The coronavirus disease COVID‐19 was first described in December 2019. The peripheral blood of COVID‐19 patients have increased numbers of neutrophils which are important in controlling the bacterial infections observed in COVID‐19. We sought to evaluate the cytotoxic capacity of neutrophils in COVID‐19 patients. 34 confirmed COVID‐19 patients (29 severe, five mild disease), and nine healthy controls were recruited from the Masih Daneshvari Hospital (Tehran, Iran) from March to May 2020. Polymorphonuclear (PMN) cells were isolated from whole blood and incubated with green fluorescent protein (GFP)‐labelled methicillin‐resistant Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) . Bacterial growth was determined by measuring the florescence of co‐cultures of bacteria and neutrophils and reported as the lag time before exponential growth. The number of viable bacteria was determined after 70 hours as colony‐forming units (CFU). The immunophenotype of tested cells was evaluated by flow cytometry. Isolated neutrophils have higher surface expression of CD16 and CD62L with negative markers for PMN‐MDSC. Bacterial growth in the presence of SA (22 ± 0.9 versus 9.2 ± 0.5 h, P < .01) and PA (12.4 ± 0.6 versus 4.5 ± 0.22, P < .01) was significantly reduced in COVID‐19 patients. After 70 h incubation of PMN with bacteria (SA and PA), CFUs were significant increased in COVID‐19 patients SA (2.6 ± 0.09 × 10 8 CFU/mL‐severe patients and 1.4 ± 0.06 × 10 8 CFU/mL‐mild patients, P < .001) and PA (2.2 ± 0.09 × 10 9 CFU/mL‐severe patients and 1.6 ± 0.03 × 10 9 CFU/mL‐mild patients, P < .001). Gentamycin proliferation assays confirmed the presence of intracellular bacteria. Reduced bacterial killing by neutrophils from COVID‐19 patients may be responsible for the high bacterial yield seen in these patients.
Background Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. Results According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. Conclusions In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25–35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.
A new coronavirus disease was described in December 2019 (COVID-19) in Wuhan City, Hubei Province, China and has reached pandemic status. According to the World Health Organization, the incubation time from being infected to symptom emergence averages 5-6 days for COVID-19 but can be up to 14 days. The mortality rate varies in different countries but is greater in elderly people and in patients with cardiovascular disease, diabetes and chronic respiratory diseases. Patients with chronic respiratory diseases often have reduced neutrophil function. We sought to measure neutrophil phagocytosis and bacterial killing in COVID-19 patients. 30 COVID-19 patients and 9 healthy individuals were recruited from the Masih Daneshvari Hospital (Tehran, Iran) from March-May 2020. Polymorphonuclear (PMN) cells were isolated from whole fresh blood and incubated with green fluorescent protein (GFP) labelled methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Phagocytosis was determined by measuring the florescence of co-cultures of bacteria and neutrophils and reported as the lag time before exponential growth. The number of viable bacteria was determined after 70 h by the Colony-Forming Unit (CFU). Bacterial phagocytosis of SA (22±0.9 versus 9.2±0.5h, p<0.01) and PA (12.4±0.6 versus 4.5±0.22, p<0.01) was significantly reduced in COVID-19 patients compared with healthy control subjects. After 70h there was a significant increase in CFU in COVID-19 subjects compared with healthy control subjects for both SA (2.6±0.09 x 108 versus 0.8±0.04 x 108 CFU/ml, p<0.001) and PA (2.2±0.09 x 109 versus 1.0±0.06 x 109 CFU/ml, p<0.001).These results suggests a defect in bacterial clearance by neutrophils in COVID-19 patients.
Background Extracellular vesicles (EVs) play a key role in cell communication and the pathogenesis of some diseases. EVs may accelerate cell death during the course of mycobacterial infection and are also considered as a new vaccine design, drug delivery, and biomarker candidates. The current study evaluates the effects of EVs from serum samples of mycobacteria-infected patients on THP-1 monocytes and PBMC cells. Method EVs were purified from the serum, then cultured separately with THP-1 monocytes and PBMCs. The cell death was determined through annexin V-FITC and PI staining. GW4869, an EVs inhibitor, was used to determine if EVs released from serum could increase THP-1 monocytes cell death. Results The cell death was significantly increased in the presence of 10 µg/ml and 5 µg/ml concentrations of the purified EVs (p < 0.05). Minimal cell death was determined in 2.5 µg/ml and 1.2 µg/ml (p < 0.05). Up to 85% of the cells were viable in the presence of the GW4869 inhibitor (p < 0.05). Conclusion Direct infection of the cells with EVs released from mycobacteria-infected patients samples, the multiplicity of infection with the EVs, and virulent or avirulent mycobacteria may change the status of the cell death. The isolated EVs from serum samples of patients with mycobacterial infection accelerated cell death, which means that they might not be considered as an optimal tool for developing drug delivery and vaccine against tuberculosis.
A new coronavirus disease was described in December 2019 in Wuhan City, Hubei Province, China and has reached pandemic status. According to the World Health Organization, the incubation time from being infected to symptom emergence averages 5-6 days for COVID-19 but can be up to 14 days. The mortality rate varies in different countries but is greater in elderly people and in patients with cardiovascular disease, diabetes and chronic respiratory diseases. Patients with chronic respiratory diseases often have reduced neutrophil function. We sought to measure neutrophil phagocytosis and bacterial killing in COVID-19 patients. 30 COVID-19 patients and 9 healthy individuals were recruited from the Masih Daneshvari Hospital (Tehran, Iran) from March-May 2020. Polymorphonuclear (PMN) cells were isolated from whole fresh blood and incubated with green uorescent protein (GFP) labelled methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Phagocytosis was determined by measuring the orescence of co-cultures of bacteria and neutrophils and reported as the lag time before exponential growth. The number of viable bacteria was determined after 70 h by the Colony-Forming Unit (CFU). Bacterial phagocytosis of SA (22±0.9 versus 9.2±0.5h, p<0.01) and PA (12.4±0.6 versus 4.5±0.22, p<0.01) was signi cantly reduced in COVID-19 patients compared with healthy control subjects. After 70h there was a signi cant increase in CFU in COVID-19 subjects compared with healthy control subjects for both SA (2.6±0.09 x 10 8 versus 0.8±0.04 x 10 8 CFU/ml, p<0.001) and PA (2.2±0.09 x 10 9 versus 1.0±0.06 x 10 9 CFU/ml, p<0.001).These results suggests a defect in bacterial clearance by neutrophils in COVID-19 patients.
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