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.     

Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation

Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1beta-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the "point of no return" in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (delta psi(m)) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-x(L)-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and delta psi(m) was followed by rhodamine 123 fluorescence. Although Bcl-x(L) expression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of delta psi(m) and cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of delta psi(m) and cell death. Together these data demonstrate that Bcl-x(L) can maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.     

Determination of the internal morphology of poly (D,L-lactide) microspheres using stereological methods

The morphological characteristics of the internal structure of poly (D,L-lactide) microspheres have been determined by stereological methods in two different formulations of microspheres, with different internal structures, prepared by using a double emulsion method. In one formulation the internal emulsion was produced by homogenisation at 3000 rpm, whilst the other was prepared at 11000 rpm. As expected the formulation prepared at the lower speed contained larger and more broadly distributed pores than that prepared at the higher speed. The porosity, pore size distribution and total internal surface area of the microspheres were obtained by stereological methods from electron microscopic measurements of the sectioned microspheres. It was found that whilst the porosity of the microspheres was 0.6 in both formulations, the preparation method gave rise to large differences in their pore size distribution characteristics. The pore size distribution was simulated by computer modelling to validate and compare alternative stereological algorithms. It was found that the Saltykov unfolding method predicts the measured pore size distribution more accurately than the Cruz-Orive unfolding method (at significance level alpha=0.1). This finding was attributed to the violation of one of the basic assumptions of the Cruz-Orive unfolding method.     

Activity of pentamidine-loaded poly (D,L-lactide) nanoparticles against Leishmania infantum in a murine model

Tumors of craniocervical junction (ccjct) may cause a variety of non specific signs and symptoms. Before the advent of computed tomography (CT) diagnosis was sometimes possible only by surgical exploration, as the available radiologic methods--plain film radiography, angiography, and myelocisternography--essentially provided only indirect information about the neural structures. Because of its beam hardening artifacts, CT remained unsatisfactory as well. These diagnostic problems have practically disappeared with the availability of magnetic resonance imaging (MRI), which is now considered the method of choice if a space-occupying lesion is suspected at the ccjct. In some cases invasive angiography may still be necessary for surgical planning, and angiography may also be needed, whenever CT or MRI suggest the presence of an aneurysm. In the first part of this overview we present strategies for the radiological examination of the ccjct.     

Plasma protein adsorption on biodegradable microspheres consisting of poly(D,L-lactide-co-glycolide), poly(L-lactide) or ABA triblock copolymers containing poly(oxyethylene). Influence of production method and polymer composition

Biodegradable particulate systems have been considered as parenteral drug delivery systems. The adsorption of plasma proteins on micro- and nanoparticles is determined by the surface properties and may, in turn, strongly influence the biocompatibility and biodistribution of both carriers. In the present study the influence of the polymer composition and the production method of microspheres on the in vitro plasma protein adsorption were investigated using two-dimensional electrophoresis (2-DE). Microparticles were prepared from poly(l-lactide) (l-PLA), poly(d,l-lactide-co-glycolide) (PLGA), and ABA triblock copolymers containing hydrophilic poly(oxyethylene) (B-blocks) domains connected to hydrophobic polyesters (A-blocks). Two different microencapsulation methods were employed, namely the w/o/w emulsion solvent evaporation method and the spray-drying technique. It could be demonstrated that the polymer composition and, especially, the encapsulation technique, influenced the interactions with plasma proteins significantly. For example, the percentages of several apolipoproteins in the plasma protein adsorption patterns of spray-dried PLGA- and l-PLA-particles were distinctly higher when compared to the adsorption patterns of the particles produced by the w/o/w-technique. Some adsorbed proteins were found to be characteristic or even specific for particles produced by the same method or consisting of identical polymers. Polyvinyl alcohol used as stabilizer in the w/o/w-technique may decisively influence the surface properties relevant for protein adsorption. The plasma protein adsorption on particles composed of ABA copolymers was drastically reduced when compared to microspheres made from pure polyesters. The adsorption patterns of ABA-particles were dominated by albumin. The plasma protein adsorption patterns detected on the different microspheres are likely to affect their in vivo performance as parenteral drug delivery systems.     

The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo

Human cytomegalovirus (HCMV)-specific CD8(+) cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vbeta segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo.     

Process variable implications for residual solvent removal and polymer morphology in the formation of gentamycin-loaded poly (L-lactide) microparticles

PURPOSE: The purpose was to determine the influence of process parameters in the precipitation with a compressed antisolvent (PCA) process on the morphology and residual dichloromethane (DCM) levels in gentamycin-loaded PLA microparticles. METHODS: The three variables studied were the rate of CO2 co-flowed during the polymer and drug post-precipitation, the post-precipitation pure CO2 flush rate, and the post-precipitation CO2 flush volume. Residual DCM levels were determined from headspace gas chromatography-mass spectroscopy (GC-MS) with single ion monitoring. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to estimate the crystallinity within microparticles. DCM was extracted from drug-loaded microparticles by both supercritical CO2 extraction and vacuum drying for up to two days to determine a lower limit for solvent removal. RESULTS: Increasing either the post-precipitations CO2 flow rate or flush volume resulted in lower residual DCM levels in the microparticle. The CO2 co-flow rate showed an opposite trend. Increasing in value resulted in a higher DCM value after precipitation. XRD and DSC analysis on these samples suggest that those produced at lower CO2 co-flow rates have a higher degree of crystallinity, which increases the diffusivity of DCM through the polymer matrix. Finally, samples subjected to extended (48 hr) CO2 extraction resulted in DCM levels on the order of one to three ppm. CONCLUSIONS: Specific PCA process conditions during microparticle formation have a strong influence on the residual solvent levels within the microparticles. Polymer morphology affects the diffusivity of solvent through the polymer matrix, which in turn determines the solvent removal rates.     

A new backbone of artificial enzymes obtained by cross-linkage of poly(ethylenimine)

Cross-linkage of the branches of poly(ethylenimine) (PEI) suppresses flexibility of the polymer as revealed by decreased affinity of the amino groups on PEI backbone towards proton or Ni(II) ion. The cross-linkage improves ability of the PEI derivative equipped with beta-cyclodextrin to deacylate an ester containing t-butylphenyl moiety.     

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) in nuclei of various rat tissues

Natural distribution of poly(adenosine diphosphate-ribose), a novel macromolecule in eukaryotes, was investigated using an indirect immunofluorescence technique. The antibody, produced in a rabbit toward poly(ADP-ribose), was most reactive with polymers having the chain length of about 25 ADP-ribose units and weakly reactive with short oligomers; it was totally inert with monomers. Immunostaining with this antibody revealed the existence of the polymer in various rat tissues. The immunostaining seems to be specific for poly(ADP-ribose), as judged by its disappearance by preabsorption of the antiserum with purified poly(ADP-ribose) or pretreatment of tissue sections with poly(ADP-ribose)-degrading enzymes. Intensification of the fluorescence by preincubation with nicotinamide adenine dinucleotide (NAD), a substrate for poly(ADP-ribose) synthesis, also supported this view. The immunofluorescence of poly(ADP-ribose) was found exclusively in the nucleus of almost all tissues tested, including liver (adult, newborn, regenerating, and hepatoma), brain, heart, intestine, pancreas, kidney, spleen, testis, thyroid gland, and skeletal muscle. Exceptions were blood cells; little fluorescence was detectable in nuclei of peripheral leukocytes. Only after preincubation with NAD, did lymphocytes and monocytes exhibit fluorescence, however, granulocytes never did exhibit fluorescence. The cells appeared to represent the first instance where poly(ADP-ribose) synthesis activity among eukaryotic cells was missing.     

Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing

Three methods were used, namely spray drying, w/o/w solvent evaporation and the aerosol solvent extraction system (ASES), for the preparation of microparticles having the same size range, to study the influence of the preparation method on polymer degradation in vitro (PBS, 37 degrees C, one month). The following five polymers of the biodegradable poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) group were selected: L-PLA, MW 81 200; DL-PLGA 75:25, MW 64-300; DL-PLGA 50:50 MW 52 600; DL-PLGA 50:50 MW 14 500, AND DL-PLGA 50:50, MW 3400, to prepare drug-free and drug-loaded microparticles. Tetracosactide was selected as model peptide. When microparticles were prepared by solvent evaporation, the mean diameter and, more markedly, the drug encapsulation efficiency tended to decrease when decreasing the molecular weight and increasing the proportion of glycolic acid in the polymer. In contrast, no direct influence of the polymer nature on these parameters was observed in spray dried microparticles. Polymer degradation was heterogenous in L-PLA and DL-PLGA 75:25 microparticles and was not influenced by the presence of the drug at a nominal loading of 1% (w/w), when prepared by the three methods (note that with ASES, only L-PLA could be used for microencapsulation). In batches made of DL-PLGA 50:50 MW 52 600, the degradation rate decreased slightly when increasing the drug loading. Only in the case of DL-PLGA 50:50 MW 14 500, the polymer degradation rate for spray dried microparticles was higher compared to that for microparticles prepared by the w/o/w solvent evaporation method. Generally, the degradation rates of the different microparticles followed the expected order: L-PLA相似文献   

Response of testicular macrophages to EDS-induced Leydig cell death

The response of testicular macrophages to massive Leydig cell death was studied by the administration of the specific Leydig cell cytotoxic ethylene dimethane sulphonate (EDS) to sham-operated (SO), short-term (STHX), and long-term (LTHX) hypophysectomized rats. EDS-killed Leydig cells showed the morphological features of the programmed cell death or apoptosis. A 2-fold increase in the number of macrophages was found on days 1-2 after treatment in both SO and STHX rats, and dead Leydig cells were completely eliminated by day 3 after treatment. Otherwise, in LTHX rats, there was a delay in the increase in the number of macrophages, and EDS-killed Leydig cells remained in the testicular interstitium for several days. These results indicate that the phagocytic capacity of the macrophage population was diminished in hypophysectomized rats, and particularly after long-term hypophysectomy.     

Is it possible to improve the quality of life of patients who have undergone pelvic evisceration?

Locally spread cancer makes up considerable percent (20-30%) in statistical structure of rectal tumors. In cases of cancer spread into the area of urine bladder triangle the operation of choice is pelvic evisceration. From 1977 to 1997 in the State research Centre of the Ministry of Health for Coloproctology pelvic evisceration in cancer spread to back wall of urine bladder in the area of triangle was carried out in 22 patients (20 male and 2 female). Mean age was 43.4 (29-56) years, 16 patients have undergone typical infralevator pelvic evisceration. There were no intraoperative lethality. Postoperative lethality made up 6.3%, complications--68.8%, 5-years survival rate--25%. Presence of two fecal fistulas on the anterior abdominal wall has decreased considerably the quality of life of the patients. Since 1993 the conception of preservation and restoration of natural passage of urine and bowel contents was adopted. In 4 cases infralevator pelvic evisceration with various types of ileocystoplasty and pull-through of colon into small pelvis with creation of smooth muscle cuff in perineal colostomy was carried out. In 2 patients evisceration was of supralevator-character cystoplasty of local tissues and performance of coloanal anastomosis were carried out. The application of reconstructive-restorative ways in coloproctology and urology considerably contributed to the improvement of the quality of life of the patients after pelvic evisceration.     

Combinations of hyperthermia (40 degrees, 45 degrees C) with radiation

Incubation of Chinese hamster cells at an elevated but sublethal temperature between fractions of radiation and/or pulses of hyperthermia at 45 degrees C strongly modified the effectiveness of cell killing. Increasing the incubation temperature from 37 to 40 degrees C between fractions of hyperthermia at 45 degrees C followed by radiation substantially enhanced cell killing; while the opposite sequence (radiation leads to incubation at 40 degrees C leads to hyperthermia at 45 degrees C) resulted in less effective cell killing than would have been expected from the independent interaction of hyperthermia and radiation alone. This suggests the use of short pulses of localized hyperthermia in the presence of physiologically tolerable fever followed by irradiation as one approach to the utilization of hyperthermia in cancer therapy.     

Reconstruction of mandibular continuity defects in dogs using poly (L-lactide) mesh and autogenic particulate cancellous bone and marrow: preliminary report

PURPOSE: This study evaluated the reconstruction of continuity defects in the canine mandible using a poly [L-lactide] (PLLA) mesh tray and particulate cancellous bone and marrow (PCBM). MATERIALS AND METHODS: Eight adult dogs were divided into two groups of four dogs each. In group A, each dog had a tray fixed with stainless steel wires on each side of the mandibular stumps with the concave surface of the tray attached to the inferior border of the mandible (U-fixation). In group B, the concave surface was attached to the superior border (inverted U-fixation). Each tray was filled with PCBM from the ilium. After the operation, the dogs were radiographed, and specimens were examined histologically at 3-, 6-, and 12-month intervals. RESULTS: All of group A showed good clinical healing and the continuity of the mandibular bone was regained within 3 months postoperatively. However, fibrous tissue had invaded through the area above the tray, resulting in a poorly shaped alveolar ridges. In group B, the dogs showed good bony regeneration with well-shaped alveolar ridges. However, two animals in this group had partial exposure of the PLLA mesh tray into the oral cavity. CONCLUSION: It is suggested that a combination of the PLLA mesh and PCBM grafts might be a useful technique for functional reconstruction of the jaw bone, specifically using method A (U-fixation) as a technique to reconstruct continuity defects of the mandible, and method B (inverted, U-fixation) as a promising method for alveolar reconstruction to make wearing dentures possible.     

Improved detection of polycyclic aromatic compounds in complex mixtures by liquid chromatographic fractionation on poly(divinylbenzene) prior to gas chromatography-mass spectrometry. Application to the analysis of diesel particulates

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6-7 kb from C9 in a head-to-head or 5' to 5' orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.     

Response to Amato's (2002) letter.

Responds to J. J. Amato's commentary (see record 2003-02090-007) on articles by M. A. Yarhouse (see record 1998-11146-011) and M. A. Yarhouse and W. Throckmorton (see record 2002-13988-007). Yarhouse contends that by claiming he is surreptitiously importing ideology in the name of science, Amato is setting this up as a false dichotomy, as though science is a completely objective enterprise and that beliefs and values can be avoided altogether. This position is a modernist view and it ignores the postmodern society and the advances in philosophy of science over the past 50 years. Yarhouse also addresses Amato's claims regarding changing same sex attraction and the concept of autonomy. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.     

The murine IgM secretory poly(A) site contains dual upstream and downstream elements which affect polyadenylation

Regulation of polyadenylation efficiency at the secretory poly(A) site plays an essential role in gene expression at the immunoglobulin (IgM) locus. At this poly(A) site the consensus AAUAAA hexanucleotide sequence is embedded in an extended AU-rich region and there are two downstream GU-rich regions which are suboptimally placed. As these sequences are involved in formation of the polyadenylation pre-initiation complex, we examined their function in vivo and in vitro . We show that the upstream AU-rich region can function in the absence of the consensus hexanucleotide sequence both in vivo and in vitro and that both GU-rich regions are necessary for full polyadenylation activity in vivo and for formation of polyadenylation-specific complexes in vitro . Sequence comparisons reveal that: (i) the dual structure is distinct for the IgM secretory poly(A) site compared with other immunoglobulin isotype secretory poly(A) sites; (ii) the presence of an AU-rich region close to the consensus hexanucleotide is evolutionarily conserved for IgM secretory poly(A) sites. We propose that the dual structure of the IgM secretory poly(A) site provides a flexibility to accommodate changes in polyadenylation complex components during regulation of polyadenylation efficiency.     

Purification and characterization of an esterase involved in poly(vinyl alcohol) degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45 degrees C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K(m) and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 microM, and 6.52 and 12.6 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.