Pyrogenic Substances Research Articles

Procedure for rapid determination of bile-tolerant gram-negative bacteria in drugs

The European Pharmacopoeia is a single reference study for the quality control of drugs. The official standards published within it provide a legal and scientific basis for quality control in the processes of drugs development, production and marketing. However, the long experience and the multitude of studies have demonstrated that medicinal forms represent unstable dispersed systems and can undergo various manifest or non-manifest changes, leading to drug incompatibilities. Along with the physico-chemical transformations, microbiological transformations can occur with the contamination of the final product with pathogenic, conditionally pathogenic or even saprophytic germs, especially medicines that do not undergo final sterilization or are not aseptically prepared. Microbial contamination of the drug can be an indicator of the production conditions; it can cause the instability of the product through the microbial decomposition of auxiliary substances, the alteration or definitive cancellation of the therapeutic effect of the active substances, the appearance of metabolites with toxic properties, especially those fractions of endotoxins on the wall cell of gram-negative bacteria with the development of allergenic, pyrogenic substances and the induction of the danger of local orgeneral infections. An important role is attributed to the group of bile-tolerant gram-negative bacteria as representatives of microorganisms that are inadmissible in one g/ml for the absence test and for the quantitative test <10 NCP/g/ml (probable number of bacteria), inadmissible in non-sterile medicinal products, herbal medicinal products for oral use and extracts used in their preparation. Due to the diversity of medicinal forms in terms of content, method of application, and therapeutic action, these preparations fulfil the indispensable conditions for the growth of microorganisms, therefore a primary concern is to avoid microbial contamination from the technological phase to the administration phase in order to prevent complications associated with bacterial infections. The given study reveals the testing of the procedure for the rapid determination of inadmissible microorganisms in non-sterile medicinal products and herbal drugs for oral use and the extracts used in their preparation, especially microorganisms of the group Enterobacteriaceae spp.

Investigation of the inhibition of bacteria and endotoxin influx by back filtration through dialyzer membranes.

We investigated the usefulness of assays using human neutrophils for radical production as well as endotoxin (ET) measurement and bacterial culture for endotoxin and bacterial influx by back filtration using dialyzers with different membrane pore diameters. Three types of dialyzers made of cellulose triacetate membrane material with different pore size FB-110EG eco, FB-110U eco, and FB-150UHβ eco were used. A circuit to generate back filtration was created. Back filtrate generated by hydraulic head pressure operation was collected. ET and bacteria were examined. Human neutrophils were exposed to back filtrate (experiments using three different membranes) and contaminated solution, and free radical production was measured using LBP-953 (Berthold) to see if there were differences in production. No bacteria were detected and the concentration of endotoxin was below the detection limit in the back filtrate from the three types of membranes and purified water. Free radical production from neutrophils in the contaminated water was highest at 4,405,750 ± 61,244cpm (counts per minute) (mean ± SD) (P < 0.01 vs FB-150UHβ eco, FB-110U-eco, and FB-110EG eco) followed by that in back filtrate via FB-150UHβ eco, FB-110U-eco, FB-110EG eco. Radical production from neutrophils was thereby higher in the back filtrate of dialyzers with larger pore-size membranes. No bacteria were observed and the concentration of ET was below the detection limit in back filtrate from any of the membranes. However, when the reverse filtrate was exposed to neutrophils, radical production increased along with pore size, suggesting the influx of small pyrogens and other pyrogenic substances.

Detection of Pyrogens in Hormonal Implants Using the LAL Test

The State Pharmacopoeia of the Russian Federation, 14th edition states that implants are a sterile dosage form, and have to be tested for pyrogens. However, it does not provide details on how the test should be performed for this dosage form.The aim of the study was to develop a LAL test procedure for detection of bacterial endotoxins (BE) in implants, using the example of a goserelin product.Materials and methods: BE extraction from the implant surface into an aqueous medium was performed with subsequent BE detection in the extract by turbidimetric kinetic test. The implant was then dissolved in dimethyl sulfoxide, and the obtained goserelin solution was tested for BEs using the gel-clot test.Results: the analysis of the Russian and foreign pharmacopoeial approaches to pyrogenic substance detection in hormonal implants helped to develop two sample preparation procedures for determination of BE content (in the extract and the implant solution). It was demonstrated that the BE content in the water extract did not exceed 0.01 EU/mL and was less than 0.07 EU per implant. The BE content in the implant solution was less than 8.3 EU per 1 mg of goserelin, which is almost eleven-fold lower than the theoretically-derived limit.Conclusions: the authors developed two test procedures for BE detection in hormonal implants using the LAL test, which could be included in manufacturers’ product files. The first procedure involves testing of the water extract from the implant surface and establishes the BE limit of no more than 20 EU/product. The second procedure involves complete dissolution of the implant in dimethyl sulfoxide and establishes the limit of not more than 97.22 EU per 1 mg of goserelin.

HISTOLOGICAL CHANGES IN THE KIDNEY STRUCTURES IN THE DYNAMICS OF THE DEVELOPMENT OF FEVER

Fever is a typical pathological process, which is characterized by a shift in the reference point of thermoregulation to a higher level of body temperature regulation under the influence of pyrogenic substances and includes three stages: temperature rise, temperature standing at a high level and its decrease. The functional state of the kidneys was investigated under the conditions of water diuresis, for which the rats through the stomach using a metal probe were injected with tap water heated to the body temperature of experimental animals in an amount of 5% of the body weight. After water loading in order to obtain plasma, the animals were euthanized by decapitation under light ether anesthesia; blood was collected in tubes with heparin. In blood plasma and urine, the concentration of creatinine was determined by the reaction with picric acid, sodium ions - by the method of photometry of the protein flame by the sulfosalicylic method with the calculation of its excretion. As evidenced by the data obtained, morphological changes during the development of aseptic fever with a hyponatric diet were characterized by: in the first stage (temperature rise) vacuolar degeneration of the epithelium of the proximal tubules and a small-focal character of changes in the properties of proteins with a shift in color to red, in the second stage (temperature at a high level) expansion of the lumen of the Shumlyansky-Bowman capsule and dystrophic changes in the epithelium of the distal tubules, in the third stage (decrease in temperature) by moderate expansion of the lumen of the Shumlyansky-Bowman capsule but not significant dystrophic changes in the epithelium of the proximal tubules. Elucidation of new mechanisms of damage in any way to the cortex of the medulla and the papilla of the kidneys in the conditions of the development of hot water.

Photochemistry after fire: Structural transformations of pyrogenic dissolved organic matter elucidated by advanced analytical techniques

Pyrogenic organic matter (pyOM) is the solid residue left after pyrolysis of organic matter, a process occurring in nature during forest fires. Upon rain events, pyrogenic dissolved organic matter (pyDOM) leaches into the rain water and is transferred to the aquatic environment where it has been identified as an important component of the global carbon cycle. Overall, pyDOM consists of highly condensed aromatic compounds (ConAC) and poorly characterized aliphatic molecules. While the ConAC are thought to be persistent in the environment, a large ConAC accumulation that would match the calculated fluxes of pyDOM formation and export has not been observed. Photo-degradation has been identified as a major pyDOM sink, and it can potentially account for the discrepancy in the global mass balance of pyDOM.Photochemistry of pyDOM has been studied to a certain extent, but neither the structural transformation pathways of pyDOM nor how they relate to pyrolysis temperature and parent feedstocks are well understood. In response to this need, we have examined the products after photo-irradiation of pyDOM derived from a temperature series (250, 400, 525, 650 °C) of oak biochars made under controlled laboratory conditions, which can be considered model pyrogenic substances. Ultrahigh resolution mass spectrometry (FT-ICR-MS), one-dimensional 1H NMR, and two-dimensional 1H-1H total correlation spectroscopy (TOCSY) NMR were used to evaluate the compositional changes that occurred with photo-irradiation.In agreement with previous studies, pyDOM aryl groups were the most photo-labile, and alkyl and O-alkyl groups were photo-produced. A new finding is that polysubstituted olefinic moieties of pyDOM were also photo-degraded. Using TOCSY we identified several small molecular weight compounds whose abundances changed non-linearly across the combustion continuum which suggests that different radical processes occur in the different char leachates. Using mass spectrometric data we observed that pyDOM from oak chars made at lower temperatures (250 and 400 °C), as well as pyDOM from a grass char prepared at 650 °C, photo-transformed to a more hydrogenated and oxygenated composition. In contrast, pyDOM from higher temperature char leachates (525 and 650 °C) transformed predominantly into smaller aliphatic compounds. To explain these differences, we propose a photo-transformation mechanism for ConAC which includes oxygenation of ConAC, ring-opening reactions, and sequential decarboxylation yielding small aliphatic N-rich molecules (marine-like composition). These structures could be further polymerized by reactive oxygen species to form straight-chain alkanes if not consumed by microorganisms. Results from these controlled experiments reveal that the extent of photo-transformation appears to be a function of production temperature and parent feedstock. This study enhances our understanding of ConAC and pyDOM, their terrestrial-to-marine transfers, geochemical stability, and eventual fate in sunlit environments. Here we also show the value of structural characterization of pyDOM, and reveal the many unknowns left to be answered for this complex and analytically challenging type of organic matter.

Ambient concentration of airborne microbes and endotoxins in rural households of southern India

Investigation of microbial communities in the indoor environment is critically important as majority of human life is spent indoors. Airborne microbial flora and their pyrogenic substances can impact human health. They have so far been insufficiently characterized. Here, we quantify bioaerosol concentration and diversity in both indoor and outdoor air of rural households in South India and statistically determine the significant predictor variables influencing them. The median concentration of bacterial and fungal aerosols in indoor air (n = 36 households) was 1031 colony forming units (CFU) m−3 and 580 CFU m−3, respectively, and in outdoor air was 742 CFU m−3 and 680 CFU m−3, respectively. Bacterial species were comprised of the four major phyla Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, and fungal species of Ascomycota, Basidiomycota, and Zygomycota, in both indoor and outdoor air. Multivariable linear regression revealed that outdoor bacterial concentration, the number of house inhabitants, indoor temperature, age of the homes and use of biomass (cow dung or firewood) as cooking fuel were significant predictor variables influencing concentrations of bacteria in the indoor air. The significant predictor variables influencing indoor endotoxin concentrations were indoor bacterial concentration, indoor meteorology (temperature and relative humidity), and use of biomass as cooking fuel. Outdoor fungal concentration was the significant predictor variable influencing fungal concentration in indoor air. Endotoxin concentrations in indoor air ranged from 1.23 to 20.7 EU m−3. Quantitative microbial risk assessment (QMRA) model revealed a probability of infection of resident women from B. anthracis to be 0.0009 to 0.010.

Validation of moist and dry heat processes used for sterilization and depyrogenation during ampoules manufacturing

Pyrogens are fever-producing substances, which are metabolic products of microorganisms and most potent pyrogens are the endotoxins. Depyrogenation can be defined as the elimination of all pyrogenic substances which is an important part of the manufacture of sterile pyrogen free pharmaceutical products. This study compared between sterilization and depyrogenation using moist heat by autoclave, as well as dry heat. After incubation period (24 hours) at 55oC-60oC the color of Geobacillus stearothermophillus spores’ control vial which didn’t sterilized in autoclave changed from blue to yellow color representing the (+ve) result, while the other sterilized vials have no color change (-ve). In addition, all examined endotoxin containing ampoules showed gel formation (+ve) when examined by Limulus Amebocyte Lysate) LAL). All ampoule groups that have autoclaved for periods of 1 hour to 4 hours showed gel formation (+ve) after LAL test, while only the last group which have autoclaving periods of 5 hours (5 cycles) showed no gel formation (-ve). Sterility of vials that contained spores of Geobacillus stearothermophillus after dry heating at 250oC for 30 min showed no color change (-ve). In addition, LAL test for endotoxin containing ampoules after dry heating at 250oC for 30 min showed no gel formation representing -ve pyrogen. So, moist heat is effective in sterilization and not for depydrogenation, whereas, dry heat is effective in sterilization as well as for depyrogenation.

Features of feverish reaction in children with herpes virus infection

One of the most persistent and striking manifestations of herpes virus infections is fever. The feverish reaction is different and depends from the child's age and the effect on the child's body of pyrogenic substances. The main biological and pathogenetic feature of herpesviruses is the long-term presence in the body of the child pathogens, which are present in different tissues. This causes the polymorphism of clinical manifestations and the severity of certain infectious diseases. The aim of our work was the analysis of modern scientific data regarding herpes infection and the observation of children in whom the fever lasted three or more days. We examined 86 children aged 1–7 years with herpesvirus infection in whom fever was observed. Verification of the diagnosis and clarification of herpes virus infection was carried out within 1–3 days from the moment of hospitalization by determining the polymerase chain reaction, Ig M and G to herpes viruses of types 1, 2, cytomegalovirus, Epstein-Barr virus and type 6 and 7 herpes viruses in blood and smears-prints of tonsils. The degree of activity of the inflammatory process in hepatocytes was evaluated by the level of alanine aminotransferases (ALT). To determine the extent of damage to hepatocytes used the indirect method — the Ritz coefficient. We used absolute and relative statistics using Microsoft Excel on an IBM PC/AT computer to evaluate the study results. In our study, 55.8% of children had pyretic fever (temperature above 39°C).The duration of the febrile reaction in 52.32% of patients was acute from 6 to 7 days. In 88.37% of children of the first 3 years of life with herpesvirus infection, a febrile reaction was accompanied by a general intoxication syndrome and manifestations of emotional lability, increased irritability, tearfulness, sleep disturbance, negative reaction to examination, and rapid fatigue. A rash on skin and mucous membranes was observed in 27.9% of children and an increase in the size of liver was recorded in 79.05% of patients.

Integrated Endotoxin Adsorption and Antibacterial Properties of Cationic Polyurethane Foams for Wound Healing.

Gram-negative bacteria, containing toxic proinflammatory and pyrogenic substances [endotoxin or lipopolysaccharide (LPS)], can lead to infection and associated serious diseases, such as sepsis and septic shock. Development of antimicrobial materials with intrinsically endotoxin adsorption activity can prevent the release of bacterial toxic components while killing bacteria. Herein, a series of imidazolium-type polyurethane (PU) foams with antimicrobial properties were synthesized. The content effects of cationic moieties on the antimicrobial activities against Gram-negative Escherichia coli and Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus as well as the endotoxin adsorption property were investigated. The obtained PU foams show slightly higher efficiency against two Gram-negative strains than for Gram-positive one and high absorbability of LPS. A wound healing test using P. aeruginosa and its isolated LPS-treated mice as the models further demonstrated that imidazolium-type PU foams combine both antibacterial and endotoxin adsorption properties and may have a potential application as an antimicrobial wound dressing in a clinical setting.

Changes in the fingerprint of oil derived substances in the sediments of the Bílina River

The aim of this study was to evaluate the changes in the presence of the crude oil derived substances in the Bilina River sediments and to verify explanatory abilities of the used analytical methods. The Bi­lina River has its source on the slopes of Krusne hory and flows to the east through surface pits in an artificial corridor in its upper stream and further continues through the open landscape of the North Bohemian brown coal basin. The primary factor determining the current appearance of the landscape is human activity and anthropogenic embankments. The area along the Bi­lina River is loaded by the opencast brown coal mining and coal processing, petroleum refineries, and chemical plants. There are major industrial operations in the basin of the River Bi­lina which are particularly co-responsible over a long term for high levels of persistent organic pollutants and inorganic pollutants in the river sediments. Twenty samples of river sediments were collected along the Bi­lina River. All samples were subjected to the determination of the total extractable hydrocarbons, the total organic carbon, the total sulphur, and the sum of the polycyclic aromatic hydrocarbons(PAH). According to Herron (1988) most of the analysed samples belong to the shales or Fe-shales where a great possibility of sorption of inorganic or organic pollutants is expected. To distinguish the river segments with the prevailing input of the petrogenic from pyrogenic substances, correlation of the total extractable hydrocarbons and PAH were applied. The ratio fluoranthen / pyren, whose value from the anthropogenically modified flow in the segment Zaluži - Bi­lina did not go beyond 0.5 which is significant for the presence of the emissions from the transport or the petroleum industry, has been proved superior to the other currently recommended ratios of PAH. The evaluation of the archival TEH data did not show any improvement of the water and sediment quality along the Bi­lina river profile.

The Bolognese surgeon Giuseppe Ruggi: how and why the aseptic surgery was introduced in Bologna in the middle half of the XIX century

BackgroundThe first reliable statistic data about perioperatory mortality were published in 1841 by the French Joseph-Francois Malgaigne (1806–1863): he referred to a mean mortality of 60% for amputations and this bad result was to be attributed mainly to hospital acquired diseases. The idea of “hospital acquired disease” although vague, included five infective nosologic entities, which at that time were diagnosed more frequently: erysipelas, tetan, pyemia, septicemia, and gangrene. Nonetheless, the suppuration with pus production was considered from most of the surgeons and doctors of that time as a necessary and unavoidable step in the process of wound healing. During the end of the eighteenth century, hospitals of the main European cities were transforming into aggregations of several wards, where the high concentration of patients created poor sanitary conditions and a consistent increase of perioperatory mortality. In 1865, Lister applied his first antiseptic dressing on the surface of an exposed fracture. These experimental attempts lead to an effective reduction of wound infections respect to the dressing with strings used previously. DiscussionLister's innovations in the field of wound treatment were based on two brand new concepts: germs causing rot were ubiquitarious and the wound infection was not a normal step in the process of wound healing. The concept of antisepsis was hardly accepted in the European surgical world: “Of all countries, Italy is the most indifferent and uninterested in experimenting this method, which has been so favorably judged from the greatest surgical societies in Germany”. This quotation from the young surgeon Giuseppe Ruggi (1844–1925) from Bologna comes from his article where he presented his first experiences on aseptic medications started the previous year in the Surgical Department of Maggiore Hospital in Bologna. In his report, Ruggi described the adopted technique and suggested that the medication should be extended to all the surgical patients of the hospital:“… this is needed to totally remove from the hospital all those elements of infection which grow in the wounds dressed with the old method”. The experimentation of this new dressing for the few treated cases was rigorous and concerned both the sterilization of surgical tools with the fenic acid (5%) and the shaving of the skin. Ruggi also observed that there was no correlation between the seriousness of the wound and its extension or way of healing: when “simple” cases that “should heal without complication” showed fever he often realized that “it was often due to a medication performed without following the rules for an accurate disinfection and dressing”. Ruggi thought that the fever was connected to “reabsorption of pyrogenic substances, which can be removed cleaning and disinfecting the wound” in cases of wounds not accurately dressed and rarely medicated. Frequent postoperative medications of the wound were able to eliminate the fever within 2 h. Ruggi's attitude toward the fine reasoning lead him to introduce the concept of immunodeficiency related to physical deterioration: “… patients treated for surgical disease may sometimes suffer from complications of medical conditions, which initially escape the most accurate investigations… The surgical operation could, in some cases, hold the balance of power”. ConclusionsThe obtained results, published in 1879, appear extremely interesting. As he wrote in 1898, for the presentation of his case record of more than 1000 laparotomies, he had started “… operating as a young surgeon without any tutor, helped only by his mind and what he could deduce from publications existing at the moment …”.

LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS-protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K GFP(uv) < 1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein-LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein-LPS aggregation.

Use of human MonoMac6 cells for development of in vitro assay predictive of adjuvant safety in vivo

Subunit vaccines composed of recombinant or purified antigens have a good safety record but are poorly immunogenic and require adjuvants to activate innate immunity and facilitate antigen specific immune response. Of the many adjuvant formulations that are under development, very few are licensed mainly due to concerns about adverse side effects. The goal of our study was to develop in vitro assays that could predict toxicity of adjuvants in vivo. Pro-inflammatory cytokines IL-β, IL-6, TNF-α, and IL-8 were measured in human primary monocytes and the monocytoid cell line, MonoMac 6 (MM6), activated with a panel of TLR agonists or with adjuvants. A 0.5EU/ml dose of Standard for endotoxin (previously shown to provide a margin between pyrogenic and non-pyrogenic substances in rabbits) was used as a comparator to establish a “safety threshold”. FSL-1, Pam3CSK4, flagellin, and R848 TLR agonists but not Alum, MF59, Poly I:C, or MPL adjuvants induced cytokines in MM6 cells above the safety threshold. To confirm the predictive value of the in vitro assays, FSL-1 and flagellin were injected intramuscularly into New Zealand White (NZW) rabbits. Both TLR agonists induced fever within 6–8h post-injection followed 24–48h later by increased C reactive protein (CRP). Importantly, an early peak in plasma prostaglandin E2 (PGE2) levels preceded rise in body temperature. In vitro production of PGE2 in monocytes and MM6 cells was found following treatments with various TLR agonists but not with alum, MF59, MPL, or Poly I:C adjuvants. Together, our studies demonstrated a strong correlation between production of pro-inflammatory cytokines above a “safety threshold” and production of PGE2in vitro and an increase in body temperature in rabbits. The developed human cell based assays could provide an important tool for early screening of new molecular moieties and adjuvant formulations and may assist in selection of safer products.

Bioactive and total endotoxins in atmospheric aerosols in the Pearl River Delta region, China

Abstract Endotoxin, a toxic and pyrogenic substance in gram-negative bacteria in atmospheric aerosols was measured over a period of one year at Nansha, Guangzhou and Hong Kong in the Pearl River Delta region, China. Atmospheric aerosols were collected by high-volume samplers. The bioactive endotoxin levels in the samples were determined using the Limulus Amebocyte Lysate (LAL) assay after extraction with pyrogen-free water while the total endotoxin levels were measured by quantifying the biomarker, 3-hydroxy fatty acids (3-OHFAs) with GC–MS. Results showed that there was no significant difference (0.19 p 10 among sites (average concentrations ranged from 0.34 to 0.39 EU m −3 ). However, Hong Kong showed a significantly lower ( p 10 (average of 17.4 ng m −3 ) compared with Nansha's 29.4 ng m −3 and Guangzhou's 32.7 ng m −3 . The bioactive endotoxins were found to be associated with the coarse mode (PM 2.5-10 ) of the particulates of natural origins while the total endotoxins were associated more with the fine mode (PM 2.5 ) of the particulates of anthropogenic origins. When normalized with particulate mass, the endotoxin loading is much higher in summer as a result of the increased growth of the bacteria when climatic conditions are favorable. The chemically determined total endotoxins were 3–4 orders of magnitude higher than the bioactive endotoxins quantified using the LAL assay. Correlation analyses between the bioactive endotoxins and 3-OHFAs with different carbon length were analyzed. Results showed that the correlations detected vary among sites and particulate sizes. Although no generalization between the total and bioactive endotoxins can be drawn from the study, the levels reported in this study suggests that the discrepancies between the two measurement approaches, and the bioactive potential of 3-OHFAs with individual carbon chains deserve further investigation.

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.

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.

Pyrogen transfer across high- and low-flux hemodialysis membranes.

The extent to which bacterial products from contaminated dialysate enter a patient's blood depends upon the type and permeability of the hemodialysis membrane in use. This study was performed to assess the transfer of pyrogenic substances across both high- and low-flux membranes (DIAPES, Fresenius Polysulfone, Helixone, Polyamide S). All experiments were carried out in the saline-saline model. The dialysate pool was contaminated either with purified lipopolysaccharide (LPS) (250 and 500 EU/mL) or with sterile bacterial culture filtrates (20 EU/mL), and in vitro dialysis was performed under diffusive and convective conditions. A significant transfer of endotoxin was observed for both low- and high-flux DIAPES challenged with either LPS or with bacterial culture filtrates. Under identical conditions, no transfer of endotoxins was detectable across Fresenius Polysulfone and Helixone upon challenge with purified LPS. With bacterial culture filtrates, endotoxin concentrations for Polyamide S and Fresenius Polysulfone were about 10% and 1%, respectively, of those measured for DIAPES, whereas no transfer of endotoxin was detectable for Helixone. Using an alternative assay (induction of interleukin-1 receptor antagonist, IL-1Ra, in whole blood), only the DIAPES membrane showed the passage of cytokine-inducing substances. Thus, when saline is present in both the blood and dialysate compartments (i.e., the situation during predialysis priming procedures), dialysis membranes differ profoundly with respect to their permeability to endotoxins.

Platelet-activating factor: a previously unrecognized mediator of fever.

Lipopolysaccharide (LPS)-induced systemic inflammation is accompanied by either hypothermia (prevails when the ambient temperature (Ta) is subneutral) or fever (prevails when Ta is neutral or higher). Because platelet-activating factor (PAF) is a proximal mediator of LPS inflammation, it should mediate both thermoregulatory responses to LPS. That PAF possesses hypothermic activity and mediates LPS-induced hypothermia is known. We asked whether PAF possesses pyrogenic activity (Expt 1) and mediates LPS fever (Expt 2). The study was conducted in Long-Evans rats implanted with jugular catheters. A complex with bovine serum albumin (BSA) was infused as a physiologically relevant form of PAF; free (aggregated) PAF was used as a control. In Expt 1, either form of PAF caused hypothermia when infused (83 pmol kg-1 min-1, 60 min, i.v.) at a subneutral Ta of 20 degrees C, but the response to the PAF-BSA complex (-4.5 +/- 0.5 degrees C, nadir) was ~4 times larger than that to free PAF. At a neutral Ta of 30 degrees C, both forms caused fever preceded by tail skin vasoconstriction, but the febrile response to PAF-BSA (1.0 +/- 0.1 degrees C, peak) was > 2 times higher than that to free PAF. Both the hypothermic (at 20 degrees C) and febrile (at 30 degrees C) responses to PAF-BSA started when the total amount of PAF infused was extremely small, < 830 pmol kg-1. In Expt 2 (conducted at 30 degrees C), the PAF receptor antagonist BN 52021 (29 micromol kg-1, i.v.) had no thermal effect of itself. However, it strongly (~2 times) attenuated the febrile response to PAF (5 nmol kg-1, i.v.), implying that this response involves the PAF receptor and is not due to a detergent-like effect of PAF on cell membranes. BN 52021 (but not its vehicle) was similarly effective in attenuating LPS (10 microg kg-1, i.v.) fever. It is concluded that PAF is a highly potent endogenous pyrogenic substance and a mediator of LPS fever.

Filtration fluid for hemodialysis treatment.

Bacterial contamination of dialysis fluid has long been recognized as a problem in hemodialyis. Cytokines released as a consequence of contaminated dialysis fluid are believed to be responsible for many acute and chronic side effects in patients undergoing renal replacement therapy. For several years now, attempts have been made to eliminate pyrogenic substances and ensure a sterile and endotoxin-free dialysis fluid. A recent dialysis fluid filter known as DIASAFE, containing a membrane based on Polysulfone (Fresenius), was tested for a period of 1,000 hours (approx. 14 weeks). Dialysis fluid samples were collected once weekly before and behind the filter and cultivated for detection of microorganisms and endotoxins. Additionally, starting after the fourth week of the study, serum samples were collected weekly and the beta2-microglobulin concentration was determined. The filter reduced microorganisms at a rate of at least 10(5) and in the majority of cases (86% of samples) by more than 106. Under clinical conditions the stability and microbiological functionality of the filters could be demonstrated for more than 1,000 hours and 150 disinfecting cycles. In four cases of endotoxin burden (> 0.5 IU/ml) in the dialysis fluid in front of the filter the concentration behind the filter was lower than 0.1 IU/ml, indicating effective reduction of endotoxins. A tendency to a reduction of beta2-microglobulin in serum from 32.5+/-3.9 mg/L to 21.5+/-5.3 mg/L was observed. These results indicate that the dialysis fluid filter used was effective, dramatically reducing the bacterial contaminants in dialysis fluid, thus protecting patients from the potentially harmful acute and long-term life-threatening consequences of contaminated dialysis fluid.

Changes in Mac-1 and CD14 Expression on Monocytes and Serum Soluble CD14 Level during Push/Pull Hemodiafiltration

Background/Aim: Employment of treated dialysate as replacement fluid raises concerns about exposure of patients to pyrogenic substances. This study was undertaken to evaluate the safety of treated dialysate as the replacement fluid for push/pull hemodiafiltration. Methods: In the present study, changes in the expressions of Mac-1 and CD14 on monocytes, which are upregulated by monocyte activation, were analyzed by flow cytometry, and the serum level of sCD14 which elevates by monocyte activation was measured by enzyme-linked immunosorbent assay (ELISA) during treatment in 7 patients on hemodialysis with regenerated cellulose (RC) membrane, polysulfone (PS) membranes and by push/pull hemodiafiltration (HDF) with PS membranes in a cross-over fashion. Results: During hemodialysis with RC, hemodialysis with PS or push/pull hemodiafiltration with PS, both Mac-1 and CD14 expressions on monocytes significantly increased by passing through the artificial kidneys, and, accordingly, the respective values downstream of the artificial kidneys were significantly higher than the predialysis values, even when the lipopolysaccharide level in dialysate was not detectable by Limulus assay. There was no significant variation in serum sCD14 levels during any of the hemodialysis with RC, hemodialysis with PS or push/pull hemodiafiltration. However, during hemodialysis with PS or push/pull hemodiafiltration with PS, changes in Mac-1 and CD14 expression on monocytes were significantly smaller than those during hemodialysis with RC. Conclusion: Monocytes are activated to a greater extent during hemodialysis with RC membranes than during push/pull HDF with PS membranes. We consider that push/pull HDF may be safer than hemodialysis with RC membrane and that it is as safe as hemodialysis with the PS membrane in terms of monocyte activation, when pyrogen-free dialysate is employed.