Assessment of Pyrogenic Response of Medical Devices and Biomaterials by the Monocyte Activation Test (MAT): A Systematic Review.

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.

Dual activation profile of monocytes is associated with protection in Mexican patients during SARS-CoV-2 disease.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been one of the most catastrophic diseases observed in recent years. It has reported nearly 550 million cases worldwide, with more than 6.35 million deaths. In Mexico, an increased incidence and mortality of this disease were observed, where the immune response has been involved in the magnitude and severity. A critical version of the disease is accompanied by hyperinflammatory responses, with cytokine and defective cellular responses. A detailed understanding of the role of molecules and cells in the immune response during COVID-19 disease may help to generate effective protection mechanisms, improving those we already have. Here we analyzed blood samples obtained from patients at the Hospital Regional de Alta Especialidad de Ixtapaluca (HRAEI), Mexico, which were classified according to living guidance for clinical management of COVID-19 by the World Health Organization: asymptomatic, mild, severe, and critical disease. We observed increased interleukin (IL)-6 levels and a T-CD8+ and T-CD4+ cell reduction correlated with the critical disease version. Importantly, here, we described a significant reduction of CD11b+CD45highCD14low monocytes during severe disease, which displayed a non-classical profile, expressing IL-10, transforming growth factor (TGF)-ß, and indoleamine 2,3-dioxygenase (IDO)1 molecule. Moreover, CD11b+CD45highCD14low monocytes obtained from infected one-dose vaccinated patients (Pfizer® vaccine) who suffered minimal symptoms showed simultaneously a dual classical and no-classical profile expressing pro- and anti-inflammatory cytokines. These results suggest that blood monocytes expressing a dual pro- and anti-inflammatory profile might be a predictive marker for protection in the Mexican population during COVID-19 disease. KEY POINTS : • Exacerbated immune response is associated with COVID-19 severe disease. • Dual monocyte activation profile is crucial for predicting protection during COVID-19. • Vaccination is crucial to induce the dual activation profile in monocytes.

Avaliação do Teste de Ativação de Monócitos (MAT) para a detecção de pirógenos em Vacina Zika Inativada experimental

Injectable products including vaccines must be safe and pyrogen free. Pyrogens are molecules that once recognized by immune system can result in inflammatory responses of IL-1β e IL-6 cytokines release, leading to fever. In order to reduce number of laboratory animals, this study aimed to evaluate Monocyte Activation Test (MAT) efficiency at pyrogen detection in Zika Vaccine. The test was performed with PyroDetect System Merck Millipore, according supplier instructions. Zika vaccine was tested pure and diluted at 1/10, 1/20 and 1/40. Pyrogen recovery was tested by using spiked and non-spiked samples. After incubation with human cryopreserved blood Cryoblood® 16-18h, 37°C and CO2 5%, the incubation mixture blood and samples was collected to quantify IL-1β levels by ELISA. The results indicate low cytokine levels at tested samples, including spiked samples. This suggests vaccine had some interferers that might hinder the monocytes response. Furthermore, standard curves resulted lower absorbance values than expected, which would lead to misestimated endotoxin concentration in the samples. To assess test accuracy the calculated endotoxin recovery must be between 80 and 120% of spiked endotoxin. The results demonstrated variable recovery to same endotoxin concentration. We concluded this test seemed instable and it did not comply with accuracy and reproducibility parameters, for Zika Vaccine in the tested condition.

As vacinas, bem como todas as formulações injetáveis para uso humano, devem ser seguras e livres de pirogênio. Os pirógenos podem ser descritos como substâncias que, quando reconhecidas pelo sistema imune inato, desencadeiam respostas inflamatórias que resultam na liberação de citocinas, como IL-1β e IL-6, e podem ocasionar diversos sintomas, entre eles a febre. Com o intuito de substituir o uso de coelhos, esse estudo buscou verificar a eficiência do Teste de Ativação de Monócitos (MAT) para sua validação analítica na detecção de pirógenos, lipopolissacarídeos (LPS) e ácido lipoteicóico (LTA), no concentrado da Vacina Zika Inativada através da quantificação do mediador inflamatório IL-1β. Para isso, foi utilizado o PyroDetect System Merck Millipore, seguindo as orientações do fabricante. O concentrado da vacina Zika foi testado puro e em diluições de 1/10, 1/20 e 1/40, com e sem a contaminação por LPS ou LTA. As amostras foram incubadas com sangue humano criopreservado Cryoblood® – Kit PyroDetect por 16-18h a 37oC e, em seguida, foi realizado um ensaio de ELISA para quantificar os níveis de IL-1β liberados no sobrenadante. Os resultados dos testes indicaram baixos níveis de detecção de IL-1β nas diluições testadas, inclusive nas amostras que foram contaminadas por endotoxina. Esses resultados sugerem que a vacina Zika possa ter algum componente que cause interferência no teste. Além disso, verificou-se menor liberação de IL-1β nos ensaios do que o esperado, o que poderia levar a resultados superestimados da concentração de endotoxina nas amostras. Para determinar a exatidão do teste, foram calculadas as taxas de recuperação de endotoxina para cada ensaio, e estas deveriam estar em uma faixa entre 80 e 120% para a validação. No entanto, os resultados demonstraram taxas de recuperação fora da faixa esperada. Sendo assim, concluímos que os parâmetros de exatidão, precisão e reprodutibilidade não foram atingidos para a Vacina Zika nas condições testadas.

Potential Involvement of Platelet-Derived Microparticles and Microparticles Forming Immune Complexes during Monocyte Activation in Patients with Systemic Lupus Erythematosus.

Microparticles (MPs) are vesicles derived from the plasma membrane of different cells, are considered a source of circulating autoantigens, and can form immune complexes (MPs-ICs). The number of MPs and MPs-ICs increases in patients with systemic lupus erythematosus (SLE). MPs activate myeloid cells by inducing IL-6 and TNF-α in both SLE and other diseases. Therefore, we propose that the recognition of MPs-ICs by monocytes rather that MPs may define their phenotype and contribute to the inflammatory process in patients with SLE. Thus, the aims of this study were to evaluate the association among circulating MPs-ICs from different cell sources, alterations observed in monocyte subsets, and disease activity in patients with SLE and to establish whether monocytes bind and respond to MPs-ICs in vitro. Circulating MPs and monocyte subsets were characterized in 60 patients with SLE and 60 healthy controls (HCs) using multiparametric flow cytometry. Patients had higher MP counts and frequencies of MPs-CD41a + (platelet-derived) compared with HCs, regardless of disease activity. MPs from patients with SLE were C1q + and formed ICs with IgM and IgG. MPs-IgG + were positively correlated with active SLE (aSLE), whereas MPs-IgM + were negatively correlated. Most of the circulating total ICs-IgG + were located on MPs. The proportion and number of non-classical monocytes were significantly decreased in patients with SLE compared with HCs and in patients with aSLE compared with patients with the inactive disease. Non-classical monocytes obtained from patients with SLE exhibited increased levels of CD64 associated with MPs-IgG +, MPs-C1q +, total circulating ICs-IgG +, and disease activity. The direct effects of MPs and MPs-IgG + on monocytes were evaluated in cell culture. Monocytes from both HCs and patients bound to and internalized MPs and MPs-IgG + independent of CD64. These vesicles derived from platelets (PMPs), mainly PMPs-IgG +, activated monocytes in vitro and increased the expression of CD69, CD64, and pro-inflammatory cytokines such as IL-1ß, TNF-α, and IFN-α. Therefore, MPs are one of the most representative sources of the total amount of circulating ICs-IgG + in patients with SLE. MPs-IgG + are associated with SLE activity, and PMPs-IgG + stimulate monocytes, changing their phenotype and promoting pro-inflammatory responses related to disease activity.

Métodos alternativos para a detecção de pirogênios em produtos e ambientes sujeitos a Vigilância Sanitária: avanços e perspectivas no Brasil a partir do reconhecimento internacional do Teste de Ativação de Monócitos / Alternative methods for the detection of pyrogens in products and environment subject to public health surveillance: advances and perspectives in Brazil based on the international recognition of the Monocyte Activation Test

Introduction: The detection of pyrogens is essential for the quality control of injectable products. The Rabbit Pyrogen Test remains widely used, despite the existence of alternative methods such as the Monocyte Activation Test (MAT). Objective: To review the use of alternative methods for pyrogen testing, pointing out advances and perspectives from the recognition of MAT by the European pharmacopoeia and its acceptance for regulatory purposes in Brazil. Method: A search was performed on the PubMed and BVS databases, with further classification, categorization by topic and critical analysis of the results. Results: Twenty-four papers were identified, addressing topics such as applications of MAT, its validation and comparisons with in vivo tests. MAT presented better results when compared to other tests, both in the evaluation of biological products and in the detection of non-endotoxin pyrogens. Limitations to diffusion include difficulties in obtaining whole human blood as a source of monocytes, for which several alternatives have been proposed. Conclusions: MAT is a promising method, with application in safety evaluation of new technologies. Its application in Brazil depends on a national implementation policy, which might include greater integration between BraCVAM, Concea and RENAMA in search for its recognition for regulatory purposes.

Introdução: A detecção de pirogênios é imprescindível no controle da qualidade de produtos injetáveis. O Teste de Pirogênio em coelhos ainda tem larga aplicação, apesar da existência de métodos alternativos como o Teste de Ativação de Monócitos (MAT). Objetivo: Revisar o uso dos métodos alternativos no teste de pirogênio, apontando avanços e perspectivas a partir do reconhecimento do MAT pela Farmacopeia Europeia e sua aceitação para fins regulatórios no Brasil. Método: Uma busca foi realizada nas bases PubMed e BVS, com posterior classificação, categorização por assuntos e análise crítica dos resultados. Resultados: Foram identificados 24 trabalhos, abordando temas como as aplicações do MAT, sua validação e comparação com testes in vivo. O MAT apresentou melhores resultados quando comparado a outros testes, tanto na avaliação de produtos biológicos como na detecção de pirogênios não-endotoxinas. Limitações para sua difusão incluem a dificuldade de obtenção de sangue total humano como fonte de monócitos, para o qual diversas alternativas têm sido propostas. Conclusões: O MAT se mostra um método promissor, com aplicação na avaliação da segurança de novas tecnologias. Sua aplicação no Brasil depende de uma política nacional de implantação, que inclua maior Integração entre BraCVAM, Concea e RENAMA na busca por seu reconhecimento para fins regulatórios.

Soluble ß-(1,3)-glucans enhance LPS-induced response in the monocyte activation test, but inhibit LPS-mediated febrile response in rabbits: Implications for pyrogenicity tests.

In the present study, we aimed to determine the influence of ß-(1,3)-d-glucans on the LPS-induced pro-inflammatory cytokine response in the Monocyte Activation Test (MAT) for pyrogens, and on the LPS-induced febrile response in the Rabbit Pyrogen Test (RPT), thus evaluating the resulting effect in the outcome of each test. It was found that ß-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same ß-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, ß-(1,3)-d-glucans significantly enhanced the LPS-induced pro-inflammatory cytokines response in MAT, insomuch that samples containing non-pyrogenic concentrations of LPS become pyrogenic. On the other hand, ß-(1,3)-d-glucans had no effect on sub-pyrogenic LPS doses in the RPT, but surprisingly, inhibited the LPS-induced febrile response of pyrogenic LPS concentrations. Thus, while ß-(1,3)-d-glucans could mask the LPS pyrogenic activity in the RPT, they exerted an overstimulation of pro-inflammatory cytokines in the MAT. Hence, MAT provides higher safety since it evidences an unwanted biological response, which is not completely controlled and is overlooked by the RPT.