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.

Adverse immunostimulation caused by impurities: The dark side of biopharmaceuticals.

Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic inflammatory reaction. Biopharmaceuticals that unexpectedly induce an inflammatory response still enter the clinic, even while meeting all regulatory requirements. Impurities (of microbial origin) in biopharmaceuticals are an often-overlooked cause of AI. This demonstrates that the current guidelines for quality control and safety pharmacology testing are not flawless. Here, based on two case examples, several shortcomings of the guidelines are discussed. The most important of these are the lack of sensitivity for impurities, lack of testing for pyrogens other than endotoxin, and the use of insensitive animal species and biomarkers in preclinical investigations. Moreover, testing for the immunotoxicity of biopharmaceuticals is explicitly not recommended by the international guidelines. Publication of cases of AI is pivotal, both to increase awareness and to facilitate scientific discussions on how to prevent AI in the future.

More than 70 years of pyrogen detection: Current state and future perspectives.

In the quality assurance of medical products, tests for sterility are essential. For parenteral pharmaceuticals, avoiding the presence of pyrogens is crucial. These fever-inducing substances (endotoxins and non-endotoxins) are not eliminated by standard sterilisation processes, and are biologically active once in the bloodstream, causing risks to human health, ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory. Over the years, various pyrogen testing methods have been introduced, namely: in the 1940s, the rabbit pyrogen test, which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test, which is an in vitro test (with the haemolymph of the horseshoe crab) that specifically detects endotoxin; and in 2010, the Monocyte-Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test. Due to the ubiquity and biological significance of pyrogens, we are currently further developing the MAT so that it can be used for other applications. More specifically, our focus is on the detection of pyrogenic contamination on medical devices, as well as on the measurement of air quality. In addition, further improvements to permit the use of cryopreserved blood in the MAT, to overcome the limitations in the availability of freshly-drawn blood from human donors, are ongoing.

Looking beyond endotoxin: a comparative study of pyrogen retention by ultrafilters used for the preparation of sterile dialyis fluid.

Sterile single-use ultrafilters are used in dialysis for the preparation of the substitution fluid given to patients undergoing dialysis treatments with high convective fluid removal. The retention of pyrogenic agents by the ultrafilters is crucial to avoiding inflammatory responses. The performance of a new single-use ultrafilter (NUF) with a positively charged flat sheet membrane of relatively small membrane area and large pore size was compared to a reference ultrafilter (RUF) with a hollow fiber membrane. Filter performance was tested with various pyrogen-contaminated dialysis fluids by direct pyrogen quantification and by measuring inflammatory responses in cell-based bioassays. The NUF completely retained oligodeoxynucleotides (ODN), whereas the RUF was fully permeable. Both filters tended to decrease biological activity of DNA in filtered bacterial lysates. The NUF reduced lipopolysaccharides (LPS) and LPS-induced biological activity by 100%, whereas the RUF produced filtrates with low but detectable levels of LPS in most cases. Peptidoglycans (PGN) were fully retained both by the NUF and the RUF. The new ultrafilter retained biologically active ODN, which has not yet been described for any other device used in dialysis, and it showed better or equal retention of LPS and PGN even with a smaller membrane surface and larger pore size.

Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

[Depyrogenation in key manufacturing processes of Reduning injection].

OBJECTIVE: To investigate the effect of removing bacterial endotoxin in the key processes of Reduning injection. METHOD: The content of bacterial endotoxins was detected by kenitic-turbidimetry and the removal efficacy was studied before and after using 0.8% of activated carbon and ultrafiltration with molecular weight cut-off of 10 x 10(3). RESULT: The adsorption rate of bacterial endotoxins was 78.7% by using activated carbon, while the removal efficacy of bacterial endotoxins was 99.6% with ultrafiltration membrane at cut-off molecular weight 10 x 10(3). CONCLUSION: The key technology can effectively guarantee the safety of Reduning injection.

[Effect on elimination of pyrogen and effectual components of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials].

OBJECTIVE: To study the elimination effect of bacterial endotoxins and the transmittance of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Panax notoginseng saponins solution before and after using the ultrafiltration. The change of the contents of active components was examined by HPLC,using notoginsennoside R1, ginsennoside Rg1, ginsennoside Rb1 and ginsennoside Rd as the mark components. RESULTS: The removal rate of bacterial endotoxin fell along with the increasing of membrane aperture. The removal rate was 20. 69% by ultrafiltration membranes of 100 KDa with polysulfone material,less than those of other ultrafiltration membranes with polysulfone material. But the removal rate of bacterial endotoxin by E membranes of blend materials was higher than those of other ultrafiltration membranes with polysulfone material. The contents of active components filtered by E membranes of blend materials was more than that of ultrafiltration membranes of 100 KDa with polysulfone material. CONCLUSION: The applicability of ultrafiltration membranes of large cut-off molecular weight and blend materials of effectual component in Panax notoginseng saponins and elimination of pyrogen is good.

Pyrogenic activity of air to characterize bioaerosol exposure in public buildings: a pilot study.

AIMS: The aim of our study was to investigate indoor air quality (IAQ) by comparing pyrogen concentration and microbiological contamination in offices in public buildings. METHODS AND RESULTS: Air samples were collected during cold and warm seasons in 39 offices in four European cities. Pyrogens were measured by the in vitro pyrogen test (IPT), moulds and bacteria by classical microbiology. In 92% of the investigated offices, pyrogen and microbial contaminations were below 150 EEU m(-3) and 10(3) CFU m(-3), respectively, whilst in 75%, moulds did not exceed 10(2) CFU m(-3). CONCLUSIONS: The IPT is a rapid, reliable tool for measuring pyrogens that could be used as an 'early warning' indicator of IAQ. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on pyrogenic compound detection in offices using IPT, which could serve for developing future indoor air guidelines.

[Effect of affinity medium and solution conditions on endotoxin removal from protein solutions].

Endotoxin removal is essential for the safety of biological products. To remove endotoxin efficiently, we used polymyxin B (PMB) affinity adsorbent to remove endotoxin from protein solutions by static adsorption. We studied the effects of spacer length and ligand density of the affinity adsorbent, pH, salt type and concentration, protein type and concentration, endotoxin concentration, and additive on endotoxin removal and protein recovery. Endotoxin content and protein concentration were determined by test and Lowry assay respectively. The results showed that PMB affinity adsorbent had high capacity, high adsorption speed, high removal efficiency and good reusability. In addition, ligand density, pH, salt concentration and the isoelectric point and hydrophobicity of protein all had remarkable influence on the endotoxin removal. Under the optimal conditions, the recoveries of hemoglobin, human serum albumin and lysozyme were 87.2%, 73.4% and 97.3%, respectively, and the corresponding endotoxin removal rates 99.8%, 97.9% and 99.7%, respectively. This study illustrated the effects of solution conditions on the efficiency of endotoxin removal and protein recovery, and would provide useful reference for the efficient removal of endotoxin from biological products.

[Biological activity of native and modified lipopolysaccharides of Pragia fontium].

Modified lipopolysaccharides (LPS) of Pragia fontium were obtained with germanium complexes (IV) of 2-aminobenzoylhydrazon of salicylic aldehyde (2-NH2-H2Bs), 2-hydroxybenzoylhydrazon salicylic aldehyde (2-OH-H2Bs) and nicotinoylhydrazon of salicylic aldehyde (H2Ns). The modification of LPS was confirmed by IR spectroscopy. Comparative investigations of pyrogenic activity of native and modified LPS showed, that only P. fontium 20125 LPS, modified by germanium complexes with 2-hydroxybenzoylhydrazon of salicylic aldehyde (2-OH-H2Bs) has lost the pyrogenic activity. In the homological reactions of double immunodiffusion in agar it was shown that all modified LPS unlike the native ones lose completely antigenic activity.

Removal of immune-stimulatory components from surfaces by plasma discharges.

Immune-stimulating microbiological components like lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan bound onto surfaces lead to severe problems when brought in contact with the organism via surgical instruments or implants. We have shown, in recent studies, that it is possible to detect different immune-stimulating components directly on the surface, via an indirect detection method, using human whole-blood and the monocyte reaction to measure the inflammatory mediator release (IL-1beta) by ELISA. With regard to the inactivation of pyrogenic substances, we present a method based on the application of a low-pressure microwave plasma discharge working at low temperatures. We found a fast (10 s to a few minutes) removal rate of the immune-stimulating competence for LPS, LTA and zymosan. To mimic the bacterial cell-wall, LPS in combination with muramyl dipeptide was employed and the decreasing rate of the inflammatory signal did not differ from pure LPS.

High sensitivity pyrogen testing in water and dialysis solutions.

BACKGROUND: The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. METHODS: In this study we adapted the IPT to the testing of dialysis solutions. RESULTS: Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. CONCLUSIONS: This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.

[Study on pyrogen in natural and cultivated edible mushrooms].

We examined endotoxin and pyrogen contents in several kinds of natural and cultivated edible mushrooms, as well as some cultivated vegetables. According to the Japanese Pharmacopoeia, 14th Ed., two types of endotoxin (gel-clot Limulus amebocyte lysate) test and the pyrogen test were performed using natural edible mushrooms collected in Aichi Prefecture and cultivated mushrooms and vegetables purchased at a market. The endotoxin contents of natural mushrooms were apparently higher than those of cultivated mushrooms or vegetables. The endotoxin contents in the cultivated mushrooms were slightly higher than those in the vegetables. Similar results were obtained in the pyrogen test.

C-reactive protein in patients on chronic hemodialysis with different techniques and different membranes.

In hemodialysis patients, C-reactive protein (CRP), an acute-phase reactant, is a sensitive and independent marker of malnutrition, anemia, and cardiovascular mortality. The aim of the present study was to evaluate CRP levels in plasma samples from long-term hemodialysis patients on different extracorporeal modalities and dialyzed with different membranes, at baseline and after 6 months. Two hundred and forty-seven patients were recruited in eight hospital-based centers. All patients had been on their dialytic modality for at least 3 months and were prospectively followed in their initial dialytic modality for 6 months. Patients were treated with conventional bicarbonate dialysis (N = 127) or hemodiafiltration (N = 120). Patients treated with conventional bicarbonate dialysis were dialyzed with different membranes: Cuprophane (N = 51), low-flux cellulose modified membrane (N = 37) and synthetic membranes (N = 39). Hemodiafiltration was performed in post-dilution mode with polysulfone (N = 66) and polyacrylonitrile (N = 54) membranes. Analysis of baseline CRP values in the clinically stable patients showed that an unexpectedly high proportion (47%) of the patients had CRP values higher than 5 mg/l (upper limit in normal subjects). The mean +/- S.D. CRP values were significantly higher (P < 0.05) in hemodiafiltration with infusion volumes < 10 l per session (14.6+/-3.1 mg/l) than in standard hemodialysis (5.1 +/- 2.1 mg/l) and hemodiafiltration with infusion volumes > 20 l per session (4.9 +/- 2.1 mg/l). These values did not significantly change after 6 months of follow-up. Concerning the membranes, the highest levels of CRP were observed in patients dialyzed with Cuprophane with a significant increase from 15.1 +/- 3.6 to 21.2 +/- 3.1 mg/l after 6 months (P < 0.05); a significant reduction of CRP levels was observed in patients dialyzed with polysulfone in the same follow-up period (from 13.5 +/- 2.9 to 8.1 +/- 2.4 mg/l; P < 0.05). The CRP increase following low volume HDF can be related to back-filtration of bacterial derived contaminants.; moreover, an important effect on CRP of the hemodialysis membrane is observed and new synthetic membranes can be used to decrease these levels.

Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

[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.

Comparative evaluation of pyrogens tests in pharmaceutical products

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a b-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.

Comparison of the reactivity of human and rabbit blood towards pyrogenic stimuli.

A comparison between humans and rabbits was performed based on stimulation of whole blood with well-known pyrogens from Gram-negative and Gram-positive bacteria, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. The reactivity was measured as release of IL-1 beta and IL-8 by ELISA. The reactivity of the two species towards LPS was found to be similar, whereas their reactivity towards LTA differed considerably. Differences between the levels of IL-1 beta and IL-8 release were observed in both species. This finding suggests that the In vitro Pyrogen Test (IPT) which uses human blood to detect contaminations, e.g. of injectable drugs, might predict the human reaction to the contamination better than the "gold standard" rabbit pyrogen test.

Characterization of a novel staphylococcal enterotoxin-like superantigen, a member of the group V subfamily of pyrogenic toxins.

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.