Journal: Frontiers in Immunology

Article Title: Omicron subvariants illustrate reduced respiratory tissue penetration, cell damage and inflammatory responses in human airway epithelia

doi: 10.3389/fimmu.2023.1258268

Figure Lengend Snippet: ( A , left) Epithelial integrity is significantly disrupted in Delta versus Omicron. TEER was measured on 2 dpi (left) using a EVOM volt-ohm-meter. TEER in Ω/cm 2 was determined for all conditions (UI, Delta, Omicron subvariants BA.5, BQ1.1, BF.7) and plotted on a dot graph. Dots represent infection experiments of pseudostratified epithelia using Delta or Omicron subvariants BA.5, BQ1.1 or BF.7 measured in triplicates. Statistical significance was calculated using One-way ANOVA with Tukey´s multiple comparisons test, means are depicted in orange. ( A , middle, right) Viral load and infectivity are significantly higher in Delta versus Omicron. ( A , middle) Viral load was determined by absolute quantification using real-time RT-PCR and a SARS-CoV-2 N standard. While Delta-infected epithelia depicted a high basolateral viral load, HAE infected with Omicron subvariants illustrated low-level infection, which corresponded to ( A , right) the infectivity of released viruses as measured by plaque assay on VeroE6/ACE2/TMPRSS2-expressing cells. (B) Delta mediates higher cell death in HAE compared to Omicron subvariants BA.5, BQ1.1 and BF7. Immunofluorescence staining of HAE cells infected for 2 days with Delta or the indicated Omicron subvariants was performed using phalloidin (orange), Höchst (blue) and a live/dead cell staining kit (red) as described. % of dead cells were calculated using the Harmony™ software and one-way ANOVA with Tukey´s multiple comparisons test. (C) LDH activity is significantly higher in Delta versus Omicron. Cytotoxicity was analyzed using the Cytotoxicity Detection Kit (LDH) from Roche according to the manufacturer´s instructions (Merck, cat# 1164493001, Austria). This kit is based on measuring LDH activity released from damaged cells. Treatment of 3D human respiratory tissue models with Delta or Omicron subvariants BA.5, BQ1.1 or BF7 revealed a significantly higher LDH activity in cells infected with Delta- versus all tested Omicron subvariants. The newest subvariants BQ1.1 and BF7 were even significantly lower in their LDH activities compared to the BA.5 subvariant, indicating less cell stress and cytotoxicity. Statistical significance was calculated using One-way ANOVA with Tukey´s multiple comparisons test, means are depicted in orange. **p<0.01; ***p<0.001; ****p<0.0001. ns not significant.

Article Snippet: Normal human bronchial epithelial (NHBE, Lonza, cat# CC-2540 S, upper respiratory tract) are available in our laboratory and routinely cultured in air liquid interface (ALI) as described ( – ).

Techniques: Infection, Quantitative Proteomics, Quantitative RT-PCR, Plaque Assay, Expressing, Immunofluorescence, Staining, Software, Activity Assay

Journal:

Article Title: Phagocytic Uptake of Encephalitozoon cuniculi by Nonprofessional Phagocytes

doi:

Figure Lengend Snippet: Kinetics of the internalization of nongerminated E. cuniculi spores by various NPP cell lines and human MDM. Two million E. cuniculi spores were added to each well of confluent cells, and the monolayers were fixed after 0, 2, 4, 6, 10, and 24 h. Intracellular spores were identified using differential immunofluorescence staining. To account for the size difference between the cells studied, internalization rates are given as the number of intracellular spores per high-power field of view (magnification, ×1,000). In confluent monolayers every field covers approximately 11.5 MRC5 cells, 50 293 cells, 60 A549 cells, and 30 macrophages. Data shown are means ± SD for three independent experiments, except for time points of >6 h for 293 cells, where the results of a single experiment are given (monolayers of 293 cells tended to detach from the coverslip after >6 h). Differences between cell lines were significant at a P value of <0.0001 (two-way analysis of variance).

Article Snippet: MRC5 cells (human lung fibroblasts; ATCC CCL 171), A549 cells (human respiratory epithelial cells; ATCC CCL 185), 293 cells (human embryonic kidney cells), MDCK cells (Madin-Darby canine kidney cells; ATCC CCL 34), and RK13 cells (rabbit kidney cells; ATCC CCL 37) were maintained under a 5% CO 2 atmosphere in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum, 2 mM l -glutamine, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 0.25 mg of amphotericin B per ml.

Techniques: Immunofluorescence, Staining

Journal: Journal of Medical Virology

Article Title: SARS‐CoV‐2 pseudovirus infectivity and expression of viral entry‐related factors ACE2, TMPRSS2, Kim‐1, and NRP‐1 in human cells from the respiratory, urinary, digestive, reproductive, and immune systems

doi: 10.1002/jmv.27244

Figure Lengend Snippet: Cell lines used in the present study

Article Snippet: A549 , Lung adenocarcinoma , ATCC CRM‐CCL‐185 , DMEM complete medium.

Techniques: Cell Culture