Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF

PURPOSE: This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres. METHODS: GM-CSF was encapsulated in PLGA/PLA microspheres by a novel silicone oil based phase separation process. Several different blends of PLGA and low molecular weight PLA were used to prepare the microspheres. The microspheres and the encapsulated GM-CSF were extensively characterized both in vitro and in vivo. RESULTS: Steady release of GM-CSF was achieved over a period of about one week without significant "burst" of protein from the microspheres. Analysis of microsphere degradation kinetics by gel permeation chromatography (GPC) indicated that low molecular weight PLA enhanced the degradation of the PLGA and thereby affected release kinetics. GM-CSF released from the microspheres was found to be biologically active and physically intact by bioassay and chromatographic analysis. Analysis of serum from mice receiving huGM-CSF indicated that the GM-CSF was biologically active and that a concentration of greater than 10 ng/mL was maintained for a period lasting at least nine days. MuGM-CSF was not detected following in vivo administration of muGM-CSF microspheres. The tissues of mice receiving muGM-CSF microspheres were characterized by infiltration of neutrophils, and macrophages which were in significant excess of those found in mice administered with placebo controls (i.e. microspheres without GM-CSF). CONCLUSIONS: This study demonstrates the influence of formulation parameters on the encapsulation of GM-CSF in PLGA/PLA microspheres and its controlled release in biologically active form. The intense local tissue reaction in mice to muGM-CSF microspheres demonstrates the importance of the mode of delivery on the pharmacologic activity of GM-CSF.     

Epitope repertoire of human CD4+ T cells on tetanus toxin: identification of immunodominant sequence segments

Sequence regions of tetanus toxin-forming CD4+ cell epitopes in 8 HLA-disparate subjects were identified. Overlapping synthetic peptides corresponding to the complete tetanus toxin sequence were used to test, in a proliferation assay, unselected blood CD4+ cells or CD4+ cell lines propagated by stimulation with tetanus toxoid. The CD4+ cell lines recognized most peptides recognized by the blood CD4+ cells and they recognized additional peptides. Their responses were stronger than those of unselected blood CD4+ cells. Two peptides were recognized by all subjects: one largely overlapped a tetanus toxin sequence region previously identified as a "universal" T cell epitope. Thirteen other peptides elicited a CD4+ cell response in 6 or 7 of the 8 subjects, and another 10 elicited responses in 5 subjects.     

Study on Ambient Air Quality of Megacity Delhi,India During Odd–Even Strategy

State Government of Delhi had adopted odd–even scheme on vehicles plying in megacity Delhi to understand and improve the air quality of Delhi. To understand the effect of odd–even scheme on the concentration of pollutants, we have analysed the concentrations of chemical constituents [organic carbon, elemental carbon, water soluble inorganic components, trace elements and stable carbon and nitrogen isotopic composition (δ¹³C_TC) and N (δ¹⁵N_TN)] of PM_2.5 and PM₁₀ along with mixing ratios of trace gases (NO _x , CO, SO₂ and NH₃) data collected at an urban site of megacity Delhi during first phase (Phase-I: winter 2016) and second phase (Phase-II: summer 2016). During the Phase-I of the scheme, mass concentrations of PM_2.5 and PM₁₀ were changed by ?13 and ?5%, respectively, whereas, concentrations of PM_2.5 and PM₁₀ were changed by +18 and +16%, respectively during the Phase-II as compared to before the implementation of the scheme. The analysis of chemical constituents of PM_2.5 and PM₁₀ reveals that the odd–even strategy marginally changed the concentrations (markers) of vehicular emission. During both the phases, mixing ratios of trace gases (NO _x , CO, SO₂ and NH₃) were reduced non-significantly during the odd–even scheme as compared to before the implementation of the scheme.     

Nebraska school nurses identify emotional maltreatment of school-age children: a replication of an Ohio study

PURPOSE: To determine how often school nurses identify emotional maltreatment using validated vignettes. DESIGN: Survey, with 16 behavior vignettes, mailed to 550 Nebraska school nurses. Four forms represented nonwhite and white, male and female children. Demographic questionnaire included. SETTING: Survey mailed with regular spring school nurses' newsletter. PARTICIPANTS: One hundred twenty-one school nurses completed and returned surveys. MAIN OUTCOME MEASURES: Each vignette was an example of emotional abuse; subjects indicated "refer" or "not refer" decisions about the vignette. All items were positive for emotional abuse. RESULTS: Sixty-four of 121 (53%) respondents correctly identified 13 or more vignettes as referable for emotional maltreatment. No influence of child gender and race on reporting decision. No nurse characteristics correlated with referral rates. CONCLUSIONS: None of the variables studied significantly influenced school nurses' identification of emotional abuse. Further research is needed to ascertain correlation between text vignettes and reporting, and to identify interventions to increase the rate of identifying emotional abuse.     

Targeted delivery of DNA encoding cytotoxic proteins through high-affinity fibroblast growth factor receptors

Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans ("low-affinity" FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.     

Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts

BACKGROUND: Subsequent to the publication of a report in 1984 entitled "Poorly Differentiated ("Insular") Carcinoma: A Reinterpretation of Langhans "wuchernde Struma," poorly differentiated insular thyroid carcinoma (PDITC) has become recognized as a distinct thyroid neoplasm. It is classified morphologically and biologically as an intermediate entity between well-differentiated (papillary and follicular) and undifferentiated (anaplastic) thyroid carcinomas. Only a few publications have addressed the findings with fine needle aspiration biopsy (FNAB). CASE: A 67-year-old female presented for evaluation of a massively enlarged thyroid gland. Fine needle aspiration biopsy of the thyroid with a 22-gauge needle showed many large, multilayered, round to oval nests of tumor cells, 0.2-0.4 mm in diameter. Rosettelike configurations of 8-15 cells, 0.025-0.050 mm in diameter, were also observed. Nests of neoplastic cells in the histologic sections were virtually identical to those in the fine needle aspiration biopsy specimens. When the patient developed metastatic cervical adenopathy one year later, a microfollicular pattern was seen on both the FNAB and histologic sections. CONCLUSION: When nests of tumor cells, 0.2-0.4 mm in diameter, are identified in a thyroid FNAB specimen, PDITC should be included in the differential diagnosis. A microfollicular pattern in a metastatic lymph node does not exclude the possibility that the primary tumor is a PDITC.     

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., I?iguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.     

Inhibition of fos-jun-DNA complex formation by dihydroguaiaretic acid and in vitro cytotoxic effects on cancer cells

The main cause of acquired inguinal hernia is weakness of Fruchaud's deep muscolofascial floor, following metabolically-determined collagen disorders. A technique for the anterior reinforcement of this structure with polypropylene mesh is described here. Following intermuscular decollement, the mesh is placed in direct contact with the surface formed by the transversalis fascia and the transversus abdominis muscle and stretched as extensively as possible. Because the posterior aspect of the inguinal canal is the true barrier to abdominal pressure, the author believe that its direct reinforcement, without interposition of the internal oblique muscle, constitutes the most correct anatomo-surgical approach to hernia repair. This is the case for both indirect hernias, in which the internal ring is reconstructed at a deeper level, and for direct hernias, in which the "tent effect" of the prosthesis is prevented. Ninety-two primary inguinal hernias (56 indirect, 29 direct and 7 direct and indirect) in 87 patients were repaired with this technique. Seventy-nine patients were followed up from 2 to 24 months. Early complications included: 7 ecchymosis, 3 seromas, 2 subcutaneous infections, 3 testicular swellings. Incision and testicular pain for longer than 6 months occurred in 2 cases. No prosthetic infections or recurrences have been detected up to the present.     

Importance of Thermophysical Properties of Thin Films in Ultra-Large-Scale Integrated Circuits and Beyond

Ultralarge-scale integrated circuits (ULSICs) are fabricated in a single crystal of silicon using submicron device structures arrayed in two dimensions, which are interconnected in the third dimension with multilevel metallizations. The latter use several alternating layers of metal and dielectric films. Currently, four-level metallizations are used in large volume ULSIC manufacturing. As the technologies advance in ULSICs and beyond, four-level metallizations are expected to increase to six-level and higher. In three-dimensional ICs (3-D ICs) and ultraperformance ICs (UPICs), in which active devices are also fabricated in the third dimension in addition to the interconnects, the total number of semiconductor, dielectric, and metal films increases significantly. The thermophysical properties of all of these films play an ever-increasing role in the overall yield, reliability, and chip size of the ICs. The thermophysical properties considered in this paper are the thermal conductivity and the stress and its variations on thermal cycling, of the SiO₂ films only. Data for these parameters are shown, with comments on their impact on the reliability of the ICs, and the role of Umklapp and point defect scattering mechanisms on the of thin films is discussed. The thermophysical properties not only are important in the current ULSICs for enhanced performance and reliability, but are even more crucial for the design and manufacturing of the next generation ICs like 3-D ICs and UPICs.