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S Miranda

Publications and source records attributed to S Miranda.

At least 19 recordsLinked to original sources

Dendritic cells therapy confers a protective microenvironment in murine pregnancy.

The fetal-placental unit is a semi-allograft and immunological recognition of pregnancy, together with the subsequent response of the maternal immune system, is necessary for a successful pregnancy. Dendritic cells (DC) show a biological plasticity that confers them special characteristics regulating both immunity and tolerance. Therapy employing DC proved to diminish the abortion in the DBA/2J-mated CBA/J females; however, the underlying mechanisms remain unknown. Here, we evaluated whether DC therapy influences the presence of immunoregulatory populations of cells at the fetal-maternal interface. To address this hypothesis, we analysed the pregnancy-protective CD8, gammadelta cell populations as well as transforming growth factor (TGF)-beta1 and progesterone-induced blocking factor (PIBF) expression at the fetal-maternal interface from abortion-prone female mice that had previously received adoptive transfer of syngeneic DC. Syngeneic DC therapy induced an increase in the number of CD8 and gammadelta cells. Additionally, an upregulation of TGF-beta1 and PIBF expression could be detected after DC transfer. We suggest that DC therapy differentially upregulates a regulatory/protective population of cells at the fetal-maternal interface. It is reasonable to assure that this mechanism would be responsible for the lower abortion rate.

Abortion, Habitual↗

Interleukin-6 and dexamethasone modulate in vitro asymmetric antibody synthesis and UDP-Glc glycoprotein glycosyltransferase activity.

The increased production of asymmetric IgG protective antibodies is one of the mechanisms proposed to explain a successful semiallogeneic pregnancy. We have previously demonstrated that IL-6 was able to enhance the synthesis of these antibodies by a murine hybridoma, while the glucocorticoid dexamethasone (DEXA) diminished it. In order to investigate the mechanism of asymmetric antibody synthesis, we investigate the role of UDP-Glc glycoprotein glucosyltransferase (GT), an endoplasmic reticulum enzyme involved in the quality control and folding of glycoproteins. Either recombinant murine rmIL-6 (0-10-40-160-320-640 ng/ml) or DEXA (0.15 microM) were added to a mouse hybridoma culture and incubated for 24 and 72 h in the first case, and for 4h in the presence of DEXA. Anti-DNP asymmetric antibodies were determined in the culture supernatants by ELISA. After harvesting, hybridoma cells were sonicated and GT activity was analysed in isolated microsomal fractions by measuring UDP((14)C)-Glc incorporation into urea-denatured thyroglobulin (urea-Tg). In the present paper, we showed that IL-6, mainly at 40 ng/ml and t=24h, was able to upregulate both in vitro GT activity (+74%) and asymmetric molecule synthesis (+227%). Lower increases were obtained employing 10 and 160 ng/ml. On the other hand, DEXA, at 0.15 microM and t=4h, showed a mild inhibition of enzyme activity (-10%) and a diminished proportion of asymmetric IgG (-49%). A direct relationship between GT activity and proportion of the asymmetric antibody synthesised was found in our hybridoma cells employing IL-6 and DEXA in different conditions, as was indicated by a correlation analysis. These results suggest that GT might be involved in the synthesis of asymmetric antibodies.

Animals↗

Asymmetric antibodies: a protective arm in pregnancy.

In normal conditions, a simple change in the pattern of cytokines towards a Th2 response is associated with the production of aggressive antibodies. This fact could not completely explain phenomena such as the fetal survival or the chronicity of certain infections. However, it has been demonstrated that Th2 cytokines increase the proportion of asymmetric antibodies, which are unable to activate effector immune mechanisms (complement fixation, clearance of antigens and phagocytosis). Investigations of asymmetrically glycosylated antibodies demonstrated that these IgG molecules have an extracarbohydrate in one of the Fab regions. This glycosylation affects their antigen interaction turning them into a functionally univalent and blocking antibodies. It has been established that their synthesis is increased under different physiopathological situations involving Th2 responses: chronic infections by extracellular microorganisms, pregnancy and allergic processes. In this review we summarize the experiments performed by our research group over the last years as well as the advances made concerning the role and mechanism of asymmetric antibodies.

Animals↗

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris.

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.

Cell Division↗

Analysis of macrophage presence in murine placenta: influence of age and parity status.

PROBLEM: Beneficial effects of multiparity status have been previously reported by different authors. However, this fact has not been fully explained. Taking into consideration the influence of the parity status on the in vitro asymmetric/protective antibodies and the fact that interleukin-6 (IL-6) is involved in the production and immune-regulatory functions of placental macrophages, the aim of this work was to compare the placental IL-6 production and tissue macrophage presence in mice with different age and parity status. METHOD OF STUDY: Three groups of mice (CBA/J x CBA/J) were analyzed: primiparous young (PY: 3.0 +/- 0.5 months old), primiparous old (PO: 8.5 +/- 0.5 months old), and multiparous old (MO: 8.5 +/- 0.5 months old, with three to four previous pregnancies). Macrophage and IL-6 were identified in placental tissue by immunohistochemistry employing anti-F4/80 or anti-IL-6 antibodies. IL-6 secretion was analyzed in the placental culture supernatants by enzyme-linked immunosorbent assay. RESULTS: The results obtained indicate that, despite the level of macrophages observed in the PO placentae was higher than in PY ones, their expression in MO placentae was very much increased, appearing like a thick layer between decidua and trophoblast. However, no significant difference was found among the groups in the tissue expression of IL-6 and in IL-6 secreted in vitro. CONCLUSIONS: Our data indicate that parity status influences the number of local macrophages and might provide evidence that could explain the known beneficial effect of multipaternity. We suggest that the number of previous pregnancies favor the production of a 'protective' population of macrophages.

Aging↗

Effect of telmisartan 80 mg once daily on 24-h blood pressure profile in patients with mild-to-moderate hypertension failing to respond to prior antihypertensive therapy.

Blood pressure is not adequately controlled in almost 50% of patients with hypertension who are in receipt of antihypertensive therapy. This multicentre, prospective, open-label trial was designed to determine whether or not once-daily telmisartan 80 mg reduced blood pressure during the last 6 h of the 24-h dosing interval in patients with mild-to-moderate hypertension who were unresponsive to previous antihypertensive therapy. The study comprised 100 patients (47 males, 53 females) who had failed to respond satisfactorily to prior treatment given for a minimum of 3 months. At screening, 24-h ambulatory blood pressure monitoring (ABPM) was conducted after the patient had been treated with the currently prescribed antihypertensive medication. Following 5 weeks of telmisartan 80 mg treatment, ABPM was repeated. Telmisartan significantly reduced mean systolic blood pressure, diastolic blood pressure (DBP) and pulse pressure compared with previous antihypertensive therapy over each time interval (24-h, morning, night-time and the last 6 h of the dosing interval [2.00 a.m.-8.00 a.m.]) analysed. In addition, more than 90% of patients responded successfully (clinic DBP <90 mmHg or a >10 mmHg reduction in clinic DBP) at the end of telmisartan treatment. In conclusion, telmisartan provides effective blood pressure control throughout the 24-h dosing interval in patients with mild-to-moderate hypertension who were unresponsive to previous antihypertensive medication.

Adolescent↗

Interleukin regulation of asymmetric antibody synthesized by isolated placental B cells.

PROBLEM: Protecting antibodies against trophoblast surface molecules were previously described. Here we analysed the synthesis of asymmetric IgG by placental B-lymphocytes. METHOD OF STUDY: B cells were isolated from human term placenta and cord blood, stimulated with anti-CD40 IgG and cocultured with transfected Fcgamma R-expressing mice Ltk-fibroblast. Interleukin-4, IL-6, IL-10, IL-11 and IL-13 were added to cultures for 14 days. Asymmetric IgG were assessed in culture supernatants by concanavalin A (Con A) fixation and enzyme-linked immunosorbent assay. RESULTS: When IL-6 was added to the cultures, the percentages of asymmetric IgG synthesized by placental B cells were: IL-6: 29 +/- 10; IL-6 + IL-10: 24 +/- 7; IL-4 + IL-10 + IL-6: 38 +/- 9. The last combination induced the highest increase in the asymmetric IgG synthesis as compared with control (19 +/- 10%, P < 0.05). Additionally, placental B cells synthesized more asymmetric IgG than umbilical cord blood B-lymphocytes (P = 0.0015). CONCLUSIONS: Isolated placental B-lymphocytes synthesized asymmetric IgG in response to Th2 interleukins, more notably IL-6 in combination with IL-4 and IL-10. The in vitro increase of protective asymmetric IgG synthesis in response to Th2-cytokines support the hypothesis that a local Th2-switch is beneficial for pregnancy outcome.

Animals↗

Enhancement of in vitro hsp72 expression by placental IL-6.

PROBLEM: To give an approach in order to elucidate the mechanism by which placental IL-6 induce modifications in the glycosylation status of immunoglobulins, in the present work, we investigate a putative relationship between a stimulus by placental IL-6 and expression of cytoplasmic "hsp70 family proteins" in an in vitro model. METHODS OF STUDY: Supernatants of cultures of placentae obtained from primiparous and multiparous AKR/J x AKR/J and AKR/J x BALB/c mouse crossbreedings were added to mouse IgGI hybridoma cultures which produced symmetric and asymmetric anti-dinitrophenol (anti-DNP) antibodies. Analyses of the expression of inducible hsp72/constitutive hsp73 in cellular lysates obtained from hybridomas cultured, in the presence of rmIL-6 or crude murine placental culture supernatants, followed by neutralization assays with anti IL-6, were performed. In addition, the level of IL-6 present in the employed placental culture supernatants was determined and compared with the placental hsp70-inducing effect. RESULTS: These experiments showed that mouse placentae were able to release IL-6 in vitro. In addition, mouse placental supernatants (PS) containing over 1,000 pg/mL of IL-6 enhanced the expression of the inducible isoform hsp72 in the employed hybridomas. This effect was abolished when the hsp70-inducing PS were previously incubated with anti-mIL6 antibody. CONCLUSIONS: These observations indicate that mouse placentae produce different titers of IL-6 and suggest that IL-6 appears to be the unique mouse placental factor able to induce in vitro hsp72 synthesis. A relationship with the increased synthesis of anti-paternal antigen asymmetric antibodies, previously observed during pregnancy, is discussed.

Animals↗

The role of oxidative stress in the toxicity induced by amyloid beta-peptide in Alzheimer's disease.

One of the theories involved in the etiology of Alzheimer's disease (AD) is the oxidative stress hypothesis. The amyloid beta-peptide (A beta), a hallmark in the pathogenesis of AD and the main component of senile plaques, generates free radicals in a metal-catalyzed reaction inducing neuronal cell death by a reactive oxygen species mediated process which damage neuronal membrane lipids, proteins and nucleic acids. Therefore, the interest in the protective role of different antioxidants in AD such as vitamin E, melatonin and estrogens is growing up. In this review we summarize data that support the involvement of oxidative stress as an active factor in A beta-mediated neuropathology, by triggering or facilitating neurodegeneration, through a wide range of molecular events that disturb neuronal cell homeostasis.

Alzheimer Disease↗

PIXE study of Cuban quaternary paleoclimate geological samples and speleothems.

PIXE elemental analysis of sediments, speleothems, and other geological formations related to the karst of the Sierra de San Carlos is presented. The similarity of the elemental composition of the sediments studied, as well as the alluvial regime which created them, indicate their common origin at each location. The Sr/Ca concentration ratio of a stalactite indicates that the average atmospheric temperature 12,000 and 18,000 years B.P. was colder than that of 6000 years B.P.

Calcium↗

The shape of the HIV/AIDS epidemic in Puerto Rico.

This study presents information on AIDS patients in Puerto Rico, including their general sociodemographic profile, some risk-related parameters, characteristics of vulnerable groups, and elements of the clinical spectrum of the disease. Data were analyzed from the Puerto Rico AIDS Surveillance Program and available studies about the HIV/AIDS epidemic in Puerto Rico. A total of 23,089 AIDS cases was reported to the Puerto Rico AIDS Surveillance Program from January 1981 through February 1999. The HIV/AIDS epidemic has affected mostly males and females between the ages of 30 and 49, though cases have also been reported for other age groups. The cumulative proportion of persons with AIDS who are women has increased tremendously, from 11.4% for the 1981-1986 period to 21.6% for the entire 1981-1999 period. In Puerto Rico the category of injecting drug users (IDUs) accounts for the majority of the AIDS cases (52%), followed by heterosexual contact (22%), and men who have sex with men (17%). The three main diagnoses for AIDS on the island are wasting syndrome (30.7%); esophageal, bronchial, and lung candidiasis (29.4%); and Pneumocystis carinii pneumonia (26.8%). According to 1994 vital statistics for Puerto Rico, AIDS was the fourth-leading cause of death. The overall reported AIDS mortality rate was 42.0 per 100,000 persons, with the rate for males, 67.8, much higher than it was for females, 17.4. AIDS is the first cause of death among persons between 30 and 39 years old. Intense efforts are needed to better understand the epidemic in Puerto Rico and its biology, social and family impacts, and financial costs.

AIDS-Related Opportunistic Infections↗

Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver.

The canalicular multispecific organic anion transporter, cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23+/-0.39 versus 1.13+/-0.15 microl/min per g of liver; P<0.05) and biliary GSH output (7.40+/-3.30 versus 2.65+/-0.34 nmol/min per g of liver; P<0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95+/-0.84 versus 5.12+/-0.47 mM; P<0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.

2,4,5-Trichlorophenoxyacetic Acid↗

Oxidative stress and upregulation of mitochondrial biogenesis genes in mitochondrial DNA-depleted HeLa cells.

The signaling mechanism through which deficitary mitochondrial function would activate nuclear genes required for mitochondrial biogenesis, has not been established. To explore the hypothesis that reactive oxygen species (ROS), a mitochondrial product, constitute part of the mitochondria-nuclei signaling pathway, we obtained HeLa cells depleted of mitochondrial DNA (rho0 cells) through exposure to ethidium bromide. We found evidences of oxidative stress in rho0 cells, employing a fluorescent probe and measuring NF-kappaB activation. Nuclear Respiratory Factor-1 (NRF-1) and Mitochondrial Transcription Factor A (Tfam) mRNA were measured by RT-PCR. For both transcription factors, rho0 cells revealed significantly higher levels of mRNA. These results support several hypothesis: that endogenous ROS enhance the expression of nuclear mitochondrial biogenesis genes NRF-1 and Tfam; that DNA deprived mitochondria lead to cellular oxidative stress, probably because of incomplete biogenesis of the mitochondrial electron transport chain, and consequently, that ROS are part of a mitochondria-nuclei regulatory signaling pathway.

Base Sequence↗

Altered modulation of the in vitro antibody synthesis by placental factors from the CBA/J x DBA/2 abortion-prone mating combination.

PROBLEM: The in vitro immunomodulating effect of placental culture supernatants (PSs) obtained from two H-2k x H-2d allogeneic crossbreedings, the CBA/J x DBA/2 abortion-prone mating combination, and the reproductively normal pregnancy CBA/J x BALB/c crossbreeding were compared, and the influence of previous deliveries was evaluated. The behavior of placentae obtained from CBA/J females with two previous pregnancies by BALB/c males was also investigated. METHOD OF STUDY: Supernatants of cultures of murine placentae were added to a mouse immunoglobulin (Ig) G1 hybridoma culture which produced anti-dinitrophenol (anti-DNP) antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies, and the proliferation of the hybridoma cells were studied. RESULTS: CBA/J x DBA/2 placental factors obtained from multiparous females induced a diminished asymmetric IgG antibody production without varying the quantity of antibody produced. In contrast, PSs obtained from the nonresorption-prone CBA/J x BALB/c mating combination with the same number of previous deliveries enhanced the production of both symmetric and asymmetric anti-DNP molecules and also increased the proportion of asymmetric blocking monoclonal antibodies (mAbs) synthesized by the hybridoma. Both of the PSs analyzed had induced similar inhibition of 3H-thymidine uptake. PSs obtained from the abortion-prone mating combination whose CBA/J females had two previous pregnancies by BALB/c males showed similar immunomodulating effects to those observed using multiparous CBA/J x BALB/c placentae. CONCLUSIONS: We propose that the placenta produces soluble factors that participate in the regulation of antibody synthesis by the mother during gestation. Such a placental immunomodulating effect appears to be altered in the CBA/J x DBA/2 abortion-prone mating combination and could be corrected by previous pregnancies by BALB/c males. These observations suggest that placental factors would be relevant to the protection of the fetus and might play an important role in the immune equilibrium between mother and fetus. Asymmetric antibody production as a Th2 responsiveness was also discussed.

Abortion, Veterinary↗

Prenatal diagnosis of Down syndrome: mothers' reflections on supports needed from diagnosis to birth.

A qualitative study involving 10 mothers who received a prenatal diagnosis of Down syndrome and chose to continue their pregnancy was presented. The time frame considered was diagnosis to delivery. We looked at how the diagnosis was presented, the decision to continue the pregnancy, and the mothers' experience with professionals from diagnosis to delivery. Mothers' suggestions to others facing the same challenges and to their health care providers were discussed as were the types of support given these mothers and what was or would have been most helpful to them. The importance of professionals reviewing their procedures and protocols for working with families facing similar circumstances was stressed.

Adult↗

Overexpression of mdr2 gene by peroxisome proliferators in the mouse liver.

BACKGROUND: In mice, fibrates induce mdr2 gene expression, and its encoded P-glycoprotein in the canalicular domain of hepatocytes, as well as increasing biliary phospholipid output. It is not known whether this effect is restricted to fibrates or is a common property of peroxisome proliferators. AIMS: To test the effect of structurally unrelated peroxisome proliferators on mdr2 gene expression and biliary phospholipid output, and to explore the molecular mechanism(s) of mdr2 gene induction. METHODS: Male CFI mice were fed on a diet supplemented with several peroxisome proliferators: phenoxyacetic acid herbicides, plasticizers, acetylsalicylic acid and partially hydrogenated fish oil. RESULTS: Increased levels of mdr2 mRNAs, assessed by Northern blot analysis, were observed in the liver of mice treated with phenoxyacetic acid herbicides: 2,4,5-trichlorophenoxyacetic acid 570+/-133%, 2,4-dichlorophenoxyacetic acid 233+/-54% (p<0.005); plasticizers: di-(2-ethylhexyl)phthalate 282+/-78%, di-(isoheptyl)phthalate 163+/-40%, phthalic acid dinonyl ester 225+/-48% (p<0.01); and partially hydrogenated fish oil 372+/-138% (p<0.005). P-glycoprotein traffic ATPase content increased in the canalicular domain of hepatocyte of mice treated with the herbicide 2,4,5-trichlorophenoxyacetic acid and with partially hydrogenated fish oil (108% and 87%, respectively, p<0.05) as well as biliary phospholipid output (106% and 74%, respectively, p<0.05). In 2,4,5-trichlorophenoxyacetic acid-fed mice we found five-fold increase on mdr2 transcription rate, assessed by nuclear run-off assay. CONCLUSIONS: Peroxisome proliferators induce mdr2 gene, its encoded P-gp in the canalicular domain of hepatocytes and increase biliary phospholipid output. The modulation of mdr2 gene might be part of the pleiotrophic response of peroxisome proliferation in mice liver and seems to be regulated mainly at a transcriptional level.

2,4,5-Trichlorophenoxyacetic Acid↗

Influence of mouse placental factors on in vitro antibody synthesis.

Placental culture supernatants (PS) obtained from various mouse crossbreedings were added to mouse IgG1 hybridoma cultures producing anti-DNP antibodies. The quantity of monoclonal antibody (mAb) produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. It was observed that the supernatants increased or diminished the production of mAb, depending on the genetic origin of the placentae. This effect was the same using placentae from primiparous or multiparous females and it was not due to modifications of the cellular proliferation of the hybridoma, as shown by 3H-thymidine uptake. It was also found that placental supernatants induced an increase in the proportion of asymmetric, blocking antibodies synthesized by the hybridoma. This effect was detected with supernatants from both allogeneic or syngeneic crossbreedings, but only when placentae were obtained from multiparous females. These observations indicate that placentae produce at least two soluble factors that participate in the regulation of antibody synthesis and suggest that these factors play an important role in the immune equilibrium between mother and fetus.

Adjuvants, Immunologic↗

Modulation of the humoral immune response by placental secretory factors.

PROBLEM: To investigate how the factors secreted by human placenta modify the quality and the quantity of the antibody produced by the hybridoma as well as its cellular proliferation. METHOD: Supernatants of cultures of human placenta (PS) were added to a mouse IgG1 hybridoma culture producing anti-DNP antibodies. The quantity of monoclonal antibody produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. RESULT: It was found that PS augmented by 40-50% the quantity of total antibody produced, increased the proportion of asymmetric (blocking) antibodies from 15% to 30%, and diminished the cellular proliferation, as measured by 3H-thymidine incorporation. CONCLUSION: These results, together with other similar observations already described in human, rat and mouse pregnancies, suggest that secretory factors produced by the placenta do modify the immune response of the mother against paternal antigens and participate in the mechanisms that make possible the survival of the allogenic fetus.

Adjuvants, Immunologic↗