation of specific immune response skewed towards Th1, as that recorded for the ODN-CpG adjuvant, may preclude triggering of Cz-mediated muscle tissue damage.

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.

Componentes celulares y fosfatasa alcalina placentaria en la infección por T. cruzi / Cellular components and placental alkaline phosphatase in Trypanosoma cruzi infection

Trypanosoma cruzi induces changes in the protein pattern of human placenta syncytiotrophoblast. Placental alkaline phosphatase (PLAP) is a glycoenzyme anchored to the membrane by a glycosyl-phosphatidylinositol molecule. PLAP activity and its presence was altered by the parasite in cultures of human placental villi and HEp2 cells with T.cruzi. The cells treated before the cultures with agents which affect PILAP or glycosyl-phosphatidylinositol (antibodies, PL-C, genistein, lithium) presented less parasitic invasion than the control ones. It was also observed a modification in the pattern of actine filaments of the host cells infected. We concluded that PLAP would participate in the process of T. cruzi invasion into placental syncitiotrophoblast cells, by a mechanism that involves hydrolysis of the glycosyl-phosphatidylinositol molecules, the activation of tyrosine kinase proteins, the increase of cytosolic calcium and the rearrangement of actine filaments of the host cells.

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.

Pedicle morphology of the immature thoracolumbar spine.

STUDY DESIGN: Human vertebral morphologic data were compiled from anatomic skeletal collections from three museums. OBJECTIVES: To quantify the morphometric characteristics of the pedicles of the immature thoracolumbar spine. SUMMARY OF BACKGROUND DATA: Little is known of pedicle morphology of the immature spine as related to pedicle screw fixation. METHODS: A total of 75 anatomic skeletal specimens were acquired from C1 to L5 in the age range of 3 to 19 years. The data were collected and analyzed using a computerized video analysis system. Each vertebral pedicle was measured in the axial and sagittal planes. The measurements included the minimum pedicle width, the pedicle angle, the distance to anterior cortex, and anteroposterior and interpedicular spinal canal diameters. RESULTS: Wide variation in pedicle morphology between specimens at each vertebral level was found in the young population. In general, compared with the average adult data, a younger spine demonstrated a near uniform reduction in the linear pedicle dimensions at each vertebral level. Pedicles from the lower lumbar vertebrae attained dimensions acceptable for standard screw sizes at an earlier age than in the thoracic vertebrae. CONCLUSIONS: The data in this study indicates that pedicle screws may be used in the adolescent spine. However, care should taken to accurately ascertain pedicle size before surgery so that improper use of screws can be avoided. Growth of the pedicles in relation to the spinal canal indicates that the increase in pedicle size is lateral to the spinal canal.

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.

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.

The role of the interosseous membrane and triangular fibrocartilage complex in forearm stability.

This study investigated the relative roles of the interosseous membrane (IOM) and triangular fibrocartilage complex (TFCC) in the transmission of force from the hand to the humerus. Our findings suggest a spectrum of forearm destabilizing injuries. The intact radius abutting the capitellum provides the primary restraint to proximal migration of the radius. After radial head excision, up to 7 mm of proximal radial migration can occur under axial compression. If the TFCC or the IOM alone is disrupted, little alteration in load or displacement is evident. When both the midportion of the IOM and TFCC are incompetent, however, further proximal radial migration occurs, the radial stump abuts the humerus, and load is shifted back to the radial column. These data suggest that the central portion of the IOM is the crucial structural subdivision within the IOM acting as a restraint to proximal radial migration. The TFCC also resists proximal radial migration and participates in load transfer. We propose that clinical migration of the radius under an axial load greater than 7 mm implies disruption of both the midportion of the IOM and TFCC.

The effect of immediate constrained digital motion on the strength of flexor tendon repairs in chickens.

Profundus tendon lacerations were repaired in the central toes of 216 chickens, and the digits were either immobilized in a cast or allowed immediate constrained motion in a tethering splint. The effect of digital motion on the early phases of tendon healing was investigated by comparing the rupture strengths of the two groups during the first 40 days after repair. The repairs in immobilized digits showed marked decreases in strength during the first 20 days, while the tendons in mobilized digits showed immediate and progressive gains in strength through the time intervals studied. By 5 days, the difference in strength between the two groups was significant (p less than 0.05), and the magnitude of this difference increased with time. The model developed in this study demonstrates that an initial loss of flexor tendon repair strength is not inevitable. Immediate constrained digital mobilization allows progressive tendon healing without an intervening phase of tendon softening.