Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs.

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-κB-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.

Levels of Cytokines in Leptospirosis Patients with Different Serovars and rfb Locus.

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 interrogansserovar 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)-1ß (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.

Barriers to the Use of Recombinant Bacterial Endotoxins Test Methods in Parenteral Drug, Vaccine and Device Safety Testing.

The Bacterial Endotoxins Test (BET) is a critical safety test that is used to detect bacterial endotoxins, which are the major contributor to fever-inducing contamination risks known as pyrogens. All parenteral therapies, including every lot of injected drugs, vaccines, medical devices, must be tested for pyrogens to ensure patient safety. Bacterial endotoxins test methods were developed as a highly sensitive detection method for bacterial endotoxins, after the discovery of a clotting cascade in horseshoe crab blood. However, horseshoe crab species are limited to some inshore coastal habitats along the Atlantic coast of the USA and others throughout Asia. Fully functional horseshoe crab clotting factors can be manufactured via recombinant protein production, and several BET methods featuring recombinant horseshoe crab proteins have now been developed for commercial use. Recombinant Bacterial Endotoxins Test (rBET) methods based on the use of recombinant Factor C (rFC) were established in the European Pharmacopoeia - however, these methods have not yet been granted compendial status in the United States Pharmacopoeia (USP). In order to facilitate dialogue between stakeholders, the Physicians Committee for Responsible Medicine hosted two virtual roundtable discussions on the perceived barriers to the use of rBET methods for US FDA requirements. Stakeholders agreed that multiple rFC-based methods have been demonstrated to have suitable analytical performance, as described in ICH Q2 on the Validation of Analytical Procedures and USP <1225> on the Validation of Compendial Procedures. United States Pharmacopoeia compendial inclusion of the rFC-based and other rBET methods was favoured, in order to reduce the additional burdens created by a lack of global harmonisation on BET testing requirements.

The monocyte activation test detects potentiated cytokine release resulting from the synergistic effect of endotoxin and non-endotoxin pyrogens.

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.

Recombinant bacterial endotoxin testing: a proven solution.

Testing of parenteral pharmaceuticals and medical devices for pyrogens (fever-inducing substances) is critical to patient safety. The original rabbit pyrogen test has largely been replaced by different bacterial endotoxin tests based on Limulus amebocyte lysate (LAL), sourced from the blood equivalent of horseshoe crabs after comparative studies to the rabbit pyrogen test. Since 2004 a bacterial endotoxin test based on recombinant factor C (rFC), the endotoxin sensor protein inside of LAL, has been used as an animal-free alternative to LAL. Likewise, numerous studies compared LAL and rFC. Here we describe the history of pyrogen and bacterial endotoxin testing and summarize the evidence presented by those studies. We demonstrate that rFC and LAL are equivalent and comparable.

Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test.

The whole blood pyrogen test invented 25 years ago, and its variant based on cryo-preserved blood one year later, brought momentum into the field of pyrogen testing, which, despite the broad application of the Limulus amebocyte lysate (LAL) assay, aka bacterial endotoxin test (BET), consumed several hundred thousand rabbits per year world-wide. The resulting international validation and lengthy acceptance and implementation process of what are called now monocyte activation tests (MATs) finally is impacting on animal numbers - at least in Europe - reducing them by more than 70% and counting. The author sees no reason for continuing any regulatory rabbit testing for pyrogens except the lack of acceptance of MATs in some regions of the world. The availability of MATs has opened also the discussion about the shortcomings of LAL/BET, namely its restriction to Gram-negative pyrogens, non-reflection of the potency of these in humans, interference and masking by many products, and animal welfare concerns for horseshoe crabs. The obvious advantages of MATs in all these respects should lead to a shift from LAL/BET to MATs. We are starting to see this for vac-cines and medical devices, but other areas like safety testing of blood transfusions, cell therapies and nanomaterials, and the assessment of air-borne pyrogens still need to grasp the opportunity provided by MATs. While the different MATs can jointly serve these needs, the whole blood MAT has some advantages as discussed here.

The Application of HL60-IL-6 Assay for in vitro Pyrogen Detection of CAR-T Cells Products.

OBJECTIVE: To detect the pyrogen in CAR-T cells product employing the HL60-IL-6 assay. METHODS: The HL60 cells were incubated with CAR-T cells injection or endotoxin standard for 48 hours. After then, the secreted cytokine interleukin-6 (IL-6) from HL60 cells was determined by ELISA. According to the four-parameter logistic curve fitted by Optical Density (OD) value corresponding to IL-6 and endotoxin standard concentration, the endotoxin equivalents of pyrogen content in the CAR-T cells products can be measured. Then, the method was validated, including the limit of detection (LOD), limit of quantitation, the recovery rate and the comparison of the determined results by HL60-IL-6 assay with that by the conventional pyrogen test, the Rabbit Pyrogen Test (RPT). RESULTS: The HL60-IL-6 assay applied to pyrogen test in CAR-T cells products has been established and validated, The LOD was 0.03 EU/mL while the LOQ was 0.07 EU/mL, the recovery rates were 121.4% and 94.5% respectively. The results determined by HL60-IL-6 assay were consistent with that by the RPT. CONCLUSION: The HL60-IL-6 assay can be employed in CAR-T cell products in vitro pyrogen test.

Reutilização do dialisador e uso único: episódios de pirogenia e bacteremia / Reutilización del dializador y uso único: episodios de reacciones pirógenas y bacteriemias / Dialyzer reuse and single use: pyrogenesis and bacteremia episodes

Resumo Objetivos Comparar a reutilização do dialisador com o uso único deste material para marcadores bioquímicos, hematológicos, episódios de pirogenias e bacteremias. Métodos Estudo longitudinal com coleta de dados retrospectiva em prontuários de pacientes em hemodiálise, em hospital público universitário. A investigação foi conduzida seis meses com a reutilização do dialisador e seis meses com uso único. Os dados foram analisados no SPSS Versão 18.0. Para comparação das médias dos exames utilizou-se teste t pareado e Wilcoxon, episódios de pirogenia e bacteremia foram analisados pelo teste de Wilcoxon e Razão de Chances (RC) como medida de força de associação. Variáveis categóricas foram analisadas pelos testes de McNemar e Exato de Fisher. O estudo foi aprovado pelo Comitê de Ética e Pesquisa. Resultados Foram analisadas 5.508 sessões de hemodiálise de pacientes predominantemente masculinos 21(62%), média de idade 58(± 14) anos, hipertensos 14(41%), tempo médio de tratamento 6±3 anos. Durante uso único identificou-se redução da ureia pós diálise, creatinina, fósforo, ferritina, hematócrito e hemoglogina em relação ao reutilizado (p < 0,05) e 91% menos risco de pirogenia comparado a reutilização do dialisador (Razão de Chance= 0,091; IC 95%: 0,002-0,625). Não houve diferença significativa na ocorrência de bacteremias. Conclusão Os resultados sugerem maior remoção de biomarcadores bioquímicos e menos episódios de pirogenias quando o dialisador é uso único.ater removal of biochemical biomarkers and fewer pyrogenics episodes when the dialyzer is a single use.

Resumen Objetivos Comparar la reutilización del dializador con el uso único de este material en marcadores bioquímicos, hematológicos, episodios de reacciones pirógenas y bacteriemias. Métodos Estudio longitudinal con recolección de datos retrospectiva en historias clínicas de pacientes en hemodiálisis, en un hospital público universitario. La investigación fue conducida seis meses con la reutilización del dializador y seis meses con un único uso. Los datos fueron analizados en SPSS Versión 18.0. Para comparar los promedios de los análisis, se utilizó el Test-T pareado y prueba de Wilcoxon, los episodios de reacciones pirógenas y bacteriemia fueron analizados mediante la prueba de Wilcoxon y Razón de Momios (RM) como medida de fuerza de asociación. Las variables categóricas se analizaron con la prueba de McNemar y la prueba exacta de Fisher. El estudio fue aprobado por el Comité de Ética e Investigación. Resultados Se analizaron 5.508 sesiones de hemodiálisis de pacientes predominantemente masculinos 21 (62 %), promedio de edad 58 (±14) años, hipertensos 14 (41 %), tiempo promedio de tratamiento 6±3 años. Durante el uso único del dializador, se identificó una reducción de la urea posdiálisis, creatinina, fósforo, ferritina, hematocrito y hemoglobina respecto al dializador reutilizado (p < 0,05) y un 91 % menos de riesgo de reacción pirógena comparado con la reutilización del dializador (Razón de Momios = 0,091; IC 95 %: 0,002-0,625). No hubo diferencia significativa en episodios de bacteriemias. Conclusión Los resultados sugieren mayor eliminación de biomarcadores bioquímicos y menos episodios de reacciones pirógenas cuando el dializador se utiliza una única vez.

Abstract Objectives To compare dialyzer reuse with its single use for biochemical, hematological markers, pyrogenesis and bacteremia episodes. Methods A longitudinal study with retrospective data collection from medical records of patients on dialysis in a public university hospital. The investigation was conducted six months with dialyzer reuse and six months with single use. Data were analyzed using SPSS Version 18.0. To compare the tests means, paired t-test and Wilcoxon were used. Pyrogenesis and bacteremia episodes were analyzed using the Wilcoxon test and Odds Ratio (OR) as association strength measures. Categorical variables were analyzed using McNemar and Fisher's Exact tests. The study was approved by the hospital's Research Ethics Committee. Results Five thousand five hundred eight dialysis sessions of predominantly male patients were analyzed, 21 (62%), with mean age of 58 (± 14) years, hypertensive 14 (41%), with mean treatment time 6 ± 3 years. During single use, a reduction in urea after dialysis, creatinine, phosphorus, ferritin, hematocrit and hemoglogin was identified in relation to reuse (p<0.05) and 91% less risk of pyrogenesis compared to dialyzer reuse (Odds Ratio=0.091; 95% CI: 0.002-0.625). There was no significant difference in the occurrence of bacteremia. Conclusion The results suggest greater removal of biochemical biomarkers and fewer pyrogenics episodes when the dialyzer is a single use.

High mobility group box 1 protein released in the course of aseptic necrosis of tissues sensitizes rats to pyrogenic effects of lipopolysaccharide.

It is still an open question as to whether or not aseptic injuries affect the generation of fever due to exogenous pyrogens including bacterial products. Therefore, in the present paper we have investigated the course of endotoxin fever in rats induced with lipopolysaccharide (LPS; given intraperitoneally in a dose of 50 µg/kg) 48 h after subcutaneous administration of turpentine oil (TRP; 0.1 mL per rat) that causes aseptic necrosis of tissues. We found that febrile response was significantly augmented in the animals pre-treated with turpentine compared to control rats (pre-treated with saline), and that observed excessive elevation of body temperature (Tb) was accompanied by enhanced release of fever mediators: interleukin-6 (IL-6) and prostaglandin E2 (PGE2) into plasma. Moreover, we found that sensitization to pyrogenic effects of lipopolysaccharide was associated with the increase in plasma level of high mobility group box 1 protein (HMGB1), one of the best-known damage-associated molecular patterns (DAMP), which was recently discovered as inflammatory mediator. Since the injection of anti-HMGB1 antibodies weakened observed hyperpyrexia in the animals pre-treated with turpentine, we conclude that HMGB1 is a plasma-derived factor released in the course of aseptic injury that enhances pyrogenic effects of LPS.

Lipopolysaccharides from Microcystis Cyanobacteria-Dominated Water Bloom and from Laboratory Cultures Trigger Human Immune Innate Response.

Massive toxic blooms of cyanobacteria represent a major threat to water supplies worldwide. Here, the biological activities of lipopolysaccharide (LPS) isolated from Microcystis aeruginosa, the most prominent cyanobacteria in water bloom, were studied. LPS was isolated from complex environmental water bloom samples dominated by M. aeruginosa, and from laboratory cultures of non-axenic as well as axenic M. aeruginosa strains PCC7806 and HAMBI/UHCC130. Employing human blood-based in vitro tests, the LPS isolated from complex water bloom revealed the priming of both major blood phagocyte population monocytes and polymorphonuclear leukocytes documented by the increased surface expression of CD11b and CD66b. This was accompanied by a water bloom LPS-mediated dose-dependent induction of tumor necrosis factor α, interleukin-1ß, and interleukin-6 production. In accordance with its priming effects, water bloom LPS induced significant activation of p38 and ERK1/2 kinases, as well as NF-κB phosphorylation, in isolated polymorphonuclear leukocytes. Interestingly, the pro-inflammatory potential of LPS from the axenic strain of M. aeruginosa was not lower compared to that of LPS isolated from non-axenic strains. In contrast to the biological activity, water bloom LPS revealed almost twice higher pyrogenicity levels compared to Escherichia coli LPS, as analyzed by the PyroGene test. Moreover, LPS from the non-axenic culture exhibited higher endotoxin activity in comparison to LPS from axenic strains. Taking the above findings together, M. aeruginosa LPS can contribute to the health risks associated with contamination by complex water bloom mass.

Staphylococcal Superantigens: Pyrogenic Toxins Induce Toxic Shock.

Staphylococcal enterotoxin B (SEB) and related superantigenic toxins produced by Staphylococcus aureus are potent activators of the immune system. These protein toxins bind to major histocompatibility complex (MHC) class II molecules and specific Vß regions of T-cell receptors (TCRs), resulting in the activation of both monocytes/macrophages and T lymphocytes. The bridging of TCRs with MHC class II molecules by superantigens triggers an early "cytokine storm" and massive polyclonal T-cell proliferation. Proinflammatory cytokines, tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 elicit fever, inflammation, multiple organ injury, hypotension, and lethal shock. Upon MHC/TCR ligation, superantigens induce signaling pathways, including mitogen-activated protein kinase cascades and cytokine receptor signaling, which results in NFκB activation and the phosphoinositide 3-kinase/mammalian target of rapamycin pathways. In addition, gene profiling studies have revealed the essential roles of innate antimicrobial defense genes in the pathogenesis of SEB. The genes expressed in a murine model of SEB-induced shock include intracellular DNA/RNA sensors, apoptosis/DNA damage-related molecules, endoplasmic reticulum/mitochondrial stress responses, immunoproteasome components, and IFN-stimulated genes. This review focuses on the signaling pathways induced by superantigens that lead to the activation of inflammation and damage response genes. The induction of these damage response genes provides evidence that SEB induces danger signals in host cells, resulting in multiorgan injury and toxic shock. Therapeutics targeting both host inflammatory and cell death pathways can potentially mitigate the toxic effects of staphylococcal superantigens.

Application of a TLR overexpression cell model in pyrogen detection.

Pyrogens are components derived from microorganisms that induce complex inflammatory responses. Current approaches to detect pyrogens are complex and difficult to replicate, thus there is a need for new methods to detect pyrogens. We successfully constructed a pyrogen-sensitive cell model by overexpressing Toll-like receptor (TLR)2, TLR4, MD2, and CD14 in HEK293 cells. Since the cytokine IL-6 is specifically released upon stimulation of the TLR2 and TLR4 signaling pathways in response to pyrogen stimulation, we used it as a read out for our assay. Our results show that IL-6 is released in response to trace amounts of pyrogens in our cell model. Pyrogen incubation times and concentrations were explored to determine the sensitivity of our cell model, and was found to be sensitive to 0.05 EU/ml of LPS and 0.05 ug/ml of LTA after stimulation for 5 hr. Our TLR overexpressing cell model, with IL-6 as readout, could be a new method for in vitro testing of pyrogens and applicable for evaluating the safety of drugs.

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

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

Pyrogens, a polypeptide produces fever by metabolic changes in hypothalamus: Mechanisms and detections.

Fever is one of the cardinal symptoms of onset of an infection or inflammation and is the common clinical indicator for medical consultation in mammalian host worldwide. Simply, fever manifested with elevation of body temperature from normal physiological range represents adaptive response of immune system on challenge with an infectious and non-infectious circumstance. Fever usually initiated in the periphery as a result of interaction of immune cells with exogenous or endogenous pyrogens. Peripheral pyrogenic signals gain access to the central nervous system via humoral and neural route. Humoral pathway was initiated with production of pyrogenic cytokines and prostaglandins from immune cells of blood as well as liver, transmitted directly to pre-optic area of hypothalamus through the circumventricular organ of brain. On the other hand an alternative pathway was initiated by the same cytokines indirectly via stimulating the vagal sensory neurons result in pyrogenic fever; so-called neuronal pathway. If the magnitude of pyrogens associated fever is very high, it will lead to severe illness ranging from septic shock to death. So it is necessary to evaluate the presence of pyrogens in implants, medical devices, drugs and biological materials to ensure safety in biomedical applications and therapeutics. Classification, route of administration, mechanism of action and detection of pyrogens and associated products are the major subject of this review.

Development and characterization of stable reporter cells for fast and sensitive detection of pyrogen.

Pyrogens are a class of heterogeneous compounds that cause fever and induce inflammatory responses in the host. Lipopolysaccharides (LPS, also known as endotoxin) is the major pyrogen in the category of drug quality control. Accurate and fast quantification of pyrogens is crucial for drug safety. In the present study, we aimed to develop a sensitive and reliable method for rapid detection of pyrogens using luciferase reporter assay. Stable human A549 luciferase reporter cells were constructed under the control of a NF-κB-responsive element or IFN-ß promoter. Our results showed that several monoclonal stable cell clones responded to 0.1 EU/ml endotoxin, which was less than human fever threshold at 0.3 EU/ml of endotoxin. Further, compared with original A549 cells, TLR4 expression on the reporter cells were significantly increased after low amount LPS stimulation. In addition, reporter cells also responded to zymosan stimulation. Therefore, these results indicated that the stable luciferase reporter cells respond to endotoxin and non-endotoxin pyrogens and have the potential to further develop into a sensitive and fast pyrogen evaluation method.

Analysis of IL-6 and IL-1ß release in cryopreserved pooled human whole blood stimulated with endotoxin.

To overcome the lack of availability of fresh human whole blood for pyrogen detection, we explored the feasibility of utilizing cryopreserved pooled human blood to detect the responses of the pro-inflammatory cytokines IL-6 and IL-1ß to LPS. Whole blood was obtained from five donors and incubated with LPS. The quantities of pro-inflammatory cytokines were measured using ELISA, and the results were compared among the samples. After the blood was cryopreserved with Dimethyl sulfoxide (DMSO) (10% v/v) and stored for 4 mo at -196℃, the detection limits of the IL-6/IL-1ß responses to LPS were 0.2/0.4 endotoxin units (EU)/ml, respectively, and IL-6/IL-1ß release increased in response to LPS in a dose-dependent manner. When these experiments were performed in three separate laboratories, the within-laboratory reproducibility of the IL-6/IL-1ß responses was 100%/86.7%, 93.3%/100%, and 86.7%/80%, and the inter-laboratory reproducibility was 92.9%/85.7%, 64.3%/63.6%, and 57.1%/66.7%, respectively. The sensitivity (the probability of correctly classifying positive samples) and specificity (the probability of correctly classifying negative samples) of the IL-6/IL-1ß tests were 81.7%/82.5% and 100%/100%, respectively. The results of this study suggest that cryopreserved pooled blood is a convenient and viable alternative for evaluating in vitro pyrogenicity. Additionally, maintaining cryopreserved pooled blood promotes safety for the user because it is released only after pretesting for infection parameters and has lower variation than fresh donations from a variety of donors.

Applicability of the Monocyte Activation Test (MAT) in the quality control of the 17DD yellow fever vaccine.

The need for alternatives to animal use in pyrogen testing has been driven by the Three Rs concept. This has resulted in the inclusion of the monocyte activation test (MAT) in the European Pharmacopoeia, 2010. However, some technical and regulatory obstacles must be overcome to ensure the effective implementation of the MAT by the industry, especially for the testing of biological products. The yellow fever (YF) vaccine (17DD-YFV) was chosen for evaluation in this study, in view of: a) the 2016-2018 outbreak of YF in Brazil; b) the increase in demand for 17DD-YFV doses; c) the complex production process with live attenuated virus; d) the presence of possible test interference factors, such as residual process components (e.g. ovalbumin); and e) the need for the investigation of other pyrogens that are not detectable by the methods prescribed in the YF vaccine monograph. The product-specific testing was carried out by using cryopreserved and fresh whole blood, and IL-6 and IL-1ß levels were used as the marker readouts. After assessing the applicability of the MAT on a 1:10 dilution of 17DD-YFV, endotoxin and non-endotoxin pyrogens were quantified in spiked batches, by using the lipopolysaccharide and lipoteichoic acid standards, respectively. The quantitative analysis demonstrated the correlation between the MAT and the Limulus amoebocyte lysate (LAL) assays, with respect to the limits of endotoxin recovery in spiked batches and the detection of no pyrogenic contamination in commercial batches of 17DD-YFV. The data demonstrated the applicability of the MAT for 17DD-YFV pyrogen testing, and as an alternative method that can contribute to biological quality control studies.

Monocyte Activation Test (MAT) as a possibility of replacement for the rabbit pyrogen test in hyperimmune sera

ABSTRACT The use of a commercial kit for the monocyte-activation test (MAT) was evaluated for assessing pyrogenic contamination of hyperimmune sera . Three batches of sera, two pyrogen free and one pyrogenic, were tested. Endotoxin spike recover indicated that sample dilutions from 1/2 to 1/10 are suitable. Kit transport and storage conditions were also evaluated, proving that an adequate cold chain must be assured to achieve good results. Furthermore, the commercial MAT kit seemed suitable to replace the rabbit pyrogen test (RPT) for pyrogen testing of hyperimmune sera, although further tests are needed to a full validation.

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

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

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.