Inhibition of SARS-CoV-2 Infection in Vero Cells by Bovine Lactoferrin under Different Iron-Saturation States.

Despite the rapid mass vaccination against COVID-19, the emergence of new SARS-CoV-2 variants of concern, such as omicron, is still a great distress, and new therapeutic options are needed. Bovine lactoferrin (bLf), a multifunctional iron-binding glycoprotein available in unsaturated (apo-bLf) and saturated (holo-bLf) forms, has been shown to exert broad-spectrum antiviral activity against many viruses. In this study, we evaluated the efficacy of both forms of bLf at 1 mg/mL against infection of Vero cells by SARS-CoV-2. As assessed with antiviral assays, an equivalent significant reduction in virus infection by about 70% was observed when either form of bLf was present throughout the infection procedure with the SARS-CoV-2 ancestral or omicron strain. This inhibitory effect seemed to be concentrated during the early steps of virus infection, since a significant reduction in its efficiency by about 60% was observed when apo- or holo-bLf were incubated with the cells before or during virus addition, with no significant difference between the antiviral effects of the distinct iron-saturation states of the protein. However, an ultrastructural analysis of bLf treatment during the early steps of virus infection revealed that holo-bLf was somewhat more effective than apo-bLf in inhibiting virus entry. Together, these data suggest that bLf mainly acts in the early events of SARS-CoV-2 infection and is effective against the ancestral virus as well as its omicron variant. Considering that there are no effective treatments to COVID-19 with tolerable toxicity yet, bLf shows up as a promising candidate.

CLEC5A expression can be triggered by spike glycoprotein and may be a potential target for COVID-19 therapy.

The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins. Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo, and in silico assays. The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor-binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Finally, CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.

In Vitro Inhibition of SARS-CoV-2 Infection by Bovine Lactoferrin

Since its emergence in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been posing a serious threat to public health worldwide as the causative agent of coronavirus disease 2019 (COVID-19). Now distributed in a pandemic pattern, this disease still lacks an effective drug treatment with low toxicity, leading pharmaceutical companies and research labs to work against time to find a candidate molecule to efficiently treat the affected patients. Due to the well-known broad-spectrum antimicrobial activity of the lactoferrin protein, we sought to verify whether its bovine form (bLf) would also be effective in vitro against SARS-CoV-2. Using an antiviral assay based on quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we found that bLf reduced progeny virus yield by up to [~]84,6% in African green monkey kidney epithelial cells (Vero E6) and [~]68,6% in adenocarcinomic human alveolar basal epithelial cells (A549) at 1 mg/mL, a concentration previously shown to have low cytotoxicity. Therefore, our preliminary data suggest that bLf has the potential to constitute a biochemical approach to fight the new coronavirus pandemic.

Relação entre polimorfismo do gene do hormônio do crescimento e características de precocidade em novilhas da raça Nelore / Relationship between polymorphism of growth hormone and precocity traits in Nellore heifers

Avaliaram-se as relações entre o polimorfismo do gene do hormônio do crescimento (GH) e as características de precocidade, em novilhas da raça Nelore. Amostras de sangue periférico foram obtidas de 181 animais de três rebanhos distintos do estado da Bahia, nas quais foi realizada a extração de DNA e a amplificação por PCR, seguidas por digestão com enzima de restrição AluI. Os fragmentos resultantes da digestão enzimática foram analisados em gel de agarose 2 por cento para determinação dos respectivos genótipos. A frequência do alelo Leu nas amostras analisadas foi estimada em 100 por cento. Em decorrência da alta incidência de homozigose para o alelo Leu, sugere-se que o restriction fragment lenght polymorphism AluI do gene GH não possa ser considerado como marcador molecular para precocidade sexual em novilhas Nelore nesses rebanhos.

The relationships between polymorphism of growth hormone gene (GH) and precocity traits in Nellore heifers were evaluated. A total of 181 animals from three different farms of Bahia state, Brazil, were blood sampled. The DNA of each animal was extracted, amplified by PCR, and digested by "AluI" restriction enzyme, and the resultant fragments were analyzed in 2 percent agarose gel for genotype identification. The frequency of Leu allele in the analyzed samples was estimated in 100 percent. Due to the high incidence of homozygose for the Leu allele, it is suggested that the restriction fragment lenght polymorphism AluI of GH gene can not be considered as a molecular marker for sexual precocity in Nellore heifers of those herds.

Atividade analgésica do extrato da Pectis jangadensis (S. Moore) / Preliminary evaluation of Pectis jangadensis (S. Moore) analgesic activity

A planta Pectis jangadensis, popularmente conhecida como "roxinha", "erva do carregador" ou "coentro da chapada" é utilizada em chás pela população matogrossense, como calmante. Não há estudo na literatura sobre qualquer atividade farmacológica dessa planta. Nosso objetivo foi avaliar atividade analgésica de seu extrato hidro-alcoólico nas doses de 100, 500 e 1000 mg/kg pelos métodos de Contorções induzidas pelo ácido acético e teste da formalina. Os nossos resultados demonstram que não houve diferença significativa entre o grupo controle e os grupos tratados com diferentes doses do extrato tanto em relação ao número de contorções induzidas pelo ácido acético quanto ao tempo de lambedura da pata, na fase 1 e 2, induzida pela formalina. Portanto, pelo menos, por via oral, parece que o extrato hidro-alcoólico da Pectis jangadensis não apresenta atividade analgésica.

"Preliminary evaluation of Pectis jangadensis (S. Moore) analgesic activity's". The plant Pectis jangadensis, popularly known as "roxinha", "erva do carregador" or "coentro da chapada" is used in teas by the matogrossense's people, as a calmative. There isn't any study in the literature about its pharmacologic activity. Our object was to evaluate its extract hidroalcoolic analgesic activity in doses of 100, 500 and 1000 mg/kg by the methods of abdominal writhing induced by acetic acid and the Formalin test. The results show that there wasn't significant difference between the control groups and the groups treated with different doses of the extract in both testes. Therefore, at least by oral, seems that the Pectis jangadensis' hidroalcoolic extract doesn't have analgesic activity.

The polymorphism in MUC1 gene in Nelore cattle.

MUC1 is a transmembrane glycoprotein expressed on the apical surfaces of the uterine epithelial tissue with predicted functions in protection and cell-cell adhesion. These properties are closely related with the repetitive region [variable number of tandem repeats (VNTR)] of the extracellullar domain and with the O-glycosylation in their serine and threonine residues. This study describes a polymerase chain reaction (PCR) protocol to analyse MUC1 bovine genetic polymorphism and demonstrates the existence of a VNTR within the sites for O-glycosylation. Oligonucleotide primers based on the Bos taurus mucin (MUC1) gene sequence GenBank AF399757 were used to amplify five VNTR MUC1 alleles from a study group of 56 pure Nelore bovines. The number of repeats varied between 10 and 24, being more prevalent than the alleles with less number of repeats. The DNA sequence analysis revealed two repeats and one of them presented 100% homology with the bovine consensus sequence already reported. The second repeat showed codons that translate to serine and proline amino acids, which are conserved in the MUC1 of several species. This study is the first description of allelic variation and the VNTR structure in the Nelore breed MUC1 gene, and we suggest that this genetic variability can be tested for association with variation in reproductive traits in Nelore cattle.