Particulate Preparations Research Articles

Particles from the Echinococcus granulosus laminated layer inhibit IL-4 and growth factor-driven Akt phosphorylation and proliferative responses in macrophages.

Proliferation of macrophages is a hallmark of inflammation in many type 2 settings including helminth infections. The cellular expansion is driven by the type 2 cytokine interleukin-4 (IL-4), as well as by M-CSF, which also controls homeostatic levels of tissue resident macrophages. Cystic echinococcosis, caused by the tissue-dwelling larval stage of the cestode Echinococcus granulosus, is characterised by normally subdued local inflammation. Infiltrating host cells make contact only with the acellular protective coat of the parasite, called laminated layer, particles of which can be ingested by phagocytic cells. Here we report that a particulate preparation from this layer (pLL) strongly inhibits the proliferation of macrophages in response to IL-4 or M-CSF. In addition, pLL also inhibits IL-4-driven up-regulation of Relm-α, without similarly affecting Chitinase-like 3 (Chil3/Ym1). IL-4-driven cell proliferation and up-regulation of Relm-α are both known to depend on the phosphatidylinositol (PI3K)/Akt pathway, which is dispensable for induction of Chil3/Ym1. Exposure to pLL in vitro inhibited Akt activation in response to proliferative stimuli, providing a potential mechanism for its activities. Our results suggest that the E. granulosus laminated layer exerts some of its anti-inflammatory properties through inhibition of PI3K/Akt activation and consequent limitation of macrophage proliferation.

Calcium polyphosphate particulates for bone void filler applications.

This study investigates the characteristics of porous calcium polyphosphate particulates (CPPp) formed using two different processing treatments as bone void fillers in non- or minimally load-bearing sites. The two calcium polyphosphate particulate variants (grades) were formed using different annealing conditions during particulate preparation to yield either more slowly degrading calcium polyphosphate particulates (SD-CPPp) or faster degrading particulates (FD-CPPp) as suggested by a previous degradation study conducted in vitro (Hu et al., Submitted for publication 2016). The two CPPp grades were compared as bone void fillers in vivo by implanting particulates in defects created in rabbit femoral condyle sites (critical size defects). The SD-CPPp and FD-CPPp were implanted for 4- and 16-week periods. The in vivo study indicated a significant difference in amount of new bone formed in the prepared sites with SD-CPPp resulting in more new bone formation compared with FD-CPPp. The lower bone formation characteristic of the FD-CPPp was attributed to its faster degradation rate and resulting higher local concentration of released polyphosphate degradation products. The study results indicate the importance of processing conditions on preparing calcium polyphosphate particulates for potential use as bone void fillers in nonload-bearing sites. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 874-884, 2017.

Synthesis of uniform poly(d,l‐lactide) and poly(d,l‐lactide‐co‐glycolide) microspheres using a microfluidic chip for comparison

Applications of poly(l-lactide) (PLA) and poly(d,l-lactide-co-glycolide) (PLGA) microspheres are widely used in the biomedical and pharmaceutical fields. The effects of PLA/PLGA on microsphere properties when using conventional particulate preparation methods are not easily defined due to the uncontrollable particle size and size distribution. This study was aimed to synthesize uniform PLA and PLGA microspheres using a phenol formaldehyde resin-based microfluidic chip, which has the advantage of being solvent-resistant, flexible, and is readily disassembled for cleaning. The proposed chip can rapidly fabricate reproducible PLA and PLGA microspheres. Uniform emulsion droplets can be achieved by hydrodynamic flow focusing. After solvent evaporation, the free-flowing PLA and PLGA microspheres have a high level of morphological uniformity and size, allowing for a clear comparison of material effects. The results indicate that the sizes of the PLA and PLGA microspheres for the various flow rates of dispersed/continuous phases are very similar. The PLA/PLGA materials do not have a significant effect on particle size, but the particle surface indicates a different morphology. The result of the cytotoxicity evaluation shows no difference between PLA and PLGA and ensures the biocompatibility of both prepared PLA and PLGA microspheres for biomedical and pharmaceutical applications in the future.

Binding constants and stability of dexamethasone-binding protein from rat heart.

A pronounced decrease in specific dexamethasone binding capacity of heart cytosol was observed. The inactivation of unbound glucocorticoid receptor was not due to the action of proteases since the rat of albumin digestion was very slow in the particulate preparations. The rate of inactivation of glucocorticoid binding capacity depends on cytosol protein concentration and temperature. Addition of hormone and glycerol to cytosol markedly slow binding capacity inactivation. The rapid loss of binding activity, especially in the cytosol with high protein concentration, makes difficult to perform equilibrium studies for simultaneous determination of binding affinity and a number of binding sites. Under various conditions the apparent dissociation constants were measured and compared with the dissociation constants, calculated from the rate of constants of association and dissociation, in order to find out optimal conditions for simultaneous determination of KD and [R0].

Effect of filtration by activated charcoal on the toxicological activity of cigarette mainstream smoke from experimental cigarettes

Activated charcoal (AC) filtration reportedly decreases the yields of smoke vapor phase constituents including some identified as human carcinogens and respiratory irritants. Non-clinical studies including chemical smoke analysis, in vitro cytotoxicity and mutagenicity (bacterial and mammalian cells), and in vivo subchronic rat inhalation studies were carried out using machine smoking at ISO conditions with lit-end research cigarettes containing AC filters. The objective was to assess whether AC filter technology would alter the established toxicity profile of mainstream smoke by increasing or decreasing any known toxicological properties, or elicit new ones. The reduced yield of vapor phase irritants from AC filter cigarettes correlated with markedly decreased in vitro cytotoxicity and in vivo morphology of the nose and lower respiratory tract. Increased yields of particulate phase constituents (e.g. polycyclic aromatic hydrocarbons) in AC filtered smoke were noted in comparison to controls in some studies. The in vitro bacterial mutagenicity of AC filtered smoke particulate preparations was occasionally increased over control levels. Laryngeal epithelial thickness was increased in some rats inhaling AC filtered smoke in comparison to controls, an effect perhaps related to higher inspiratory flow. When tested under more intense Massachusetts Department of Public Health smoking conditions, AC filter associated reductions in vapor phase constituent yields were smaller than those seen with ISO conditions, but the effect on in vitro cytotoxicity remained.

Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged alpha(2A)-adrenergic receptor (AR) expression was driven by the endogenous mouse alpha(2A)AR gene locus. Exploiting this mouse line, we evaluated alpha(2A)AR trafficking and alpha(2A)AR-mediated inhibition of Ca(2+) currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the alpha(2A)AR subtype. We discovered a more rapid desensitization of Ca(2+) current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of alpha(2A)AR evoked by clonidine than by guanfacine. Clonidine-induced alpha(2A)AR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of alpha(2A)AR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-alpha(2A)AR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

Indian Traditional Medicine Jasada Bhasma and Other Zinc-Containing Nanoparticles Alleviate Reactive Oxygen Species-Mediated Cell Damage in Saccharomyces cerevisiae

ABSTRACT Indian traditional medicine Jasada Bhasma is a unique particulate preparation of zinc, which has been used by traditional practitioners for the treatment of various diseases such as diabetes, eye diseases, etc. This paper is an attempt to understand the mode of action of this and related types of particulate matter in biology. The biological activity of Jasada Bhasma has been tested on Saccharomyces cerevisiae wild strain, chosen as a model eukaryotic system. Results show that Jasada Bhasma– treated yeast cells were able to maintain a higher level of growth over control. DNA and enzyme studies, together with advanced microscopic studies of cellular and nuclear morphology, suggest a protective action against cellular damage mediated by reactive oxygen species (ROS). Further, a nanoparticulate-size fraction of the Bhasma showed effects comparable to the unfractionated mass, at much lower dosage levels. In comparative studies, qualitatively similar effects were also exhibited by other compounds of zinc. Our findings provide a preliminary justification of the potential usefulness of Jasada Bhasma as a therapeutic agent and provide a first level understanding the mechanism of Bhasma action through free radical control.

Vaccine progress

Despite the recent development of new anti-mould agents, there remains a significant incidence of invasive aspergillosis in the most immunocompromised hosts and the response to these agents is still dismal. There is a need for a different approach: prevention by vaccination. We have demonstrated that a hyphal sonicate of Aspergillus fumigatus was capable of conferring protection against subsequent invasive pulmonary aspergillosis in corticosteroid immunosuppressed mice. Subcutaneous vaccination was superior to nasal vaccination. Mice exposed intranasally to viable conidia were noted to respond serologically to a 19 kDa protein. This protein was identified as the allergen Asp f 3 by mass spectrometry. Vaccination with recombinant Asp f 3 was protective. Truncated forms of Asp f 3 that lacked either one of the two known IgE binding sites were cloned and also demonstrated protection against aspergillosis. Although all of these recombinant proteins required an adjuvant (TiterMax) for efficacy, a particulate preparation of rAsp f 3 was also found to be protective without requiring adjuvant. At least two T-cell epitopes (11-mer and 13-mer) have been identified in Asp f 3. There are homologues of Asp f 3 in other Aspergillus species as well as in other moulds (Coccidioides posadasii, Penicillium citrinum) and yeasts (Candida albicans, C. boidinii, Saccharomyces cerevisiae). Asp f 3, truncated non-allergenic versions of Asp f 3, and T-cells epitopes of Asp f 3 are potential candidates for vaccines potentially capable of protecting immunocompromised hosts against invasive aspergillosis.

Report of “24th Symposium on Particulate Preparations and Designs”

Report of “24th Symposium on Particulate Preparations and Designs”

Advances in environmental and occupational disorders 2006

In 2006, there continued to be significant contributions to the Journal in the area of environmental and occupational allergy. In addition to very strong clinical observations, there were a number of studies that examined the effect of environmental agents on cellular and molecular processes in allergy, and molecular approaches were also used in investigations of allergen structure and biology. The Journal also received a number of very strong epidemiologic studies examining several aspects of environmental and occupational allergy. Thus, this particular area of our specialty thrives as a result of clinical, mechanistic, epidemiologic, and translational observations. This article reviews a number of these papers presented in the Journal in 2006.

Pulmonary Toxicity Screening Studies in Male Rats with M5 Respirable Fibers and Particulates

M5 fiber is a high-strength, high-performance organic fiber type that is a rigid rod material and composed of heterocyclic polymer fibers of type PIPD. The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled M5 respirable fibers and particulates in rats. Using a pulmonary bioassay and bridging methodology, the acute lung toxicity of intratracheally instilled M5 particulates and that of its fibers were compared with a positive control particle type, quartz, as well as a negative control particle type, carbonyl iron particles. Moreover, the results of these instillation studies were bridged with data previously generated from inhalation studies with quartz and carbonyl iron particles, using the quartz and iron particles as the inhalation/instillation bridge material. For the bioassay experimental design, in the bronchoalveolar lavage studies, the lungs of rats were intratracheally instilled with 0.5 or 0.75 mg/kg of M5 particulate or 1 or 5 mg/kg of the following control or particle types: (1) M5 long fiber preparation, (2) silica–quartz particles, and (3) carbonyl iron particles. Phosphate-buffered saline (PBS)-instilled rats served as additional controls. Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers, cell proliferation methods, and histopathological evaluation of lung tissue at 24 h, 1 wk, 1 mo and 3 mo post instillation exposure. The bronchoalveolar lavage results demonstrated that lung exposures to quartz particles, at both concentrations but particularly at the higher dose, produced significant increases vs. controls in pulmonary inflammation and cytotoxicity indices. Exposures to M5 particulate and M5 long fiber preparation produced transient inflammatory and cell injury effects at 24 h postexposure (pe) as well as at 24 h and 1 wk pe, respectively, but these effects were not sustained when compared to quartz-silica effects. Exposures to carbonyl iron particles and PBS resulted in only minor short-term and reversible lung inflammation, likely related to the effects of the instillation procedure. Histopathological analyses of lung tissues revealed that pulmonary exposures to M5 particulate and in particular, the M5 long fiber preparation in rats produced some inflammatory responses, observed up to 1 wk postexposure. These responses were often associated with the presence of M5 long fiber in the airways or in the proximal alveolar regions but appeared to be reversible at 1 and 3 mo postexposure. In contrast, pulmonary exposures to silica–quartz particles in rats produced a dose-dependent lung inflammatory response characterized by neutrophils and foamy (lipid-containing) alveolar macrophage accumulation and evidence of early lung tissue thickening consistent with the development of pulmonary fibrosis. Based on our results, we conclude the following: (1) It was very difficult to produce M5 fibers into a respirable fibrous form; these findings suggest that aerosol exposure concentrations of respirable fibrous M5 in the workplace are likely to be rather low. (2) The particulate and long fiber preparations of M5 that were tested produced a moderate amount of pulmonary inflammatory activity, more active than our negative control, carbonyl iron particles, but substantially less active in terms of inflammation, cytotoxicity, and fibrogenic effects than the positive control particle type, silica–quartz particles. Thus, based on the results of this study, we would expect that inhaled M5 respirable fibers have a low risk potential for producing adverse pulmonary effects.

Activity Report of Division of Particulate Preparation and Design

Activity Report of Division of Particulate Preparation and Design

Flavonoids as antagonists at A1 adenosine receptors

This study aimed to investigate the potential for flavonoid action at A(1) adenosine receptors in vitro. In a radioligand binding assay for A(1) adenosine receptor occupancy in particulate preparations from guinea-pig cerebral cortex, flavonoids competed in concentration-dependent manners with Hill slopes typically not different from unity. Of the flavonoids tested, quercetin showed highest affinity (pKi value of 5.33). At a concentration of 28 mm, quercetin evoked a rightward shift in the N(6)-cyclopentyladenosine-induced inhibition of electrically evoked contractions of the guinea-pig isolated ileum, allowing the calculation of a pK(i) value of 4.71. These data suggest, therefore, that flavonoids represent an additional dietary source of A(1) adenosine receptor antagonists (beyond the methylxanthines, caffeine and theophylline).

Comparative Effectiveness of Cervical Transforaminal Injections with Particulate and Nonparticulate Corticosteroid Preparations for Cervical Radicular Pain

Cervical transforaminal epidural injections of corticosteroids have been used in the treatment of radicular pain. Particulate agents have been associated with rare adverse neurological outcomes. It is unknown whether nonparticulate preparations are any less effective than particulate preparations. Therefore, a study was designed to determine whether there is a basis for promoting a theoretically safer nonparticulate corticosteroid preparation. Volunteer patients were randomized to receive a single cervical transforaminal epidural injection with one of two corticosteroid preparations. This study was undertaken in a private practice setting. Those with single-level, unilateral radicular pain with advanced imaging demonstrating single-level neural compression. Patients received a single cervical transforaminal epidural injection with either dexamethasone or triamcinolone. Ratings were obtained by an independent unbiased assessor at 4 weeks via a telephone interview. A visual analog pain scale was used preprocedurally and a verbal integer scale was used at 4 weeks to assess the severity of the patient's radicular pain. As a secondary outcome measure, a patient-specified functional outcome measure was obtained. Both groups exhibited statistically and clinically significant improvements in pain at 4 weeks. Although the triamcinolone group exhibited a somewhat greater improvement, the difference between groups was not significantly different. The study found that the effectiveness of dexamethasone was slightly less than that of triamcinolone, but the difference was neither statistically nor clinically significant. A theoretically safer nonparticulant agent appears to be a valid alternative to particulate agents that have been used to date, and which have been associated with hazard.

Activity Report of SPTJ Research Division of Particulate Preparation and Design (2003-2004)

Activity Report of SPTJ Research Division of Particulate Preparation and Design (2003-2004)

A β-(1 → 3)-arabinopyranosyltransferase that transfers a single arabinopyranose onto arabino-oligosaccharides in mung bean ( Vigna radiate) hypocotyls

Arabinopyranosyltransferase (Ara pT) activity that results in the transfer of a single arabinopyranose (Ara p) residue from UDP-β- l-arabinopyranose (UDP-Ara p) to exogenous (1 → 5)-linked α- l-arabino-oligosaccharides labeled with 2-aminobenzamide (2-AB) at their reducing ends was identified in a particulate preparation obtained from 3-day-old mung bean ( Vigna radiate L. Wilezek) hypocotyls. The transferred Ara residue was shown to be β-(1 → 3)-linked to O-3 of the non-reducing terminal Ara f residues of the oligosaccharide using nuclear magnetic resonance spectroscopy together with glycosyl composition and glycosyl linkage composition analyses. The 2AB-labeled arabino-octasaccharide was the most effective acceptor substrate analyzed, although arabino-oligosaccharides with a degree of polymerization between 4 and 7 were also acceptor substrates. Maximum Ara pT activity was obtained at pH 6.5–7.0, and 20 °C in the presence of 25 mM Mn 2+ and 0.5% Triton X-100.

Identification of elongating �-1,4-galactosyltransferase activity in mung bean ( Vigna radiata ) hypocotyls using 2-aminobenzaminated 1,4-linked �-d-galactooligosaccharides as acceptor substrates

Galactosyltransferase (GalT) activity that results in the transfer of galactose (Gal) from UDP-Gal to exogenous (1-->4)-beta-galactooligosaccharides labeled with 2-aminobenzamide (2AB) at their reducing ends was identified in a particulate preparation obtained from 2-day-old mung bean (Vigna radiata L. Wilezek) hypocotyls. The enzymes responsible were shown, by high-performance anion-exchange chromatography and normal-phase liquid chromatography-electrospray ionization mass spectrometry, to transfer up to eight Gals to the non-reducing end of 2AB-labeled galactooligosaccharide. Using 1H nuclear magnetic resonance spectroscopy, and beta-galactosidase and endo-beta-(1-->4)-galactanase treatments of the enzymatically formed 2AB-labeled galactooligosaccharides, the newly incorporated Gal residues were shown to be beta-(1-->4) linked. Time-course studies indicated that at least two different types of GalT isoform are involved in the elongation of the acceptor substrates. 2AB-labeled galactoheptaose was the most effective acceptor substrate analyzed, although galactooligosaccharides with a degree of polymerization between 4 and 6 were also acceptor substrates. 2AB-labeled penta- and heptasaccharides (RG5 and RG7) generated from rhamnogalacturonan I (RG-I) were not acceptor substrates, suggesting that the GalTs were not capable of adding Gal residues directly to the RG-I backbone. Maximum GalT activity was obtained at pH 6.5 and 20 degrees C in the presence of 25 mM Mn2+ and 0.75% (w/v) Triton X-100. The enzyme had an apparent Km of 20 microM for 2AB-labeled galactoheptaose and 32 microM for UDP-Gal. The characteristics of the enzyme in mung bean microsomal membranes and the usefulness of fluorogenic 2AB-labeled galactooligosaccharides for the assay of GalT are discussed.

Design of microcapsules with hydrogel as a membrane component and their preparation by a spouted bed

This paper describes particulate design and preparation to control drug release thermosensitively or in a delayed mode from microcapsules (MCs) that were prepared by coating with a membrane incorporating hydrogel as a particulate or chemical component. The particulate preparation was carried out by a spouted bed coating process. The microcapsules releasing drug with a positive thermosensitivity were prepared by dispersing the newly developed latex particles in the ethylcellulose matrix membrane. The latex particles were synthesized so as to have a thermoinsensitive core and a thermosensitive poly( N-isopropylacrylamide) hydrogel shell. Poly( N-isopropylacrylamide) with a lower critical solution temperature (LCST) at 32 °C in water shrank at higher temperatures and highly swelled at lower temperatures than LCST, leading to so-called positively thermosensitive release. On the other hand, when the acrylic ter-polymer of latex particles has a hydrophilic component, 2-hydroxyethyl methacrylate, the water uptake rate of microcapsules could be controlled by its content in the ter-polymer and membrane thickness. The release was characterized by a delayed mode; then the lag time was controlled by the 2-hydroxyethyl methacrylate content and the membrane thickness. This content was also related to the mechanical flexibility of membrane that was critical in the release rate control after the release of drug starts.

Quantitative measurement of glutamate receptor subunit protein expression in the postnatal rat spinal cord

Glutamate is the major excitatory neurotransmitter in the CNS and its effects on neurons are dependent on the type and composition of glutamate receptors with which it interacts. In this study, the protein expression levels of several ionotropic glutamate receptor subunits ( N-methyl- d-aspartate (NMDA) subunits NR1, NR2A, NR2B, and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor subunits GluR1, GluR2, GluR4) were quantified in particulate preparations from rat spinal cord at various ages after birth. We found that all six subunits showed high expression in the early postnatal period, followed by a subsequent decline as the rats matured to adults. The levels of two subunits (NR2A and GluR4) were found to initially increase during the first postnatal week prior to the decline to adult levels. The high levels of expression observed of these subunits in the early postnatal period may have implications for mechanisms of neural injury and cell death in the immature nervous system that involve cation influx through ionotropic glutamate receptors.

Immunobiology of a Synthetic Luteinizing Hormone Receptor Peptide 21–41

Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function.