Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Tityus serrulatus Scorpion Venom: In Vitro Tests and Their Correlation with In Vivo Lethal Dose Assay.

Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO) recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms' quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating-and discarding-poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms.

Anti-SARS-CoV-2 equine F (Ab′)2 immunoglobulin as a possible therapy for COVID-19

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab′)2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab′)2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Determination of the relationship among cell proliferation, metabolic activity and stage of pregnancy by AgNORs as markers in bovine placenta / Determinación de la relación entre la proliferación celular, la actividad metabólica y la etapa de preñez por los AgNOR como marcadores en la placenta bovina

SUMMARY: Nucleolus Organizer Regions (NORs) are defined as nucleolar components containing argyrophilic proteins selectively stained by silver methods (AgNORs). Several investigations have shown the AgNOR quantity and area represent a valuable parameter of cell kinetics, since they reflect the level of activity and cellular proliferation. This article addresses an evaluation of the functional activity and relation between days of pregnancy and proliferative capacity of trophoblastic mononucleate and binucleate cells from bovine placentomes. Both the number and size of AgNORs were determined in different phases of gestation by silver nitrate staining in conventional histological slides. The results showed a significant increase (from 1 to 12 AgNORs) in the number of AgNORS per trophoblastic mononucleate cell in the 3rd trimester, with predominance of 4-6 AgNORs/cell. In the 1st and 2nd trimesters, the number ranged between 1 and 9 AgNORs/cell, with predominance of 1-3 AgNORs. No significant differences were observed between the 2nd and 3rd trimesters, but in the first, in binucleate cells (19-27 and 10-18 AgNORs/cell, respectively) - this number was higher than the one registered in trophoblastic mononucleate cells in the same period. Thus, AgNORs can be used as markers of the proliferative placental cell cycle and established a relation between number of AgNORs and days of gestation. This relation can be used for diagnoses and prognoses of several placental pathologies, including pregnancy losses from manipulated embryos.

RESUMEN: Las Regiones Organizadoras de Nucléolos (NOR) se definen como componentes nucleolares que contienen proteínas argirofílicas teñidas selectivamente por métodos de plata (AgNOR). Varias investigaciones han demostrado que la cantidad y el área de AgNOR representan un parámetro importante de la cinética celular, ya que reflejan el nivel de actividad y proliferación celular. Este trabajo analiza la actividad funcional y la relación entre los días de preñez y la capacidad proliferativa de las células trofoblásticas mononucleadas y binucleadas de placentomas bovinos. Tanto el número como el tamaño de los AgNOR se determinaron en diferentes fases de la gestación mediante tinción con nitrato de plata en portaobjetos histológicos convencionales. Los resultados mostraron un aumento significativo (de 1 a 12 AgNOR) en el número de AgNORS por célula mononucleada trofoblástica en el tercer trimestre, con predominio de 4-6 AgNOR / célula. En el primer y segundo trimestre, el número osciló entre 1 y 9 AgNOR / célula, con predominio de 1-3 AgNOR. No se observaron diferencias significativas entre el 2do y 3er trimester; en el primer trimestre, en células binucleadas (19-27 y 10-18 AgNORs / célula, respectivamente) - este número fue superior a la cantidad registrada en células mononucleadas trofoblásticas en el mismo período. Por tanto, los AgNOR se pueden utilizar como marcadores del ciclo celular placentario proliferativo y se establece una relación entre el número de AgNOR y los días de gestación. Esta relación puede ser útil en el diagnóstico y pronóstico de varias patologías placentarias, incluidas las pérdidas de preñeces de embriones manipulados.

Preparedness against pandemic influenza: production of an oil-in-water emulsion adjuvant in Brazil

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Preparedness against pandemic influenza: production of an oil-in-water emulsion adjuvant in Brazil

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4–8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

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.

Slc11a1 (Nramp1) alleles interact with acute inflammation loci to modulate wound-healing traits in mice.

Lines of mice were obtained by selective breeding for maximum (AIRmax) or minimum (AIRmin) acute inflammation. They present distinct neutrophil influx and show frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) alleles. This gene is involved in ion transport at the endosomes within macrophages and neutrophils, interfering in their activation. Homozygous AIRmax and AIRmin sublines for the Slc11a1 gene were produced to examine the interaction of this gene with the acute inflammatory loci. The present work investigated wound-healing traits in AIRmax and AIRmin mice, in F(1) and F(2) intercrosses, and in Slc11a1 sublines. Two-millimeter ear punches were made in the mice and hole closure was measured during 40 days. AIRmax mice demonstrated significant tissue repair while AIRmin mice did not. Significant differences between the responses of male and female mice were also observed. Wound-healing traits demonstrated a correlation with neutrophil influx in F(2) populations. AIRmax( SS )showed higher ear-wound closure than AIRmax( RR ) mice, suggesting that the Slc11a1 S allele favored ear tissue repair. QTL analysis has detected two inflammatory loci modulating ear wound healing on chromosomes 1 and 14. These results suggest the involvement of the acute inflammation modifier QTL in the wound-healing phenotype.