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Pyrogens are fever-inducing substances routinely investigated in health products through tests such as the Rabbit Pyrogen Test (RPT), the Limulus Amebocyte Lysate (LAL), and the Monocyte Activation Test (MAT). However, the applications of the MAT for medical devices and biomaterials remain limited. This work aimed to overview the studies evaluating the pyrogenicity of medical devices and biomaterials using the MAT, highlighting its successes and potential challenges. An electronic search was performed by December 2023 in PubMed, Scopus, and Web of Science, identifying 321 records which resulted in ten selected studies. Data were extracted detailing the tested materials, MAT variants, interferences, and comparisons between methods. Methodological quality was assessed using the ToxRTool, and the results were synthesized descriptively. The selected studies investigated various materials, including polymers, metals, and natural compounds, employing the different biological matrices of the MAT. Results showed the MAT's versatility, with successful detection of pyrogens in most materials tested, though variability in sensitivity was noted based on the material and testing conditions. Challenges remain in optimizing protocols for different material properties, such as determining the best methods for direct contact versus eluate testing and addressing the incubation conditions. In conclusion, the MAT demonstrates significant potential as a pyrogen detection method for medical devices and biomaterials. However, continued research is essential to address existing gaps, optimize protocols, and validate the test across a broader range of materials. [ABSTRACT FROM AUTHOR]

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Copyright of Zeitschrift für Rheumatologie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Pyrogens are fever-inducing substances routinely investigated in health products through tests such as the Rabbit Pyrogen Test (RPT), the Limulus Amebocyte Lysate (LAL), and the Monocyte Activation Test (MAT). However, the applications of the MAT for medical devices and biomaterials remain limited. This work aimed to overview the studies evaluating the pyrogenicity of medical devices and biomaterials using the MAT, highlighting its successes and potential challenges. An electronic search was performed by December 2023 in PubMed, Scopus, and Web of Science, identifying 321 records which resulted in ten selected studies. Data were extracted detailing the tested materials, MAT variants, interferences, and comparisons between methods. Methodological quality was assessed using the ToxRTool, and the results were synthesized descriptively. The selected studies investigated various materials, including polymers, metals, and natural compounds, employing the different biological matrices of the MAT. Results showed the MAT’s versatility, with successful detection of pyrogens in most materials tested, though variability in sensitivity was noted based on the material and testing conditions. Challenges remain in optimizing protocols for different material properties, such as determining the best methods for direct contact versus eluate testing and addressing the incubation conditions. In conclusion, the MAT demonstrates significant potential as a pyrogen detection method for medical devices and biomaterials. However, continued research is essential to address existing gaps, optimize protocols, and validate the test across a broader range of materials.

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.

To develop and validate a novel reporter gene assay (RGA) to detect pyrogen, HL60 cells were transfected with an NF-κB-RE plasmid containing the luciferase gene to generate stably transfected cells. Through stimulation with pyrogens, a signal was obtained that was dose-dependent with the concentration of pyrogen. Using the cells, we selected and optimized the parameters and found that the optimal conditions may be with 5 × 105/ml cells that were seeded and incubated with pyrogen for 3–6 h in IMDM medium with 2% FBS. Based on the optimized parameters, a novel RGA was developed. Then, the RGA was validated and the results showed that the linearity was greater than 0.95 between the signals and the concentrations of pyrogen, the recoveries of pyrogen were all between 50% and 200%, and the precision was less than 35%. There was no difference in the sensitivity, specificity or reproducibility between RGA and BET, and the results from RGA and MAT and RPT were consistent. Furthermore, the RGA can be applied to the pyrogen detection of monoclonal antibodies. Due to its advantages including a fast detection speed, high sensitivity, convenient mode of operation and wide-pyrogen spectrum detection, RGA is promising as a supplementary method to detect pyrogen. [ABSTRACT FROM AUTHOR]

Pyrogens are classified in two groups, endotoxin pyrogens and non-endotoxin pyrogens (NEPs). The presence of either in parenteral pharmaceuticals or medical devices can cause severe harm to subjects, and when occurring in combination, synergistic potentiation effects can occur. As the standard in vitro pyrogen test, the Limulus Amebocyte Lysate (LAL) assay can detect LPS only, an endotoxin, but not NEPs. We tested whether the Monocyte Activation Test (MAT) that measures IL-6 induction, is suited for detecting synergistic pyrogen effects. Here we show that MAT reliably detects the NEPs heat-killed Staphylococcus aureus, R848 and lipoteichoic acid, in addition to LPS. When combinations of these pyrogens were tested, a potentiation of IL-6 production was seen beyond an additive effect, apparently reflecting on in-vivo synergisms. The current study therefore demonstrates that MAT not only is a reliable and reproducible assay for the sensitive detection of both endotoxin and non-endotoxin pyrogens, but also for identifying synergistic effects when parenteral drugs are contaminated with multiple pyrogens. [ABSTRACT FROM AUTHOR]

All parenteral medicinal products require, among others, to be tested for pyrogen contaminants. Pyrogens are a group of heat-stable molecules that can induce a fever reaction at deficient concentrations when applied parenterally, which can lead to hypotension, shock with organ failure, and death. Pyrogen testing has to be carried out independently from sterility tests, as pyrogens can also be released by pathogens killed in the sterilization process. The "golden standard" of pyrogen testing has been the rabbit pyrogen test (RPT). Since the EU Directive 2010/63 ("Protection of Experimental Animals "), re-approvals of the employment of the RPT have been rejected in the European Union since the end of 2021. That makes the RPT obsolete from the end of 2026 in the EU, and alternative pyrogen testing methods have to be found not to endanger drug safety. Tests like the Limulus Amebocyte Lysate (LAL) or the Recombinant Factor c (RFc) test do not encompass the whole spectrum of pyrogens. The Monocyte Activation Test (MAT) is the only alternative pyrogen test method. The classical MAT described in the European Pharmacopoeia, chapter 2.6.30, is based on measuring one secreted cytokine from human immune cells in response to pyrogens by enzyme-linked immunosorbent assay (ELISA). We have developed an advanced MAT where we measure two cytokines simultaneously. Our system is based on nucleic acid amplification techniques (NAT) employing digital PCR (dPCR) and the human lymphoblastic cell line HL-60. HL-60 cells express a plethora of cytokines and chemokines in response to pyrogens, from which we chose to measure IL-1b and TNFa, both of them involved in the fever response and required in the EP2.6.30. Moreover, simultaneous measurement of a housekeeping gene ensures data accuracy. Drug safety relies on tests for measuring pyrogen contaminations. Alternative methods like the LAL, which requires the hemolymph of the horseshoe crab, are not entirely satisfying because of the limitation of the test to the sole measurement of lipopolysaccharide (LPS) and the limited resource of this endangered species. Similarly, available MATs rely on measuring one secreted cytokine by ELISA. These limitations make them inferior to our NAT-based dPCR system, where we can simultaneously measure the gene expression of two cytokines. This makes the NAT-MAT a reliable, animal-free alternative method to detect pyrogenic contaminants and ensure drug safety. [ABSTRACT FROM AUTHOR]

Phytohemagglutinin (PHA) is a plant mitogen that can agglutinate human leukocytes and erythrocytes. PHA is mainly derived from red kidney beans and can act as an exogenous pyrogen. When entering into the blood circulation, exogenous pyrogens principally interact with monocytes and macrophages and induce the release of pro-inflammatory cytokines. Monocytes and macrophages are the cells that fight against foreign invaders and acts as a primary line of immune defence. Similar to PHA, the chemical 2,4,6-trinitrophenol (TNP) also acts as an exogenous pyrogen. The study focused on the in vitro interaction of PHA and TNP with the human monocyte/macrophage cell model THP-1. The exposure and associated change in cellular morphology, organelle function, mechanism of cell death, inflammatory signalling and expression of inflammation-related genes were analyzed in different time periods. It was observed that PHA and TNP induce dose and time-dependent toxicity to monocytes/macrophages where the mechanism of cell death was different for PHA and TNP. Both PHA and TNP can evoke immune signalling with increased expression of inflammatory genes and associated activation of intracellular signalling cascades. [ABSTRACT FROM AUTHOR]

Fever is a frequent and important symptom in patients with rheumatological diseases and can be an expression of activity of the underlying rheumatological disease. There is great variability in the incidence of fever as a symptom of the disease between individual diseases. The growing understanding of the molecular signatures of the diseases can help to explain these discrepancies: A genetic overactivation of potently pyrogenic cytokines is the reason why fever is nearly always present in autoinflammatory syndromes. In contrast, fever is less common in polyarthritis and myositis and mostly limited to severe courses of disease. In the diagnostic work-up of fever, frequent differential diagnoses, such as infections, malignancies, side effects of drugs and hypersensitivity reactions should be considered. This article provides an overview of the physiology of the development of fever, describes the relevance of fever in individual rheumatological diseases and proposes a workflow for the clinical clarification of rheumatological patients who present with fever., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Fever can be due to infectious or noninfectious causes and results from the body's natural response to exogenous or endogenous pyrogens. Laboratory tests including complete blood count, differential blood count, C‑reactive protein, erythrocyte sedimentation rate and procalcitonin do not have sufficient sensitivity and specificity to definitively detect or rule out an infectious (bacterial, viral, parasitic) cause of fever. Blood cultures should be carried out when bacteremic or septic illnesses are suspected. Fever is not always present in infections and can be absent, especially in older and immunocompromised patients. If fever is suspected, core temperatures should be taken, e.g., rectally, orally or invasively. Depending on the clinical situation, infectious causes must be excluded as the most likely cause of an acutely occurring fever. The investigation of long-standing fever (fever of unknown origin, FUO) can be complex and some infectious diseases should first be ruled out, whereby a syndromic classification often helps to clarify the cause of the fever., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF- κ B-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays., (Creative Commons Attribution license.)

Wildfire alters hydrologic and geomorphic systems, promoting increases in runoff and erosion relative to unburned areas. As a result, pyrogenic carbon (PyC) produced by wildfires can experience substantial lateral redistribution from overland flow. Since landscape position helps to determine the fate of PyC, it is critical to understand the geomorphic factors that govern its lateral redistribution as well as the sensitivity of those factors to soil burn severity, which controls the magnitude of many wildfire‐induced hydrologic and geomorphic changes. In this study, we quantified the spatial distribution of near‐surface (0–5 cm) PyC on three hillslopes roughly 1 year after the 2018 Buzzard Fire in the Gila National Forest, New Mexico, USA. We then use hydrologic monitoring data, terrain analysis, and rainfall‐runoff modeling to explain the observed spatial distribution of PyC. Near‐surface PyC concentrations decreased from averages of roughly 12–8 g/kg as slope and unit stream power increased by factors of ∼2 and 4, respectively, on a hillslope burned at low severity. This suggests that susceptibility to runoff‐driven erosion was a dominant control on the redistribution of PyC. In contrast, the spatial distribution of PyC in areas burned at moderate to high severity was independent of slope and unit stream power. We attribute this pattern to intense runoff and lack of canopy cover, which promoted erosion of PyC regardless of slope and landscape position. Results demonstrate that how wildfire‐induced hydrogeomorphic changes can modulate the importance of terrain attributes in controlling the spatial distribution of PyC in upland landscapes. Key Points: Soil burn severity impacted relationships between the spatial distribution of pyrogenic carbon (PyC) on hillslopes and hydrogeomorphic variablesNear‐surface PyC on a low burn severity hillslope is best correlated with topographic slope following 1 year of erosionNear‐surface PyC on moderate/high burn severity hillslopes is not correlated with slope or unit stream power after 1 year of erosion [ABSTRACT FROM AUTHOR]

Aerosols supply bioaccessible iron to marine biota which could affect climate through biogeochemical feedbacks. This paper review progresses in research on pyrogenic aerosol iron. Observations and laboratory experiments indicate that the iron solubility of pyrogenic aerosol can be considerably higher than lithogenic aerosol. Aerosol models highlight a significant contribution of pyrogenic aerosols (~20%) to the atmospheric supply of dissolved iron into the ocean. Some ocean models suggest a higher efficiency of pyrogenic iron in enhancing marine productivity than lithogenic sources. It is, however, challenging to quantitatively estimate its impact on the marine biogeochemical cycles under the changing air quality and climate. [ABSTRACT FROM AUTHOR]

Dating lime mortar samples using the radiocarbon (14C) method can be difficult. This is because the contamination is similar to the primary dating material (CaCO3) and consequently difficult to remove. Mortar can also have late-in-formation pyrogenic carbonate from interactions with the environment after the initial hardening phase, such as recrystallization, fire damage or delayed hardening. When 14C dating a system of primary dating material, contamination and late-in-formation pyrogenic carbonate, one approach is multi-fraction dating with conclusiveness criteria. If a sample has sufficient contamination or late-in-formation pyrogenic carbonate, the criteria evaluate the result inconclusive. To improve inconclusive results from such samples, this study investigates sample preparation by thermal decomposition. Here samples that were inconclusively dated by the authors' traditional method, sequential dissolution with 85% phosphoric acid, are investigated further. This study finds that CO2 thermally decomposed at low temperatures contains some late-in-formation pyrogenic carbonate. By rejecting CO2 decomposed at low temperatures, Kastelholm castle and Kimito church in Finland are conclusively and accurately dated. Furthermore, a preheating method removes some late-in-formation carbonate, but not enough for a conclusive result. Finally, thermal decomposition finds difficulty in discerning binder carbonate from limestone and marble contamination. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The human body has a perfect thermoregulatory system to meet the needs of normal life activities. The central regulation of body temperature is mainly explained by the theory of "setting point (setpoint, SP)". Fever is a positive but nonspecific response of the body to infections and other pyrogens, which causes immune cells to release cytokines, leading to a brain protein-mediated rise in body temperature. Cytokines can be roughly divided into 2 categories: proinflammatory cytokines and anti-inflammatory cytokines. IL-1, TNF-α, and IL-6 are proinflammatory cytokines, whereas IL-4 and IL-10 are anti-inflammatory cytokines. IL-2 is a cytokine that can both activate and inhibit immunity. IL-8 is a neutrophil chemotactic factor, and IFN is a cytokine that plays a key role in the proper induction and maintenance of innate and acquired immunity. This article reviews the pathophysiological characteristics of fever and the cytokines related to fever (IL-2, 4, 6, 8, 10, IFN, TNF, etc.). [ABSTRACT FROM AUTHOR]

Background: Modern medicine is not the choice of patients with "shimetere" in the Gurage community owing to their perception of 'parenteral medication use severely aggravates the disease'. For this reason, the root part of Polygala sadebeckiana Gürke is commonly utilized as traditional medicine in the management of the disease. The aim of this study was to evaluate the antimicrobial activity of Polygala sadebeckiana Gürke extract on bacterial isolates from wound samples of patients with "Shimetere"., Methods: The agar well diffusion method was used to evaluate antibacterial activity, and the agar dilution method was utilized to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MICs). The crude extract was tested against isolated bacteria at concentrations of 25, 50, 75 and 100 mg/mL in triplicate (3x). The positive controls were azithromycin (15 µg) and cloxacillin disk (5 µg), and the negative control was dimethylsulfoxide (5%). The group mean comparisons were made using one-way ANOVA at a significance level of p < 0.05, and the results are presented as the mean ± standard deviation. The presence of secondary metabolites from crude extract was checked by standard testing procedures., Results: S. aureus and S. pyrogen were the two identified bacteria from 9 (60%) and 3 (20%) wound samples, respectively. All identified bacterial strains were susceptible to the reference antibiotics. Tannins and saponins were the most abundant secondary metabolites found in the crude extracts. The average inhibition zones of the plant extracts with 100, 75, 50 and 25 mg/mL concentrations were 27, 20.33, 15.25, and 11.96 mm (p < 0.000) for S. aureus and 30.02, 24.50, 19.07, and 15.77 mm (p < 0.000) for S. pyrogen bacteria, respectively. The MIC and MBC of the crude extract were 1.67 and 10 mg/mL for S. aureus and 0.98 and 4 mg/mL for S. pyrogen., Conclusion: Polygala sadebeckiana Gürke contained significant tannins and saponins as secondary metabolites and had antibacterial activities against isolated bacteria (S. aureus and S. pyrogen) from "Shimetere". The potential mechanism of antibacterial action of the plant extract was cell wall synthesis inhibition., (© 2024. The Author(s).)

Leptospirosis has a wide spectrum of clinical manifestations ranging from mild to severe disease. The cytokine response is considered one of the key drivers for this varying manifestation. The different cytokine response observed in patients with leptospirosis could be due to the variation of infecting serovars. Since the rfb locus codes for the lipopolysaccharide synthesis of the bacterial cell wall, which also determines the serovar, this locus may play a role in driving a specific cytokine response in the host. We investigated 12 commonly used cytokine profiles in serum samples of culture, microscopic agglutination test (MAT), or polymerase chain reaction (PCR)-positive patients with leptospirosis. The sequences of the rfb locus in culture-positive samples were generated from whole genome sequencing and serovar status was drawn from original data published. Isolated cultures were subjected to whole genome sequencing using the PacBio RS II system, and the resulting data were used to determine the species. The recovered genomic data were annotated with the Rapid Annotation using Subsystem Technology (RAST) subsystem, and the rfb locus was extracted. The cytokine analysis was carried out using the Qiagen human ELISA kit. Eighteen samples were found to be positive by culture, while the other 7 samples were positive by PCR or MAT. Infections from Leptospira interrogans serovar Autumnalis (5), Pyrogens (3), Icterohaemorrhagiae (1) Leptospira borgpetersenii (all 7 samples clustered in same clonal group with serovar status not determined), Leptospira weilii (1 with serovar status not determined), and Leptospira kirschneri serovar Grippotyphosa (1) were included in the analysis. Three patients [infected with Leptospira interrogans serovar Autumnalis (2) and Pyrogens (1)] and 2 MAT-positive patients (highest titer against serovar Bratislava of L.interrognas ) were reported to have severe clinical manifestations, while the rest had mild to moderate symptoms. Although the serum cytokine concentration of patients with severe clinical manifestation was comparatively higher, a statistically significant difference was observed only for interleukin (IL)-1β ( P  < 0.05). IL-10/tumor necrosis factor-alpha (TNF-α) ratio was high in patients with severe complications. In general, patients infected with L. interrogans showed higher concentration of cytokines compared to L. borgpetersenii .

Pyrogens, classified as bacterial endotoxins and non-endotoxin pyrogens (NEPs), induce fever or shock when released into the bloodstream or spinal fluid. Recently, a monocyte-activation test (MAT) involving human cell culture has been developed to detect pyrogens in injectable products. To evaluate the sensitivity of MAT, a reference standard endotoxin was used as a positive control; however, the reactivity differed between the endotoxins and NEPs, necessitating positive controls for NEPs. This study aimed to explore a preparation method for heat-killed Staphylococcus aureus (HKSA) as a positive control for NEPs in MAT. Because S. aureus forms grape-like clusters, nine types of glass filters with pore sizes of 0.5-2.7 µm were evaluated to obtain a uniform bacterial suspension. The suspension was then heat-treated to kill the bacteria, resulting in HKSA samples. Serial dilutions of HKSA were tested by MAT using peripheral blood mononuclear cells. The interleukin-6 concentrations in the culture supernatant were measured by enzyme-linked immuno-sorbent assay to assess pyrogenic activities of HKSA. The pore sizes of the glass filters affected the uniformity of HKSA, and GF/C filter was selected for HKSA preparation. Repeated filtration improved uniformity, and a uniform suspension of HKSA was obtained through double filtration using a GF/C filter. Despite the decrease in HKSA activity as filtration frequency increased, the detection limit remained consistently unchanged. This suggests that repeated filtration can adjust the activity of HKSA to a baseline level and that a uniform suspension of HKSA exhibiting low variation is suitable as a positive control in MAT.

The ELISA-based monocyte activation test (MAT) facilitates the replacement of the rabbit pyrogen test (RPT) for the detection of Innate Immune Response-Modulating Impurities (IIRMIs) in injectable drugs by activation of monocytes in human peripheral blood mononuclear cells (PBMCs). We describe the use of a triple-color IL-1β/IL-6/TNF-α FluoroSpot assay as a sensitive tool for quantification of the frequencies of IIRMI-activated monocytes as well as determination of the relative amount of pyrogenic cytokine(s) produced by each activated cell., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)