Estudo da resposta imune humoral em mulheres com neoplasia ovariana. -

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+ CD25+ Foxp3+ T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-gamma-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.

Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast to T. gondii, N. caninum represents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused by N. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, by analogy with T. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

The mechanism of action of a novel benzo[c]phenanthridine alkaloid, NK314 and the cellular responses

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that is currently in clinical trials as an antitumor compound, based on impressive activities in preclinical models. However, its mechanism of action is unknown. The present investigations were directed at determining the mechanism of action of this agent and cellular responses to NK314. My studies demonstrated that NK314 intercalated into DNA, trapped topoisomerase IIα in its cleavage complex intermediate, and inhibited the ability of topoisomerase IIα to relax super-coiled DNA. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIα, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. This indicates topoisomerase IIα is the target of NK314 in the cells. NK314 caused phosphorylation of the histone variant, H2AX, which is considered a marker of DNA double-strand breaks. DNA double-strand breaks were also evidenced by pulsed-field gel electrophoresis and visualized as chromosomal aberrations after cells were treated with NK314 and arrested in mitosis. Cell cycle checkpoints are activated following DNA damage. NK314 induced significant G2 cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G2 checkpoint pathway was activated in response to NK314, which can be abrogated by the Chk1 inhibitor UCN-01. Cell cycle checkpoint activation may be a defensive mechanism that provides time for DNA repair. DNA double-strand breaks are repaired either through ATM-mediated homologous recombination or DNA-PK-mediated non-homologous end-joining repair pathways. Clonogenic assays demonstrated a significant decrease of colony formation in both ATM deficient and DNA-PK deficient cells compared to ATM repleted and DNA-PK wild type cells respectively, indicating that both ATM and DNA-PK play important roles in the survival of the cells in response to NK314. The DNA-PK specific inhibitor NU7441 also significantly sensitized cells to NK314. In conclusion, the major mechanism of NK314 is to intercalate into DNA, trap and inhibit topoisomerase IIα, an action that leads to the generation of double-strand DNA breaks, which activate ATM and DNA-PK mediated DNA repair pathways and Chk1 mediated G2 checkpoint pathway. ^

Pluronic F127-modified liposome-containing tacrolimus-cyclodextrin inclusion complexes:improved solubility, cellular uptake and intestinal penetration

Objective The aim of this study was to investigate Pluronic F127-modified liposome-containing cyclodextrin (CD) inclusion complex (FLIC) for improving the solubility, cellular uptake and intestinal penetration of tacrolimus (FK 506) in the gastrointestinal (GI) tract. Methods Molecular modelling was performed to screen the optimal CD for the solubilization of FK 506. FLIC was prepared by thin-lipid film hydration with the inclusion complex solutions followed by membrane extrusion. Dilution tests were conducted in simulated gastric fluids and phosphate-buffered solution of sodium taurocholate to investigate the solubility improvement of FK506. The cellular uptake of nanocarriers was studied in Caco-2 cells, and intestinal mucous membrane penetration in the GI tract was evaluated in Sprague-Dawley rats. Key findings The results showed that β-CD had the strongest binding energy with the guest molecule among the CDs. The prepared FLIC has an average diameter of 180.8 ± 8.1 nm with a spherical shape. The solubility and cellular uptake of FK 506 was greatly improved by the incorporation of CD complexes in the Pluronic F127-modified liposomes. Intestinal mucous membrane penetration was also significantly improved by the preparation of FLIC. Conclusion With improved drug solubility and intestinal mucous membrane penetration, FLIC shows a promising oral delivery system for FK 506. © 2013 Royal Pharmaceutical Society.

System design and resource management in the next-generation integrated WLAN and 3G cellular networks

Next-generation integrated wireless local area network (WLAN) and 3G cellular networks aim to take advantage of the roaming ability in a cellular network and the high data rate services of a WLAN. To ensure successful implementation of an integrated network, many issues must be carefully addressed, including network architecture design, resource management, quality-of-service (QoS), call admission control (CAC) and mobility management. ^ This dissertation focuses on QoS provisioning, CAC, and the network architecture design in the integration of WLANs and cellular networks. First, a new scheduling algorithm and a call admission control mechanism in IEEE 802.11 WLAN are presented to support multimedia services with QoS provisioning. The proposed scheduling algorithms make use of the idle system time to reduce the average packet loss of realtime (RT) services. The admission control mechanism provides long-term transmission quality for both RT and NRT services by ensuring the packet loss ratio for RT services and the throughput for non-real-time (NRT) services. ^ A joint CAC scheme is proposed to efficiently balance traffic load in the integrated environment. A channel searching and replacement algorithm (CSR) is developed to relieve traffic congestion in the cellular network by using idle channels in the WLAN. The CSR is optimized to minimize the system cost in terms of the blocking probability in the interworking environment. Specifically, it is proved that there exists an optimal admission probability for passive handoffs that minimizes the total system cost. Also, a method of searching the probability is designed based on linear-programming techniques. ^ Finally, a new integration architecture, Hybrid Coupling with Radio Access System (HCRAS), is proposed for lowering the average cost of intersystem communication (IC) and the vertical handoff latency. An analytical model is presented to evaluate the system performance of the HCRAS in terms of the intersystem communication cost function and the handoff cost function. Based on this model, an algorithm is designed to determine the optimal route for each intersystem communication. Additionally, a fast handoff algorithm is developed to reduce the vertical handoff latency.^

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris): Morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n = 190) and manatees (n = 56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immunocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included T$\rm\sb{h}$, T$\rm\sb{c/s}$, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating T$\rm\sb{h}$ cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Immunocytes of the Atlantic bottlenose dolphin (Tursiops truncatus) and West Indian manatee (Trichechus manatus latirostris) : morphologic characterizations and correlations between healthy and disease states under free-ranging and captive conditions

Interest in the health of marine mammals has increased due, in part, to the attention given to human impact on the marine environment. Recent mass strandings of the Atlantic bottlenose dolphin (Tursiops truncatus) and rising mortalities of the endangered Florida manatee (Trichechus manatus latirostris) have raised questions on the extent to which pollution, infectious disease, "stress," and captivity influence the immune system of these animals. This study has provided the first in-depth characterization of immunocytes in the peripheral blood of dolphins (n=180) and manatees (n=56). Immunocyte morphology and baseline values were determined in clinically normal animals under free-ranging, stranded and captive living conditions as well as by age and sex. Additionally, immuocyte population dynamics were characterized in sick animals. This was accomplished with traditional cytochemical techniques and new lymphocyte phenotyping methodology which was validated in this study. Traditional cytochemical techniques demonstrated that blood immunocyte morphology and cell numbers are similar to terrestrial mammals with some notable exceptions. The manatee heterophilic granulocyte is a morphologically unique cell and probably functions similarly to the typical mammalian neutrophil. Eosinophils were rarely found in manatees but were uncommonly high in healthy and sick dolphins. Basophils were not identified. Manatees had higher total lymphocyte numbers compared to dolphins and most terrestrial mammals. Lymphocyte subsets identified in healthy animals included Th, Tes, B and NK cells. Dolphin and manatee T and B cell values were higher than those reported in man and most terrestrial mammals. The manatee has extraordinarily high absolute numbers of circulating Th cells which suggests an enhanced immunological response capability. With few exceptions, immunocyte types and absolute numbers were not significantly different between free-ranging, stranded and captive categories or between sex and age categories. The evaluation of immunocyte dynamics in various disease states demonstrated a wide variation in cellular responses which provided new insights into innate, humoral and cell-mediated immunity in these species. Additionally, this study demonstrated that lymphocyte phenotyping has diagnostic significance and could be developed into a potential indicator of immunocompetence in both free-ranging and captive dolphin and manatee populations.

Gender mismatch between donor and recipient is a factor of morbidity but does not condition survival after cardiac transplantation

We intended to evaluate the influence of sex mismatch between donor and recipient, which is still under much debate, on survival and comorbidities after cardiac transplantation. From November 2003 to December 2013, a total of 258 patients were transplanted in our center. From these, 200 receptors were male (77.5%) and constituted our study population, further divided into those who received the heart from a female donor (Group A) - 44 patients (22%) and those who received it from a male donor (Group B) - 156 (78%). Median follow-up was 4.2 ± 3.0 years (1-10 years). The two groups were quite comparable with each other, except for body mass index, systolic pulmonary artery pressure, and transpulmonary gradient, which were significantly lower in Group A. A low donor/recipient weigh ratio (<0.8) was avoided whenever possible. Hospital mortality was not different in the two groups. During follow-up, global survival was similar, as was survival free from acute cellular rejection and cardiac allograft vasculopathy. However, patients in Group A had decreased survival free from serious infections and malignant tumors. Allocation of female donors to male receptors can be done safely, at least in receptors without pulmonary hypertension and when an adequate donor/recipient weigh ratio is ensured.

Gender mismatch between donor and recipient is a factor of morbidity but does not condition survival after cardiac transplantation

We intended to evaluate the influence of sex mismatch between donor and recipient, which is still under much debate, on survival and comorbidities after cardiac transplantation. From November 2003 to December 2013, a total of 258 patients were transplanted in our center. From these, 200 receptors were male (77.5%) and constituted our study population, further divided into those who received the heart from a female donor (Group A) - 44 patients (22%) and those who received it from a male donor (Group B) - 156 (78%). Median follow-up was 4.2 ± 3.0 years (1-10 years). The two groups were quite comparable with each other, except for body mass index, systolic pulmonary artery pressure, and transpulmonary gradient, which were significantly lower in Group A. A low donor/recipient weigh ratio (<0.8) was avoided whenever possible. Hospital mortality was not different in the two groups. During follow-up, global survival was similar, as was survival free from acute cellular rejection and cardiac allograft vasculopathy. However, patients in Group A had decreased survival free from serious infections and malignant tumors. Allocation of female donors to male receptors can be done safely, at least in receptors without pulmonary hypertension and when an adequate donor/recipient weigh ratio is ensured.

Tratamiento del rechazo mediado por anticuerpos en pacientes trasplantados renales revisión sistemática

Antecedentes: El trasplante renal es la mejor alternativa terapéutica para la enfermedad renal crónica terminal. Los medicamentos inmunosupresores previenen el rechazo. El rechazo mediado por anticuerpos es frecuente y disminuye la función y duración del injerto. Objetivo: Evaluar sistemáticamente la evidencia disponible relacionada con la eficacia y seguridad del tratamiento para el rechazo mediado por anticuerpos en pacientes trasplantados renales. Metodologia: Revisión sistemática en bases de datos MEDLINE, EMBASE, Scopus y Biblioteca virtual de la salud. Literatura gris google scholar, google academico, www.clinicaltrialsregister.eu, and https://clinicaltrials.gov/. Búsqueda manual referencias artículos pre-seleccionados así como de revisiones previamente publicadas. Se siguieron las recomendacioes guia PRISMA para la identificacion de artículos potenciales, tamizaje y selección teniendo en cuenta los criterios de inclusion. Extracción datos de acuerdo a las variables, revisión calidad de los artículos elegidos utilizando evaluación riesgo de segos de Cochrane. Resultados: Se seleccionaron 9 ensayos clínicos publicados entre 1980 y 2016, incluyeron 222 pacientes (113 brazo de intervención y 109 en el control), seguimiento promedio 16 meses. Intervenciones evaluadas plasmaféresis, inmunoadsorción y rituximab. Hubo una amplia heterogeneidad en la definición de criterios de inclusión, criterios diagnósticos de rechazo y medidas de evaluación de eficacia de las intervenciones. Tres estudios encontraron diferencias estadísticamente significativas entre los grupos de tratamiento. Conclusiones: La evidencia sobre la eficacia de los tratamientos del rechazo mediado por anticuerpos en injertos renales es de baja calidad. Son necesarios ensayos clínicos controlados para poder definir el tratamiento óptimo de estos pacientes.