Recombinant Bacillus Calmette-Guérin Expressing SARS-CoV-2 Chimeric Protein Protects K18-hACE2 Mice against Viral Challenge.

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

Avaliação do perfil de infiltração e relevância do uso de estabilizantes celulares nas amostras de líquido cefalorraquidiano recebidas no Laboratório de Marcadores Celulares do Centro de Hematologia e Hemoterapia de Santa Catarina / Infiltration profile evaluation and relevance of the use of cellular stabilizers in cerebrospinal fluid samples received by Cell Labelling Laboratory in Hematology and Hemotherapy Center of Santa Catarina

Objetivo: O presente estudo pretende analisar o perfil e as condições das amostras de líquido cefalorraquidiano (LCR) recebidas no setor de Marcadores Celulares do Centro de Hematologia e Hemoterapia de Santa Catarina (HEMOSC). Métodos: Os dados foram obtidos retrospectivamente através do sistema HemoSis e dos planos de trabalho contidos no laboratório num intervalo de 12 meses. Resultados: Das 117 amostras avaliadas, observa-se que 54% das provenientes do interior do estado de Santa Catarina são pouco viáveis, enquanto que apenas 9% das amostras da grande Florianópolis apresentaram este problema. No total de amostras avaliadas, quatro laudos foram inconclusivos devido à baixa viabilidade. Em relação ao perfil das amostras, somam-se LCR referentes a 65 pacientes sem predomínio de sexo, majoritariamente em idade adulta e se sobressaem as investigações de Linfoma Não-Hodgkin B e leucemias agudas. Dos pacientes, nove revelaram amostras infiltradas. Entre as neoplasias que acometem o sistema nervoso central, há um predomínio de infiltração por doenças linfoides nas amostras recebidas pelo laboratório. Além disso, a baixa viabilidade celular em algumas amostras pode estar associada a resultados inconclusivos ou de baixa confiabilidade. Conclusão: É preciso adequar as coletas do LCR e inserir o uso dos estabilizantes na rotina para evitar resultados inconclusivos e possíveis recoletas de amostra.

Effects of perioperative hypothermia on healing of anastomosis of the colon in rats.

OBJECTIVE: We evaluated the hypothesis that induced perioperative hypothermia in rats causes adverse effects on the healing of colonic anastomosis. MATERIALS AND METHODS: Forty-eight Wistar rats were divided into eight groups of six animals that underwent laparotomy, sigmoid section, and anastomosis. Four groups were operated under normothermic conditions (36 ± 1 °C) and four under hypothermic conditions (32 ± 1 °C). The reoperations were performed on days 3, 7, and 14 post-surgery, and two groups where SHAM reoperated on day 3. We evaluated anastomotic bursting pressure and tissue hydroxyproline content; performed a histological analysis of inflammatory parameters and healing (inflammatory cell infiltrate, edema, fibrin, collagen deposition and apoptotic cells) with categorization scores = 0, 1, 2, 3; and examined the relative quantification gene expression (cDNA) of inflammatory cytokines [interleukin 1 (IL-1), interleukin 6 (IL-6), and interleukin 10 (IL-10)] and growth factors [vascular endothelial growth factor and insulin-like growth factor 1 (IGF-1)] by reverse transcription polymerase chain reaction. RESULTS: Both of the hypothermic groups showed lower anastomotic burst pressure on days 7 and 14 post-surgery, reduced hydroxyproline content on day 14, reduction of inflammatory infiltrates and edema at day 3, and less collagen deposition on day 14. In animals that were hypothermic, the cytokine gene expression showed reduced IL-1 on day 3, reduced IL-6 on days 7 and 14, and reduced IL-10 on days 7 and 14 and a reduction in the growth factor IGF-1 on day 7. CONCLUSION: Perioperative hypothermia had detrimental effects on the healing of colonic anastomosis in rats.

Selection of TcII Trypanosoma cruzi population following macrophage infection.

BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, which exhibits a high genetic variability. TcI, TcII, or mixed TcI/TcII strains may be found during acute human infection while mainly TcII parasites are present at the chronic stage of disease. In a previously studied Chagas disease outbreak, we identified mixed TcI/TcII strains in the vector Triatoma tibiamaculata and only TcII strains in infected humans, indicating that T. cruzi populations may be selected within the human host. METHODS: Utilizing molecular typing and cell biology techniques, we investigated the interaction of TcI, TcII, and mixed TcI/TcII strains with macrophages, an important cell population implicated in controlling protozoan infection. RESULTS: TcII but not TcI strains were selected by both human and murine macrophages in vitro and by peritoneal cavity cells in vivo. Biological analysis revealed that, compared with TcI, TcII strains display higher infective and multiplicative ability as well as lower doubling time inside macrophages. However, TcI and TcII strains present similar susceptibility to interferon-γ-activated macrophages in vitro. CONCLUSIONS: Taken together, our results reveal the existence of an intracellular selection process in macrophages that favors TcII, but not TcI, when infection occurs with vector-derived mixed TcI/TcII strains.