Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

Stability of the pstS transcript of Escherichia coli

The pst operon of Escherichia coli is composed of five genes that encode a high-affinity phosphate transport system. As a member of the PHO regulon, pst transcription is activated under phosphate shortage conditions. Under phosphate-replete conditions, the pst operon also functions as a negative regulator of the PHO genes. Transcription of pst is initiated at the promoter located upstream to the first gene, pstS. Immediately after its synthesis, the primary transcript of pst is cleaved into shorter mRNA molecules. The transcription unit corresponding to pstS is significantly more abundant than the transcripts of the other pst genes due to stabilisation of pstS mRNA by a repetitive extragenic palindrome (REP) structure downstream to the pstS locus. The presence of the REP sequence also results in an increased level of PstS proteins. However, the surplus level of PstS proteins produced in the presence of REP does not contribute to the repressive role of Pst in PHO expression.

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

Transcriptional regulation of the Na(+)/H(+) exchanger NHE3 by chronic exposure to angiotensin II in renal epithelial cells

Angiotensin II (Ang II) exerts an acute bimodal effect on proximal tubule NHE3: while low doses stimulate the exchanger, high doses inhibit it. In the present study, we have investigated the chronic effects of Ang II on NHE3 expression and transcriptional regulation. Treatment of a tubular epithelial cell line, OKP, with Ang II 10(-11) M significantly increased NHE protein expression and mRNA levels, without evidence of bimodal effect. No change in mRNA half-life was detected, but transient transfection studies showed a significant increase in NHE3 promoter activity. Binding sites for Sp1/Egr-1 and AP2 transcription factors of the NHE3 proximal promoter were mutated and we observed that the Sp1/Egr-1 binding site integrity is necessary for Ang II stimulatory effects. Inhibition of cytochrome P450, PI3K, PKA and MAPK pathways prevented the Ang II stimulatory effect on the NHE3 promoter activity. Taking all the results together, our data reveal that chronic Ang II treatment exerts a stimulatory effect on NHE3 expression and promoter activity. The Ang II up-regulation of the NHE3 promoter activity appears to involve the Sp1/Egr-1 binding site and the interplay of several intracellular signaling pathways. (C) 2011 Elsevier Inc. All rights reserved.

Synthesis and characterization of phosphate glass microspheres for radiotherapy applications

Class microspheres containing the radioisotope (32)P, a beta(-) particle emitter, and half-life of 14.3 days, can be easily introduced in specific human organs such as liver, pancreas. and uterus to kill cancer cells. In the present work phosphate glass microspheres were produced with different compositions and particle size distribution in the range of 20- 30 mu m. Two different thermal processes were used to spherodize glass particles originally with irregular shapes. Samples were characterized by X-rays diffraction to check the amorphous structure, energy dispersive X-rays fluorescence spectroscopy to determine the final glass composition, and Fourier transformed infrared spectroscopy to determine the structural groups in the glass structure. The dissolution rate of glass samples in water was determined at 90 degrees C, and in simulated body fluid (SBF) at 37 degrees C. Classes with dissolution rates close to 10(-5) g/(cm(2) day) were obtained, which make them suitable for the present application. Scanning electron microscopy was used to evaluate the shape of the microspheres before and after the dissolution tests. The cytotoxicity tests showed that these microspheres can be used for biological applications. (C) 2008 Elsevier B.V. All rights reserved.

Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells

Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m) = 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.

End-of-life practices in seven Brazilian pediatric intensive care units

Objective: To evaluate the incidence of life support limitation and medical practices in the last 48 hrs of life of children in seven Brazilian pediatric intensive care units (PICUs). Design. Cross-sectional multicenter retrospective study based on medical chart review. Setting: Seven PICUs belonging to university and tertiary hospitals located in three Brazilian regions: two in Porto Alegre (southern region), two in Sao Paulo (southeastern region), and three in Salvador (northeastern region). Patients. Medical records of all children who died in seven PICUs from January 2003 to December 2004. Deaths in the first 24 hrs of admission to the PICU and brain death were excluded. Interventions: Two pediatric intensive care residents from each PICU were trained to fill out a standard protocol (K = 0.9) to record demographic data and all medical management provided in the last 48 hrs of life (inotropes, sedatives, mechanical ventilation, full resuscitation maneuvers or not). Student`s t-test, analysis of variance, chi-square test, and relative risk were used for comparison of data. Measurements and Main Results. Five hundred and sixty-one deaths were identified; 97 records were excluded (61 because of brain death and 36 due to <24 hrs in the PICU). Thirty-six medical charts could not be found. Cardiopulmonary resuscitation was performed in 242 children (57%) with a significant difference between the southeastern and northeastern regions (p =.0003). Older age (p = .025) and longer PICU stay (p = .001) were associated with do-not-resuscitate orders. In just 52.5% of the patients with life support limitation, the decision was clearly recorded in the medical chart. No ventilatory support was provided in 14 cases. Inotropic drug infusions were maintained or increased in 66% of patients with do-not-resuscitate orders. Conclusions. The incidence of life support limitation has increased among Brazilian PICUs but with significant regional differences. Do-not-resuscitate orders are still the most common practice, with scarce initiatives for withdrawing or withholding life support measures.