Granulocyte and monocyte adsorptive apheresis induces apoptosis of neutrophils and release of a novel chemoattractant for desensitization of interleukin-8 response - In vitro and in vivo results.

OBJECTIVE: Apoptotic cells participate in maintenance of homeostasis of the adaptive immune system. Granulocyte/monocyte adsorptive apheresis (GMA) performed with an Adacolumn has been shown to have clinical efficacy together with immunomodulatory effects for immune-mediated disorder cases, such as inflammatory bowel disease (IBD) or psoriatic arthritis. Although induction of apoptosis in neutrophils by GMA has been observed, the detailed mechanism remains unclear. METHODS: To focus on phagocytosis-induced cell death (PICD) that induces apoptotic neutrophils, a comparative study utilizing a GMA-carrier (leukocyte adsorbing carrier for Adacolumn) and yeast particles was performed with in vitro and in vivo examinations. RESULTS: L-selectin was significantly (P = 0.0133) shed, reactive oxygen species (ROS) production was promoted (P = 0.0019), and apoptosis induction was enhanced (P = 0.0087) by peripheral blood co-cultured with the GMA-carrier or yeast particles as compared to incubated blood alone. Furthermore, degranulation of myeloperoxidase, elastase, and lactoferrin was increased by both treatments, while the highest level of interleukin-1 receptor antagonist release was found with GMA-carrier treatment (P = 0.0087) as compared to the yeast particles. Plasma from blood treated with the GMA-carrier showed chemotactic activity, and suppressed neutrophil migration to IL-8 and LTB4. In vivo results demonstrated that neutrophil chemotaxis to IL-8 was desensitized (P = 0.0078) in rabbits following GMA apheresis, while CXCR1 and CXCR2 expressions in neutrophils were reduced by exposing peripheral blood to the GMA-carrier. CONCLUSIONS: GMA may regulate the immune system in patients with an immune-mediated disorder by inducing a biological response of neutrophils with a PICD-like reaction.

Simultaneous in situ visualization and quantitation of dual antigens adsorbed on adjuvants using high content analysis.

Aim: Traditional antigenicity assay requires antigen recovery from the particulate adjuvants prior to analysis. An in situ method was developed for interrogating vaccine antigens with monoclonal antibodies while being adsorbed on adjuvants. Materials & methods: The fluorescence imaging-based high content analysis was used to visualize the antigen distribution on adjuvant agglomerates and to analyze the antigenicity for adsorbed antigens. Results: Simultaneous visualization and quantitation were achieved for dual antigens in a bivalent human papillomavirus vaccine with uniquely labeled antibodies. Good agreement was observed between the in situ multiplexed assays with well-established sandwich enzyme-linked immunosorbent assays. Conclusion: The streamlined procedures and the amenability for multiplexing make the in situ antigenicity analysis a favorable choice for in vitro functional assessment of bionanoparticles as vaccine antigens.

An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses.

A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

Effect of hemoadsorption during cardiopulmonary bypass surgery - a blinded, randomized, controlled pilot study using a novel adsorbent.

BACKGROUND: Cardiopulmonary bypass (CPB) surgery initiates a systemic inflammatory response, which is associated with postoperative morbidity and mortality. Hemoadsorption (HA) of cytokines may suppress inflammatory responses and improve outcomes. We tested a new sorbent used for HA (CytoSorb™; CytoSorbents Europe GmbH, Berlin, Germany) installed in the CPB circuit on changes of pro- and anti-inflammatory cytokines levels, inflammation markers, and differences in patients' perioperative course. METHODS: In this first pilot trial, 37 blinded patients were undergoing elective CPB surgery at the Medical University of Vienna and were randomly assigned to HA (n = 19) or control group (n = 18). The primary outcome was differences of cytokine levels (IL-1ß, IL-6, IL-18, TNF-α, and IL-10) within the first five postoperative days. We also analyzed whether we can observe any differences in ex vivo lipopolysaccharide (LPS)-induced TNF-α production, a reduction of high-mobility box group 1 (HMGB1), or other inflammatory markers. Additionally, measurements for fluid components, blood products, catecholamine treatment, bioelectrical impedance analysis (BIA), and 30-day mortality were analyzed. RESULTS: We did not find differences in our primary outcome immediately following the HA treatment, although we observed differences for IL-10 24 hours after CPB (HA: median 0.3, interquartile range (IQR) 0-4.5; control: not traceable, P = 0.0347) and 48 hours after CPB (median 0, IQR 0-1.2 versus not traceable, P = 0.0185). We did not find any differences for IL-6 between both groups, and other cytokines were rarely expressed. We found differences in pretreatment levels of HMGB1 (HA: median 0, IQR 0-28.1; control: median 48.6, IQR 12.7-597.3, P = 0.02083) but no significant changes to post-treatment levels. No differences in inflammatory markers, fluid administration, blood substitution, catecholamines, BIA, or 30-day mortality were found. CONCLUSIONS: We did not find any reduction of the pro-inflammatory response in our patients and therefore no changes in their perioperative course. However, IL-10 showed a longer-lasting anti-inflammatory effect. The clinical impact of prolonged IL-10 needs further evaluation. We also observed strong inter-individual differences in cytokine levels; therefore, patients with an exaggerated inflammatory response to CPB need to be identified. The implementation of HA during CPB was feasible. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01879176, registration date: June 7, 2013.

Effect of Cellulose Acetate Beads on the Release of Transforming Growth Factor-ß.

Transforming growth factor-ß (TGF-ß) is released by activated platelets and induces the differentiation of T-helper 17 from naïve T cells. Contact between blood and cellulose acetate (CA) beads induces cytokine release, although their inflammatory effects on TGF-ß release are unclear. We aimed to clarify the effect of CA beads on the release of TGF-ß in vitro. We incubated peripheral blood with and without CA beads and measured platelets and TGF-ß. Compared with blood samples incubated without beads, the platelet count and amount of TGF-ß significantly decreased in blood samples incubated with CA beads. In conclusion, CA beads inhibited the release of TGF-ß from adsorbed platelets. The biological effects of this reduction of TGF-ß release during platelet adsorption to CA beads need further clarification.

It takes two to tango: Understanding the interactions between engineered nanomaterials and the immune system.

The immune system represents our primary defense system against foreign intrusion, including pathogens as well as particles. In order to understand the potential toxicity of engineered nanomaterials of ever increasing sophistication, it is necessary to understand the sophistication of the immune system with its multiple, specialized cell types and soluble mediators. Moreover, it is important to consider not only material-intrinsic properties of the pristine nanomaterial, but also the acquired, context-dependent 'identity' of a nanomaterial in a living system resulting from the adsorption of biomolecules on its surface. The immune system has evolved to recognize a vast array of microbes through so-called pattern recognition; we discuss in the present review whether engineered nanomaterials with or without a corona of biomolecules could also be sensed as 'pathogens' by immune-competent cells.

Enhanced humoral and cell-mediated immune responses generated by cationic polymer-coated PLA microspheres with adsorbed HBsAg.

Surface-engineered particulate delivery systems for vaccine administration have been widely investigated in experimental and clinical studies. However, little is known about charge-coated microspheres as potential recombinant subunit protein antigen delivery systems in terms of adsorption and related immune responses. In the present study, cationic polymers, including chitosan (CS), chitosan chloride (CSC), and polyethylenimine (PEI), were used to coat PLA microspheres to build positively charged surfaces. Antigen adsorption capacity was enhanced with increased surface charge of coated microspheres. In macrophages, HBsAg adsorbed on the surface of cationic microspheres specifically enhanced antigen uptake and augmented CD86, MHC I, and MHC II expression and IL-1ß, IL-6, TNF-α, and IL-12 release. Antigens were more likely to localize independent of lysosomes after phagocytosis in antigen-attached cationic microsphere formulations. After intraperitoneal immunization, cationic microsphere-based vaccine formulations generated a rapid and efficient humoral immune response and cytokine release as compared with aluminum-adsorbed vaccine and free antigens in vivo. Moreover, microspheres coated with cationic polymers with relatively high positive charges and higher antigen adsorption exhibited strong stimulation of the Th1 response. In conclusion, PLA microspheres coated with cationic polymers may be a potential recombinant antigen delivery system to induce strong cell and humoral immune responses.

Antibody responses in mice to particles formed from adsorption of a murine monoclonal antibody onto glass microparticles.

Immunogenicity of therapeutic monoclonal antibodies (mAbs) is a concern because of the effects of anti-drug antibodies (ADAs) on therapeutic efficacy. Particulate matter has been suggested as a potential contributing factor to immunogenicity. In this study, we investigated ADA levels in mice in response to administration of a murine immunoglobulin G (IgG)2c/κ mAb (mAb1) that was generated in C57BL/6J mice. Particles of mAb1 were formed by adsorbing the protein to glass microparticles. Formulations containing microparticles were administered subcutaneously to mice of either the syngeneic strain, C57Bl/6J, or the allogeneic strain, BALB/c. ADA levels were measured using an isotype-specific enzyme-linked immunosorbent assay method. Whereas BALB/c mice showed strong IgG1 and IgG2b responses against both the particulate and native mAb1 samples, adsorption of mAb1 to particles rendered it slightly more immunogenic than its native, soluble form. In BALB/c mice, immunoglobulin M (IgM) was produced after the first week of injections and then faded gradually. In contrast, C57BL/6J mice showed moderate IgM, IgG1, IgG2b, and IgG3 responses to injections of glass particle-adsorbed mAb1. ADA responses were higher in the allogeneic BALB/c mice, which do not produce mAbs of the IgG2c/κ isotype. Thus, the presence of both foreign epitopes and particles may be important in inducing ADA responses.

Estudo da aplicação da tirosinase vegetal no tratamento de efluentes e verificação da genotoxicidade do efluente tratado em células vegetais / Study on tyrosinase application in the treatment of plant effluent and verification of genotoxicity of the treated effluent into plant cells

Os problemas ambientais relacionados à crescente atividade industrial têm gerado preocupações aos órgãos governamentais e entidades de proteção ambientais, sendo necessários estudos de base que busquem novas alternativas para a recuperação de áreas poluídas e a solução de problemas operacionais relacionados com as técnicas empregadas. Um dos compostos mais encontrados em diversos efluentes industriais, principalmente de indústrias bioquímico-farmacêuticas, é o fenol que provoca um impacto danoso no ambiente devido ao fato de ser um poluente tóxico. O presente trabalho propõe, portanto, avaliar a oxidação e destruição do fenol através da utilização da enzima tirosinase extraída de vegetais, cujos resultados podem ser úteis para o tratamento de outros compostos fenólicos como o hormônio 17β-estradiol ou os que se encontram nos efluentes procedentes da produção de azeite ("águas de vegetação") após a recuperação dos polifenóis importantes como antioxidantes. A tirosinase tem a capacidade de transformar fenóis em produtos menos solúveis em água e menos danosos, permitindo assim uma agressão menor ao ambiente. Outro método de remoção do fenol também foi avaliado utilizando queratina extraída de penas de galinha, quitina e quitosana como bioadsorventes. A atividade enzimática foi determinada espectrofotometricamente com soluções de fosfato de potássio e L-tirosina. Para determinar a concentração de fenol aps a oxidação foi utilizada a Cromatografia Líquida de Alta Eficiência (HPLC). Para estudar a adsorção do fenol aplicou-se o método colorimétrico a partir das soluções de tampão borato, 4 aminoantipirina e ferricianeto de potássio e as absorbâncias foram lidas em espectrofotômetro UV-Vis a 546nm, enquanto a determinação de polifenis presentes na "água de vegetação" foi realizada pelo método Folin-Ciocalteu. A quantidade de tirosinase nas batatas das variedades Ágata e Galette di Bologna apresentou-se muito baixa a ponto de modificarmos a matéria prima para...

Environmental problems related to growing industrial activity have generated concerns among government entities and environmental protection, being necessary more baseline studies that seek new alternatives for the recovery of polluted areas and solution of problems related to the operational techniques employed. One of the compounds most commonly found in many industrial effluents, mainly from biochemical and pharmaceutical industries, is phenol, which causes a detrimental impact on the environment due to its toxicity. Therefore, this work proposes the oxidation and destruction of phenol using the enzyme tyrosinase, extracted from plants, whose results could be useful in the future for the treatment of other phenolic compounds such as 17β-estradiol hormone or those found in the effluent coming from the production for olive oil ("vegetation water") after polyphenols recovery. Such an enzyme has the ability of transforming phenols into products less soluble in water and less dangerous, thereby allowing for a minor impact on the environment. Another method of phenol removal was also evaluated using keratin extracted from chicken feathers, chitin and chitosan as phenol biosorbents. Potassium phosphate buffer and L-tyrosine solutions were used for the determination of enzymatic activity, the high performance liquid chromatography (HPLC) for the determination of phenol concentration after oxidation, and a colorimetric method making use of solutions of borate buffer, 4-aminoantipyrine and potassium ferricyanide as well as reading of the absorbance at 546nm to investigate phenol biosorption, while the presence of polyphenols in "vegetation water" was determined by the Folin-Ciocalteu method. The presence of the tyrosinase in potato varieties Agata and Galette di Bologna was shown to be very low, thus suggesting to change the biosorbent material. So, additional tests were done on apples, kiwi, banana and mushroom, but only the last showed a considerable activity...

Polymethylmethacrylate strengthens antibody response in hemodialysis patients not responding to hepatitis B vaccine: preliminary data.

AIM: Most chronic uremic patients undergoing dialysis show a diminished antibody response to anti-hepatitis B vaccination (anti-HBv) and at this regard high sCD40 serum levels seem to inhibit the immunocompetent response. A simple and feasible technique for removing soluble sCD40 by adsorption is standard bicarbonate dialysis performed by polymethylmethacrylate (PMMA) dialyzers able to remove serum high molecular weight toxins. Aim of the study was to assess whether PMMA dialyzers could enhance the long-term response to anti-HBv in non-responder (NR) ESRD patients. METHODS: The study involved 5 ESRD patients (mean age 65 years) who had been on maintenance thrice-weekly dialysis for 42.4±15.8 months. They had been previously defined NR to anti-HBv for their low antibody titres (<10 IU/L) despite a previous full course of vaccination using protocols recommended for patients on dialysis. After a period of three months of treatment with PMMA dialyzers, keeping all the parameters of dialysis efficiency (the filter surface of 2.1 m(2), KT/V, dialysis time, QB) unchanged compared to previous treatments, all patients received a new booster dose of Fendrix vaccine (20 µg). Hepatitis B markers were checked at month 1, 3, 12 and 18 after administration of the booster dose and returning to the previous membranes used. RESULTS: Three of five patients showed a strong antibody response (>1000 IU/L) that lasted effectively over time even after 18 months despite discontinuation of PMMA. CONCLUSION: In conclusion our preliminary results confirm that PMMA membranes enhance immune response to anti-HBv in NR HD patients.

The mode of actions of the Adacolumn therapeutic leucocytapheresis in patients with inflammatory bowel disease: a concise review.

Patients with active inflammatory bowel disease (IBD) have elevated and activated myeloid leucocytes which infiltrate the colonic mucosa in vast numbers. Myeloid leucocytes such as the CD14(+) CD16(+) monocytes are major sources of tumour necrosis factor (TNF)-α, and therefore selective granulocyte/monocyte (GM) adsorption (GMA) should promote remission or enhance efficacy of pharmacological therapy. However, studies in IBD have reported both impressive as well as disappointing efficacy outcomes, indicating that patients' demographic factors might determine responders or non-responders to GMA. Nonetheless, this non-drug intervention has an excellent safety profile, and therapeutic GMA is expected to expand. In this review, attempts have been made to compile an update on the mode of actions (MoA) of the Adacolumn GMA. The MoA of GMA appears to be more than adsorption of excess neutrophils and TNF-producing CD14(+) CD16(+) monocytes per se. Adsorbed GMs release interleukin (IL)-1 receptor antagonist, hepatocyte growth factor and soluble TNF receptors, which are anti-inflammatory. Additionally, a sustained increase in lymphocytes including the regulatory CD4(+) CD25(+) T cells (lymphocyte sparing) is seen post-GMA. The impact of GMA on the immune system is potentially very interesting in the context of treating immune-related diseases. Future studies are expected to add intriguing insights to the MoA of GMA.

In vivo adsorption of autoantibodies in myasthenia gravis using Nanodisc-incorporated acetylcholine receptor.

Autoantibodies directed against the skeletal muscle acetylcholine receptor (AChR) play a critical role in the pathogenesis of the autoimmune disease, myasthenia gravis (MG). The pathogenic importance of anti-AChR antibodies is substantiated clinically by the often dramatic clinical improvement that follows removal of circulating antibodies utilizing extracorporeal plasma exchange (PE). Unfortunately, the effects of PE are non-specific as immunoglobulins (IgG) and other plasma proteins are removed in addition to anti-AChR IgG. In this study, we have successfully incorporated the AChR protein purified from Torpedo californicus into a Nanodisc (ND) membrane scaffold protein/phospholipid structure. We go on to demonstrate the effectiveness of this ND-AChR complex, administered intravenously, in the in vivo down-modulation of anti-AChR antibodies and subsequent amelioration of clinical disease in the experimental murine model of MG. These results provide proof-of-principle for the in vivo antigen-specific reduction of pathogenic anti-AChR antibodies utilizing ND-AChR particles. Further development of this strategy may provide an effective, antigen-specific, and readily accessible acute therapy for exacerbating MG or myasthenic crisis.

Characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes and their application to the development of impedimetric immunosensors.

This paper describes the characterization of the gold-catalyzed deposition of silver on graphite screen-printed electrodes (SPEs) using electrochemical impedance spectroscopy (EIS) and the application of this approach to the development of impedimetric immunosensors. After applying -0.1 V for 45 s, the amount of electrodeposited silver quantitatively changes the magnitude of two elements of the electrical equivalent circuit: the interface capacitance, Ci, and the charge-transfer resistance, R(CT). Better correlations have been found when considering the R(CT) since this parameter is almost exclusively dependent on the amount of deposited silver under these experimental conditions. This approach has been successfully applied to the development of an impedimetric immunosensor for aflatoxin M(1). The R(CT) magnitude shows good correlation with the amount of gold immobilized on the electrode surface after a competitive assay and thus, with the toxin concentration. This approach has been found sensitive in a wide range of concentrations, from 15 to 1000 free-AFM(1) ppt with a limit of detection of 12 ppt.

Towards an understanding of the adjuvant action of aluminium.

The efficacy of vaccines depends on the presence of an adjuvant in conjunction with the antigen. Of these adjuvants, the ones that contain aluminium, which were first discovered empirically in 1926, are currently the most widely used. However, a detailed understanding of their mechanism of action has only started to be revealed. In this Timeline article, we briefly describe the initial discovery of aluminium adjuvants and discuss historically important advances. We also summarize recent progress in the field and discuss their implications and the remaining questions on how these adjuvants work.

[Coupled plasma filtration adsorption: rationale and perspectives in septic shock]. / Filtration et adsorption plasmatiques couplées: principe et perspectives dans le traitement du choc septique.

PURPOSE: Septic shock is the leading cause of multiple organ failure and mortality in intensive care units. This condition seems to be related to an overproduction of both pro- and anti-inflammatory mediators, leading to an immunological dysfunction. Therapeutic strategies aimed at reducing blood and tissue concentrations of these mediators have been developed. One of these techniques, based on extra-renal filtration, is called "coupled plasma filtration and adsorption". SOURCE: English and French language articles published between 1984 and 2008 were identified through a computerized Medline search. Keywords and MeSH terms used were: "coupled plasmafiltration", "coupled plasma filtration", "adsorption", "blood purification", "cytokines", "hemofiltration", "sepsis". Relevant publications were retrieved and scanned for additional sources. Nine publications were found. PRINCIPAL FINDINGS: Coupled plasma filtration adsorption is an extra-corporeal treatment, based on non-specific cytokine adsorption on a specially designed resin cartridge, coupled with hemofiltration. Experimental studies and the first clinical trials have shown interesting results regarding hemodynamics and respiratory parameters. However, these trials failed to demonstrate any improvement in outcome. CONCLUSION: Additional evidence is needed to clarify the role of this blood purification technique for the treatment of septic shock.

High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles.

Poly(hydroxyethyl methacrylate) (PHEMA) nanoparticles with an average size of 150 nm in diameter and with a poly-dispersity index of 1.171 were produced by a surfactant free emulsion polymerization. Specific surface area of the PHEMA nanoparticles was found to be 1779 m(2)/g. Reactive imidazole containing 3-(2-imidazoline-1-yl)propyl(triethoxysilane) (IMEO) was used as a pseudo-specific ligand. IMEO was attached covalently onto the nanoparticles. PHEMA-IMEO nanoparticles were used for the affinity binding of immunoglobulin-G (IgG) from human plasma. To evaluate the degree of IMEO loading, the PHEMA nanoparticles were subjected to Si analysis by using flame atomizer atomic absorption spectrometer and it was estimated as 64.5 mg/g of polymer. The nanoparticles were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). IgG binding onto the PHEMA nanoparticles was found to be 5.2 mg/g. Much higher binding values (up to 843 mg/g) were obtained for the PHEMA-IMEO nanoparticles. IgG could be repeatedly bound and eluted on PHEMA-IMEO nanoparticles without noticeable loss in the IgG binding capacity.