Induction of T Cell Anergy by Liposomes with Incorporated Major Histocompatibility Complex (MHC) II/Peptide Complexes

Purpose. The aim of this study was to use small unilamellar liposomeswith incorporated MHC II/peptide complexes as a carrier system formultivalent antigen presentation to CD4 + T cells. Methods. Purified peptide pre-loaded MHC II molecules wereincorporated into small unilamellar liposomes and tested for their ability toactivate A2b T cells. The outcome of T cell activation by such liposomesin the absence of accessory cells was tested via flow cytometry and aT cell anergy assay. Results. Provided the presence of external co-stimulation,MHC II/peptide liposomes were able to induce proliferation of the A2b T cellclone. More importantly incubation of these T cells withMHC II/peptide liposomes in the absence of co-stimulation did not induceproliferation, however, a MHC/peptide ligand-density dependent downregulation of the TCR was observed. Interestingly, when T cells afterincubation with the MHC II/peptide liposomes were restimulated withtheir specific antigen in the presence of professional APC, these cellswere anergic. Conclusions. We propose MHC II/peptide liposomes as a novel meansto induce T cell anergy. The possibility to prepare tailor-madeliposomal formulations may provide liposomes with an important advantagefor applications in immunotherapy.     

罗哌卡因乳酸羟基乙酸共聚物微球的制备及体外释药研究

目的:优化罗哌卡因乳酸羟基乙酸共聚物微球制备工艺,并考察其粉粒学特征和体外释药特性。方法:以乳酸羟基乙酸共聚物为载体,采用W/O/W乳剂-扩散溶剂挥发法制备微球,以微球的粒径、药物包封率、载药量及微球形态等重要粉粒学特征为考察指标,通过正交分析试验优化微球制备工艺,并进行体外释药研究。结果:以优化处方制备的制剂,外观光滑圆整,平均粒径为(2.525±0.047)μm,粒径在1.8～5.0μm的占总数的80%以上,载药量(6.067±0.312)%,包封率(58.05±1.169)%。其体外释药曲线可用Higuchi方程拟合,192h累积释药率达82%,t1/2=60.16h。结论:罗哌卡因乳酸羟基乙酸共聚物微球具有明显的缓释性。     

Protective effect of poly(I): Poly(C) against cephaloridine nephrotoxicity in the rat

The effect of poly(I):poly(C) during cephaloridine nephrotoxicity in the rat was determined by measurement of urinary enzyme excretion. Urinary alkaline phosphatase, lactate dehydrogenase and muramidase levels were lower in rats receiving a combination of poly(I): poly(C) and cephaloridine as compared to the levels of these enzymes in the urines of rats receiving cephaloridine alone. Poly(I): poly(C) alone did not cause an elevation of these enzymes and incubation of the enzymes in urine with poly(I): poly(C) did not inhibit the enzyme activity. It is concluded that poly(I): poly(C) acted to protect kidney cells from damage by cephaloridine.     

Novel Approach for Delivery of Insulin Loaded Poly(lactide-co-glycolide) Nanoparticles Using a Combination of Stabilizers

Insulin stability during microencapsulation and subsequent release is essential for retaining its biological activity. The successful delivery of insulin relies on the proper selection of stabilizers in addition to other parameters. Attempts were made to address the problem with a few combination of stabilizers for maintaining the integrity of insulin during formulation and delivery. Insulin loaded nanoparticles with different stabilizers such as pluronic F68, trehalose, and sodium bicarbonate were prepared by the double emulsion evaporation method using two different copolymer ratios of poly(DL-lactide-co-glycolide) (50:50 and 85:15). The presence of stabilizers in the nanoparticles resulted in an increase in the particle size but a reduction of encapsulation efficiency. Insulin release rate was comparatively higher for the batches containing stabilizers when compared with controls for both the copolymer ratios. Also the presence of stabilizers resulted in sustained release of insulin resulting in prolonged reduction of blood glucose levels in streptozotocin induced diabetic rats. From the in vitro and in vivo studies, we concluded that a combination of stabilizers results in beneficial effects without compromising the advantages of delivery systems.     

Preparation and Characterization of Innovative Protein-coated Poly(Methylmethacrylate) Core-shell Nanoparticles for Vaccine Purposes

Purpose This study aims at developing novel core-shell poly(methylmethacrylate) (PMMA) nanoparticles as a delivery system for protein vaccine candidates. Materials and Methods Anionic nanoparticles consisting of a core of PMMA and a shell deriving from Eudragit L100/55 were prepared by an innovative synthetic method based on emulsion polymerization. The formed nanoparticles were characterized for size, surface charge and ability to reversibly bind two basic model proteins (Lysozyme, Trypsin) and a vaccine relevant antigen (HIV-1 Tat), by means of cell-free studies. Their in vitro toxicity and capability to preserve the biological activity of the HIV-1 Tat protein were studied in cell culture systems. Finally, their safety and immunogenicity were investigated in the mouse model. Results The nanoparticles had smooth surface, spherical shape and uniform size distribution with a mean diameter of 220 nm. The shell is characterized by covalently bound carboxyl groups negatively charged at physiological pH, able to reversibly adsorb large amounts (up to 20% w/w) of basic proteins (Lysozyme, Trypsin and HIV-1 Tat), mainly through specific electrostatic interactions. The nanoparticles were stable, not toxic to the cells, protected the HIV-1 Tat protein from oxidation, thus preserving its biological activity and increasing its shelf-life, and efficiently delivered and released it intracellularly. In vivo experiments showed that they are well tolerated and elicit strong immune responses against the delivered antigen in mice. Conclusions This study demonstrates that these new nanoparticles provide a versatile platform for protein surface adsorption and a promising delivery system particularly when the maintenance of the biologically active conformation is required for vaccine efficacy.     

Poly Ⅰ:C抗瘤特性及与顺铂的协同抗瘤作用

本文观察了国产 Poly I∶C 在小鼠的抗瘤特性及与顺铂的协同抗瘤作用。Poly I∶C对 S_(180)、Lewis 肺癌和小鼠肝癌均有明显抑瘤作用。Poly I∶C 在体内和体外均增加 NK、巨噬细胞活性。Poly I∶C 在体外没有明显细胞毒作用。实验提示 Poly I∶C 通过免疫调节产生抗瘤作用。Poly I∶C 与顺铂具有协同抑瘤作用,但没有增加顺铂的毒性。     

Lymphatic Uptake and Biodistribution of Liposomes After Subcutaneous Injection: III. Influence of Surface Modification with Poly(ethyleneglycol)

Purpose. The aim of the present paper was to assess the effect of inclusion of distearoylphosphatidylethanolamine-poly(ethyleneglycol) (DSPE-PEG) into liposomal bilayers on the lymphatic uptake and lymph node localization of liposomes after subcutaneous administration. Methods. [³H]-Cholesteryloleylether labeled liposomes of various composition and sizes were injected s.c. into the dorsal side of the foot of rats. At several time-points after injection, blood levels of liposomes were determined. Lymphatic uptake from the s.c. site of injection and lymph node localization in regional lymph nodes were determined at the end of the 52 h observation period. Results. The results demonstrate that inclusion of DSPE-PEG into several types of liposomes has only a modest effect on lymphatic uptake. Also lymph node localization is only slightly affected by PEG-mediated steric stabilization. Conclusions. Factors other than the presence of a steric barrier are more important in determining lymphatic uptake from the s.c. injection site. The observation that lymph node localization was only slightly affected by PEG-coating strongly suggests that macrophage uptake is not the only important mechanism of lymph node localization of s.c. administered liposomes.     

Effect of Liposome Characteristics and Dose on the Pharmacokinetics of Liposomes Coated with Poly(amino acid)s

Long-circulating liposomes, such as PEG-liposomes, are frequently studied for drug delivery and diagnostic purposes. In our group, poly(amino acid) (PAA)-based coatings for long-circulating liposomes have been developed. These coatings provide liposomes with similar circulation times as compared to PEG-liposomes, but have the advantage of being enzymatically degradable. For PEG-liposomes it has been reported that circulation times are relatively independent of their physicochemical characteristics. In this study, the influence of factors such as PAA grafting density, cholesterol inclusion, surface charge, particle size, and lipid dose on the circulation kinetics of PAA-liposomes was evaluated after intravenous administration in rats. Prolonged circulation kinetics of PAA-liposomes can be maintained upon variation of liposome characteristics and the lipid dose given. However, the use of relatively high amounts of strongly charge-inducing lipids and a too large mean size is to be avoided. In conclusion, PAA-liposomes represent a versatile drug carrier system for a wide variety of applications.     

Genetic polymorphism of C452T (T127I) in human gamma-glutamyl hydrolase in a Japanese population

We investigated the genotype distribution and allele frequency of C452T polymorphism of gamma-glutamyl hydrolase (GGH) gene, which causes the decreased enzymatic activity affecting the efficacy of methotrexate (MTX), in a Japanese population. The polymerase chain reaction-restriction fragment length polymorphism assay was applied to determine the genotype of C452T polymorphism in 269 Japanese healthy individuals. The genotype distribution was as follows: C/C, 89.2% (n=240); C/T, 10.4% (n=28); T/T, 0.4% (n=1). The frequency of C and T allele was 0.944 and 0.056, respectively. The obtained genotype distribution was well agreed with those expected by Hardy-Weinberg equilibrium. The genotype distribution and allele frequency in a Japanese population were found to be similar to those of African-Americans but significantly different from Caucasians. Although the frequency of variant T allele in a Japanese population is not so high as compared to Caucasians, determination of C452T polymorphism of GGH may be useful for monitoring of efficacy and side-effects of MTX for treatment of diseases such as rheumatoid arthritis or childhood acute leukemia. To our knowledge, this is the first report about the examination of C452T polymorphism of GGH in a Japanese population.     

Enhancing Initial Release of Peptide from Poly(d,l-lactide-co-glycolide) (PLGA) Microspheres by Addition of a Porosigen and Increasing Drug Load

The objective of this study was to evaluate formulation variables such as drug load and addition of a porosigen in achieving an increased initial release of peptide from poly(d,l-lactide-co-glycolide) (PLGA) microspheres by altering carrier characteristics. Leuprolide acetate-loaded PLGA microspheres were prepared by a solvent-extraction–evaporation process and were characterized for their drug load (HPLC assay), bulk density (tapping method), size distribution (dynamic light scattering), specific surface area (Brunauer–Emmett–Teller [BET] analysis), surface morphology (scanning electron microscopy), in vitro drug release (at 37°C), and in vivo efficacy (suppression of rat serum testosterone). Increasing the drug load, and adding various amounts of calcium chloride to organic and aqueous phases of the emulsion during processing yielded particles with increased porosity, lower bulk density, higher specific surface area, and accordingly higher initial release. In an animal model, these formulations showed a faster onset of testosterone suppression compared to microspheres without higher drug load or calcium chloride. The approaches employed in this study were found to be effective in avoiding the therapeutic lag phase usually observed with microencapsulated macromolecular drugs.     

Induction of potent and long-lasting CD4 and CD8 T-cell responses against hepatitis C virus by immunization with viral antigens plus poly(I:C) and anti-CD40

Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.     

Tetanus Toxoid Loaded Nanoparticles from Sulfobutylated Poly(Vinyl Alcohol)-Graft-Poly(Lactide-co-Glycolide): Evaluation of Antibody Response After Oral and Nasal Application in Mice

Purpose. Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. Methods. Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. Results. Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 × 10³ (IgG) and 2 × 10³ (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. Conclusions. Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.     

Induced Pluripotent Stem Cells (iPSCs) Provide a Potentially Unlimited T Cell Source for CAR-T Cell Development and Off-the-Shelf Products

Pharmaceutical Research - Chimeric antigen receptor T (CAR-T) cell therapy has been increasingly conducted for cancer patients in clinical settings. Progress in this therapeutic approach is...     

In vitro effect of inosiplex on T lymphocytes. I. Influence on T cells with receptors for IgG (T gamma)

Inosiplex, a complex of inosine and 2-hydroxypropyldimethyl ammonium-4-(acetylamino)benzoate, 1:3 molar ratio, originally developed for antiviral use, is now under wider investigation because of its immunopotentiating properties. This compound can have some actions on T cells at various stages of differentiation, thus promoting an enhancement of their blastogenic responses to varied mitogenic agents (PHA, Con A, PWM, MLC, tetanus toxoid, and viral antigens). Our studies demonstrated that under the influence of inosiplex human peripheral blood T lymphocytes bearing Fc IgG receptors have an augmented receptor avidity for SRBC which result in an increased E active rosette formation, and that T cells preincubated with the drug at the appropriate concentrations express more Fc IgG receptors. Even though T gamma cells exert "in vitro" immunoregulatory properties, the increase in percentage of T gamma lymphocytes do not correlate with a potentiation of the Con A-induced suppressor activity of T cells. Moreover, the lymphocytes treated with the substance in the absence of Con A exert helper functions, increasing the mitogenic responses of the second culture PHA--treated lymphocytes. These data appear to suggest a pro-proliferative inosiplex-induced effect which could mask a concomitant suppressor cell induction.     

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid–polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene—5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.     

Selection of a Ligand-Binding Neutralizing Antibody Assay for Benralizumab: Comparison with an Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Cell-Based Assay

Assessment of anti-drug antibodies (ADAs) for neutralizing activity is important for the clinical development of biopharmaceuticals. Two types of neutralizing antibody (NAb) assays (competitive ligand-binding assay [CLBA] and cell-based assay [CBA]) are commonly used to characterize neutralizing activities. To support the clinical development of benralizumab, a humanized, anti–interleukin-5 receptor α, anti-eosinophil monoclonal antibody, we developed and validated a CLBA and a CBA. The CLBA and CBA were compared for sensitivity, drug tolerance, and precision to detect NAbs in serum samples from clinical trials. The CLBA was more sensitive (27.1 and 37.5 ng/mL) than the CBA (1.02 and 1.10 μg/mL) in detecting NAbs to benralizumab for the polyclonal and monoclonal ADA controls, respectively. With the same polyclonal ADA control, the CLBA detected 250 ng/mL of ADA in the presence of 100 ng/mL of benralizumab, whereas the CBA detected 1.25 μg/mL of ADA in the presence of 780 ng/mL of benralizumab. In 195 ADA-positive samples from 5 studies, 63.59% (124/195) and 16.9% (33/195) were positive for NAb as measured by the CLBA and the CBA, respectively. ADA titers were strongly correlated (Pearson’s correlation coefficient r?=?0.91; n?=?195) with CLBA titers. Moreover, the CLBA titer correlated with CBA percentage inhibition in the CBA-positive samples (Spearman’s coefficient r?=?0.50; n?=?33). Our data demonstrated advantages of the CLBA in various aspects and supported the choice of the CLBA as a NAb assay for the phase III trials.     

Poly(acrylic acid) microspheres loaded with lidocaine: Preparation and characterization for arterial embolization

A new embolic agent, poly(acrylic acid) microspheres (PMs), was synthesized and the cytocompatibility was proved by mouse L929 fibroblast cells. An analgesic drug, lidocaine, was loaded on the PMs to relief pain caused by embolization. PMs and lidocaine loaded microspheres (LMs) were characterized by investigating infrared spectrum, morphology, particle size, and equilibrium water contents (EWC). A series of tests were employed to evaluate the elasticity of PMs, LMs and Embosphere?, including once compression, twice compression, and stress relaxation test. The pressures of PMs and LMs passing through a catheter were measured on line by our new designed device. Drug release was studied with T-cell apparatus. The properties of PMs and LMs were proved to be suitable for embolization. Both PMs and LMs in this study might be potential embolic agents in the future.     

Cytotoxicity in human cancer cells and mitochondrial dysfunction induced by a series of new copper(I) complexes containing tris(2-cyanoethyl)phosphines

Over the last few years a lot of research has been done to develop novel metal-based anti-cancer drugs, with the aim of improving clinical effectiveness, reducing general toxicity, and broadening the spectrum of activity. The search for novel metal-based antitumour drugs other than Pt agents includes the investigation of the cytotoxic activity of copper(I/II) compounds. Among these copper agents, particular attention has been recently devoted to hydrophilic copper(I) species bearing phosphines because of their noteworthy stability in aqueous media together with their remarkable in vitro cytotoxic activity. In this study we report on the synthesis, characterization and cytotoxic assays of a series of Cu(I) complexes with tris(2-cyanoethyl)phosphine (PCN) and bis(2-cyanoethyl)phenylphosphine (PCNPh). They were prepared by reaction of [Cu(CH₃CN)₄]⁺ or CuX₂ precursors with the pertinent phosphine in acetone or acetonitrile solutions producing compounds of the following formulation: [Cu(PCN)₂]⁺ 2, [Cu(CH₃CN)(PCN)]⁺ 3, [Cu(X)(PCN)] (X = Cl, 4; Br, 5), and [Cu(PCNPh)₂]⁺ 6. The new copper(I) complexes were tested for their cytotoxic properties against a panel of several human tumour cell lines. Cellular copper uptake rate was correlated with cell growth inhibition in 2008 human ovarian cancer cells. Moreover, copper(I)-PCN complexes were evaluated for their ability to alter the most relevant mitochondrial pathophysiological parameters such as respiration, coupling, ATP-synthetase activity and membrane potential in isolated mitochondria. These data were correlated with changes in mitochondrial membrane potential and production of reactive oxygen species (ROS) in drug-treated 2008 cells.