A novel alternative for pyrogen detection based on a transgenic cell line.

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted 'reduction, replacement and refinement' principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

High molecular pyrogens present in plant extracts interfere with examinations of their immunomodulatory properties in vitro.

The widely accepted strategy to justify the use of medicinal plant extracts in diseases with inflammatory background is their examination on in vitro models using immune cells. It is also a key initial step of research for active principles, which could be then isolated and tested on more advanced models, becoming new pharmacologically active lead molecules. The crucial aspect which has not been so far addressed in this context, is the presence of pyrogens in plant preparations. The aim of this study was the examination of pyrogens interference with in vitro evaluation of anti-inflammatory activity of plant extracts using human primary neutrophils model together with introduction of effective method of interfering factors elimination. The obtained results showed that chosen plant extracts contained pyrogens, which were responsible for concentration-dependent stimulation of pro-inflammatory cytokines production by human neutrophils in vitro in the same extent as LPS did. The ultrafiltration method was successfully applied for pyrogens elimination, which effectiveness was confirmed using LAL test. The determined interference of pyrogens implies the necessity of their consideration and removal when in vitro studies include direct addition of plant extracts to the cell culture, what can be obtained by ultrafiltration, which does not affect extract composition.

Engineered Pyrogenic Materials as Tools to Affect Arsenic Mobility in Old Mine Site Soil of Mediterranean Region.

The application of pyrogenic materials in immobilization processes of metalloids represents a burning issue in environmental and waste applications and management. The main objective of this study was to characterize the effect of biomass pretreatment by Cu, Fe and Mg blending and pyrolysis temperature on As sorption efficiency as a model of anionic metalloids from model solutions and As immobilization in old mine soil by pyrogenic materials. The physico-chemical characterization of engineered materials produced in slow pyrolysis process at 400 and 700°C from metal-blended hard wood chips (30% w/w) showed increasing of surface areas (1.4-1.8-fold), changes in pH, and more than 50% decrease in total C content. The batch sorption processes of As ions by Cu-modified pyrogenic materials (CuPM), Fe-modified pyrogenic materials (FePM), and Mg-modified pyrogenic materials (MgPM) showed increasing uptake in order CuPM700 (Qmax 2.56 mg g-1) < CuPM400 (Qmax 3.88 mg g-1) < FePM700 (Qmax 5.90 mg g-1) < MgPM700 (Qmax 7.42 mg g-1) < MgPM400 (Qmax 9.59 mg g-1) < FePM400 (Qmax 10.55 mg g-1). Engineered pyrogenic materials produced at 400°C showed higher immobilization effect on soluble As in soil pore water of old mine site soil from Mediterranean area. FePM400 reduced mobility of arsenic > 3.2 times and MgPM400 > 5 times compared to control. Promising pyrogenic material MgPM400 showed immobilization effect also on additional heavy metals (Cd, Cu, Fe, Mn, Pb, Sr, Zn) present in studied soil.

A Novel Reporter Gene Assay for Pyrogen Detection.

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signaling molecule that causes the excessive secretion of various pyrogen-induced pro-inflammatory factors. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. The RGA could detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 µg/ml, and 1 µg/ml, respectively. The method had good precision and accuracy and could be applied to many molecules (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, Haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, group A and group C meningococcal conjugate vaccine, diphtheria, tetanus, pertussis [acellular, component], poliomyelitis [inactivated] vaccine, and imject alum adjuvant). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.

Purification of ADCs by Hydrophobic Interaction Chromatography.

Antibody-drug conjugate (ADC) in vitro potency has been shown to be dependent on drug load, with higher drug load providing lower IC50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance, dose-limiting toxicity, etc. Developing a preparative HIC process for ADC purification to isolate species with a specific drug loading involves several steps including conjugation optimization, resin selection, solubility studies gradient screening, and step gradient development (buffer selection). In this chapter, the rationale and general considerations for developing a preparative hydrophobic interaction chromatography (HIC) method are described for isolation of an example ADC with specific drug load, e.g., two monomethyl auristatin E (MMAE) payloads (E2).

Self-Sterilizing Laser-Induced Graphene Bacterial Air Filter.

Nosocomial infections transmitted through airborne, droplet, aerosol, and particulate-transported modes pose substantial infection risks to patients and healthcare employees. In this study, we demonstrate a self-cleaning filter comprised of laser-induced graphene (LIG), a porous conductive graphene foam formed through photothermal conversion of a polyimide film by a commercial CO2 laser cutter. LIG was shown to capture particulates and bacteria. The bacteria cannot proliferate even when submerged in culture medium. Through a periodic Joule-heating mechanism, the filter readily reaches >300 °C. This destroys any microorganisms including bacteria, along with molecules that can cause adverse biological reactions and diseases. These molecules include pyrogens, allergens, exotoxins, endotoxins, mycotoxins, nucleic acids, and prions. Capitalizing on the high surface area and thermal stability of LIG, the utility of graphene for reduction of nosocomial infection in hospital settings is suggested.

A comparison of pyrogen detection tests in the quality control of meningococcal conjugate vaccines: The applicability of the Monocyte Activation Test.

The meningococcal C conjugate vaccine (MenCC) is an interesting model with which to test the efficacy of the Monocyte Activation Test (MAT) as an alternative method of pyrogen testing in the quality control of vaccines. The MenCC that has been produced by Bio-Manguinhos in Brazil is in the final development stage, and, as recommended in the guidelines for MenCC production, its pyrogen content must be determined by using the Limulus Amoebocyte Lysate (LAL) assay and the Rabbit Pyrogen Test (RPT). This represents an ideal opportunity to compare LAL and RPT data with data obtained by using a MAT system with cryopreserved whole blood and IL-6/IL-1ß as marker readouts. In order to assess the compatibility of the MAT with MenCC, endotoxin and non-endotoxin pyrogen content was quantified by using MenCC samples spiked with lipopolysaccharide (LPS), lipoteichoic acid or zymosan standards. The presence of the aluminium-based adjuvant interfered with the MAT, increasing the readout of IL-1ß in LPS-spiked MenCC batches. This infringed the product-specific validation criteria of the test, and led to IL-6 being chosen as the more suitable marker readout. No pyrogenic contaminants were identified in the MenCC batches tested, demonstrating consistency among the different systems (MAT, RPT and the LAL assay). In conclusion, the introduction of the MAT during MenCC development could contribute to the elimination of animal tests post-licensing, ensuring human protection based on an effective non-animal based method of quality control.

Preclinical safety and efficacy evaluation of 'BioCaS' bioactive calcium sulfate bone cement.

A new bioactive calcium sulfate-based formulation (named 'BioCaS') has been developed for bone filler applications. This is a self-setting injectable cement where the preset form comprises bassanite obtained from the uniform submicron-sized precursor crystals of gypsum, modified with hydrogen orthophosphate ions. The results of the safety and efficacy evaluation of BioCaS cement, done as per the International Standards and guidelines, are presented in this paper. The study plan consisted of in vitro screening tests of cytotoxicity and haemolysis and in vivo biocompatibility evaluation, including an acute systemic toxicity test (in mice), an intracutaneous reactivity test (in rabbits), a pyrogen test (in rabbits) and a maximization sensitization test (in guinea pigs). The efficacy of the material in healing bone defects was investigated by implanting it in artificially created defects in rabbit femora, with clinically established hydroxyapatite porous ceramic as the control, followed by histological analysis at 12, 26 and 52 weeks. Set BioCaS cement consisted of hydrogen orthophosphate incorporating low-dimensional gypsum crystal lattices, the bioactivity of which has been identified by immersion in simulated body fluid. BioCaS was proved to be non-cytotoxic and non-haemolytic in the screening tests. In the live/dead assay, human osteoblast-like human osteosarcoma cells adhered well and spread on the surface of the material, attaining typical morphology and affirming the bone cell compatibility of the material. In the biocompatibility evaluation there were no acute systemic effects and the material proved non-pyrogenic. There was no intracutaneous erythemic or oedematous reactivity and no hypersensitivity observed in the Magnusson-Kligman method. The material satisfied the biocompatibility requirements. Bone implantation study revealed BioCaS to be osteoconductive and its efficacy of healing the experimental bone defects in rabbit femora is on a par with that of hydroxyapatite ceramic. The material resorbed at a pace matching that of new bone formation. This property of osteotransductivity will help the defect to heal and gain strength faster.

Hemocompatibility testing according to ISO 10993-4: discrimination between pyrogen- and device-induced hemostatic activation.

Next to good hemocompatibility performance of new medical devices, which has to be tested according to the ISO 10993-4, the detection of pyrogen-contaminated devices plays a pivotal role for safe device application. During blood contact with pyrogen-contaminated devices, intense inflammatory and hemostatic reactions are feared. The aim of our study was to investigate the influence of pyrogenic contaminations on stents according to the ISO 10993-4. The pyrogens of different origins like lipopolysaccharides (LPS), purified lipoteichoic acid (LTA) or zymosan were used. These pyrogens were dried on stents or dissolved and circulated in a Chandler-loop model for 90 min at 37°C with human blood. Before and after circulation, parameters of the hemostatic system including coagulation, platelets, complement and leukocyte activation were investigated. The complement system was activated by LPS isolated from Klebsiella pneumoniae and Pseudomonas aeruginosa and by LTA. Leukocyte activation was triggered by LPS isolated from K. pneumoniae, LTA and zymosan, whereas coagulation and platelet activation were only slightly influenced. Our data indicate that pyrogen-contaminated devices lead to an alteration in the hemostatic response when compared to depyrogenized devices. Therefore, pyrogenicity testing should be performed prior to hemocompatibility tests according to ISO 10993-4 in order to exclude hemostatic activation induced by pyrogen contaminations.

Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test.

Threats of pyrogenicity were discovered more than a century ago. Measures to determine the safety of parenterals and, more recently, medical devices and cell therapies for human use have been in place for 70 years. Currently, there are three testing possibilities available: the Rabbit Pyrogen Test, the Limulus Amebocyte Lysate test (Bacterial Endotoxin Test), and test systems using human whole blood or human monocytes, called Monocyte Activation Test (MAT). The MAT is based on the human fever reaction and thus most closely reflects the human situation. Unfortunately, regulations and testing guidelines are not fully harmonized, despite formal international validation. Furthermore, data showing that the MAT is capable of covering the totality of possible pyrogens relevant to humans were not included in the MAT validations of the last decade. For this review we collate evidence from published literature, unpublished data of our own, and results from the international validation study to show that there is overwhelming scientific evidence to conclude that the whole blood MAT reliably detects non-endotoxin pyrogens. Therefore, further validation exercises do not seem warranted.

[Depyrogenation test regarding inox and glass containers in the preparation of parenteral nutrition mixtures]. / Test de dépyrogénation des conteneurs en inox et en verre pour la préparation des mélanges pour nutrition parentérale.

INTRODUCTION: The preparation of parenteral nutrition mixture (PNM) in an open chamber requires the use of intermediate containers sterile and non-pyrogenic. A sterilization of containers by moist heat in large autoclaves is the suitable method. However, sterilization by moist heat is not a depyrogenation method. In our study, we report the validation of a sterilization and depyrogenation method for containers by dry heat using a convection oven. MATERIALS AND METHODS: Sterilization and depyrogenation of material by dry heat have been audited by the reduction of at least three logarithms of original endotoxin rate. The containers were initially artificially contaminated with a suspension of endotoxin for 16 hours. Contaminated containers were placed in an oven with revolving heat at 250 °C for 1 hour. After treatment with dry heat, the residual endotoxin levels in the containers were determined by a kinetic chromogenic method. RESULTS: After treatment with dry heat, the average log reductions of endotoxin levels were respectively, for glass and steel containers, 4.78 ± 0.07 and 4.87 ± 0.03. DISCUSSION AND CONCLUSION: The present validation study confirms the effectiveness of treatment with dry heat for sterilization and depyrogenation of glass and steel containers. This method of sterilization and depyrogenation meets the microbiological quality requirements for the preparation of MNP.

Detection of pyrogenicity on medical grade polymer materials using rabbit pyrogen, LAL and ELISA method.

The objective of the study is to detect the pyrogenicity of five medical grade gelatinous polymer materials, intended for the manufacturing of capsule for pharmaceutical applications, by an indigenously developed ELISA, LAL and rabbit pyrogen assays. The ELISA methodology includes the incubation of the sample extract with blood from a healthy donor at 37°C. Any pyrogen present in the extract induces the IL-1ß which can be determined by ELISA. The rabbit pyrogen and LAL assays were performed as per standards. The result of the ELISA method indicated that all the materials extract induced high level of IL-1ß as a marker for pyrogenicity. The rise in temperature of rabbit pyrogen was above 0.5°C in all materials extract. LAL assay induced an endotoxin level above 0.5EU. All the five polymer materials were found pyrogenic in all the assays. The ELISA method is very sensitive because the lowest limit of detection was 10pg/ml endotoxin. Hence it can be concluded that the ELISA method will be an added advantage for the quality control release of a batch of medical products and improving the existing methodologies in the context of reduction and replacement in the use of animal models.

[Biological activity of native and modified lipopolysaccharides Rahnella aquatilis].

The results of the comparative toxicity studies of native lipopolysaccharide (LPS) of Rahnella aquatilis 96U037 and that modified by tin complexes indicates that, due to the modification of LPS by tin complex with benzoylhydrazone of 4-dimethylaminobenzaldehyde, a decrease of its toxicity was observed that led to disappearance of the pyrogenic effect. All obtained derivatives lost completely the antigenic activity both in homologous and heterologous systems which may indicate to the interaction of modifying complexes with certain groups being the components of antigenic determinant. The paper is presented in Ukrainian.

Sterilized recombinant spider silk fibers of low pyrogenicity.

We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.

A novel nasal delivery system of a Chinese traditional medicine, Radix Bupleuri, based on the concept of ion-activated in situ gel.

The purpose of this study was to develop a nasal in situ gel system for Radix Bupleuri employing gellan gum as a polymer. Radix Bupleuri in situ gel containing 0.2 mL essential oil extracted from 450 g Radix Bupleuri, proper solubilizing agents and gellan gum (0.5% w/v) was prepared and characterized. The antipyretic effect produced by in situ gel formulation was investigated in fevered rabbits and compared to an intranasal solution. The resulting in situ gel was a clear and light-yellow liquid, with viscosity of 346 mPa x s and caproic acid content of 1.31 +/- 0.01 mg/mL. Intranasal administration of this preparation to fevered rabbits decreased body temperature markedly (1.1 degree C at the doses of oil from 1.5 g Bupleuri/body) and the effect could last for 20-30 h. The results suggest that Radix Bupleuri in situ gel can be greater effective than the solution in the treatment of fever.

Crystal structure of the streptococcal superantigen SpeI and functional role of a novel loop domain in T cell activation by group V superantigens.

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved approximately 15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the alpha3-beta8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of SpeI at 1.56 A resolution. Although the alpha3-beta8 loop in SpeI is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of SpeI indicates that TCR beta-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the alpha3-beta8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on Vbeta-skewing analysis of human T cells activated with SpeI and structural models, we propose that the alpha3-beta8 loop is positioned to form productive intermolecular contacts with the TCR beta-chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by SpeI, and likely all other group V SAgs.

Persistent organic pollutants and sedimentary organic matter properties: a case study in the Kishon River, Israel.

The Kishon River, the second largest coastal river in Israel, has been severely polluted for several decades. Sediments from upstream and downstream sites of the river were analyzed, lipid-extracted and evaluated for phenanthrene uptake. Total polycyclic aromatic hydrocarbon (PAH) concentration in downstream sediments was 299 microg kg(-1), consisting mostly of petrogenic-derived PAHs. Downstream sedimentary lipids were found to be dominated by fresh and decomposed petroleum-derived n-alkanes. The total PAH concentration in upstream sediments was 173 microg kg(-1), consisting mostly of pyrogenic-derived PAHs, whereas lipids from these sediments were mostly vegetation-derived. Spectroscopic data suggested an exceptionally high aromatic content in downstream humic acid, which originated from PAHs attached to its structure. Sorption data suggested that upstream sedimentary cuticle-derived lipids function as a sorption domain, while downstream sedimentary lipids, consisting of shorter-chain-length petroleum-derived alkanes, compete with phenanthrene for sorption sites.

Release of TNF-alpha and IL-1beta from porcine brain endothelium corresponds to the pyrogenic potential of three marketed formulations of amphotericin.

BACKGROUND: Formulations of amphotericin include a deoxycholate suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (L-Amph). Fever is most frequent with d-Amph, intermediate with Ablc, and lowest with L-Amph. OBJECTIVE: To determine if the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1) from brain endothelium corresponds to the incidence of amphotericin fever. RESULTS: Release of TNF-alpha and IL-1beta after L-Amph treatment was similar to negative controls while after d-Amph treatment release was similar to lipopolysaccharide. Ablc treatment produced intermediate pyrogen release.NF-kappaB expression, a transcriptional regulator for TNF-alpha and IL-1beta genes, corresponded to this secretion pattern. TNF-alpha release was elevated 2 hours (p = 0.0021) after treatment while significant elevations in IL-1beta required 6 hours (p = 0.0009). CONCLUSION: Results from this in vitro study suggest that amphotericin fever may be directly mediated by brain endothelium. These experiments also suggest that amphotericin fever is initially mediated by TNF-alpha.

[Characterization of the lipopolysaccharide from Rahnella aquatilis 1-95].

The lipopolysaccharide from the freshwater bacterium Rahnella aquatilis 1-95 has been isolated and investigated for the first time. The structural components of the lipopolysaccharide molecule: lipid A, core oligosaccharide, and O-specific polysaccharide were isolated by mild acidic hydrolysis. In lipid A, 3-hydroxytetradecanoic and tetradecanoic acids were found to be the predominant fatty acids. In the core oligosaccharide, galactose, arabinose, fucose, and an unidentified component were shown to be the major monosaccharides. The O-specific polysaccharide consists of a regularly repeating trisaccharide unit with the acyl and phosphate following structure: [structure: see text] groups have been shown to be responsible for the toxic and pyrogenic properties of the lipopolysaccharide of R. aquatilis.