Journal: mBio

Article Title: Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium

doi: 10.1128/mbio.03233-21

Figure Lengend Snippet: Temporal progression of innate immune proteins. (A and B) Average Log 2 FC values of selected innate immunity (A) and cytosolic nucleic acid-sensing (B) proteins. All proteins shown were significantly changed (FDR of <0.05 by one-way ANOVA), with the following exceptions: LCN-2 ( P = 0.05004), S100A8 ( P = 0.061), and S100A9 ( P = 0.0513). (C to E, G, and H) qRT-PCR of the indicated mRNAs isolated from cIECs. Each point represents an individual mouse. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (by one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [uninfected mice]). Zbp1 data (C) did not pass a normality test and were not subjected to further statistical analysis. (F) LCN-2 in stool samples measured by ELISA. Each point (at individual time points) represents an individual mouse; samples at each day postinfection are from the same mice, collected over time. Data are from 3 biological repeats. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (by RM one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [0 DPI]). (I) z scores of ferritin (FTL1/FTH1) and MT2 protein abundances across the entire C. rodentium infection time course (4 to 48 DPI). Means ± SEM are shown.

Article Snippet: IFN-γ levels were determined using an IFN-γ Femto-HS high-sensitivity mouse uncoated ELISA kit (Thermo Fisher Scientific), and IL-22 levels were determined using an IL-22 mouse uncoated ELISA kit (Thermo Fisher Scientific), in both cases according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Isolation, Control, Enzyme-linked Immunosorbent Assay, Infection

Journal: mBio

Article Title: Citrobacter rodentium Infection Induces Persistent Molecular Changes and Interferon Gamma-Dependent Major Histocompatibility Complex Class II Expression in the Colonic Epithelium

doi: 10.1128/mbio.03233-21

Figure Lengend Snippet: IFN-γ and cIEC MHCII expression are not required for protective immunity against C. rodentium . (A) Quantification of the indicated immune cell types in colonic tissue during C. rodentium infection in WT mice. Each point represents an individual mouse, and data are from two biological repeats. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (by one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [uninfected mice]). (B and C) Quantification (B) and representative flow cytometry plots (C) of EpCAM + cIECs expressing MHCII (anti-IA/IE staining) in uninfected or C. rodentium -infected WT mice. Each point represents an individual mouse. Data are from two biological repeats. ****, P < 0.0001 (by one-way ANOVA with Dunnett’s posttest for multiple comparisons to the control group [uninfected mice]). (D and E) Quantification (D) and representative flow cytometry plots (E) of EpCAM + cIECs expressing MHCII (anti-IA/IE staining) in uninfected or C. rodentium -infected Ifn γ −/− mice. Each point represents an individual mouse. Data are from two biological repeats. (F) C. rodentium colonization of Ifn γ −/− mice. Each line represents an individual mouse. Data are from two biological repeats. (G) C. rodentium colonization at 10 DPI following reinfection of WT and Ifn γ −/− mice initially mock infected (UI+CR) or infected with C. rodentium (CR+CR). Each point represents an individual mouse. Data are from two biological repeats. (H) ELISA measurement of anti- C. rodentium IgG in the serum (left) and colonic explants (right) of the mice shown in panel G, 10 days after the second infection. ns, not significant ( P > 0.05); **, P < 0.01 (by unpaired two-tailed Student’s t test between the indicated groups). UI+CR colonic explant data (right) were not statistically analyzed due to the high number of samples falling below the limit of detection (LoD).

Article Snippet: IFN-γ levels were determined using an IFN-γ Femto-HS high-sensitivity mouse uncoated ELISA kit (Thermo Fisher Scientific), and IL-22 levels were determined using an IL-22 mouse uncoated ELISA kit (Thermo Fisher Scientific), in both cases according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Control, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Vaccines

Article Title: Adenovirus DNA Polymerase Loses Fidelity on a Stretch of Eleven Homocytidines during Pre-GMP Vaccine Preparation

doi: 10.3390/vaccines10060960

Figure Lengend Snippet: The VSS quality control. ( a ) The VSS established from clone 3BG1.1AC11.1AE1 was tested for genetic stability over five blind passages in the M9.S suspension cells (VSS+5), and the tHIVconsv6 protein expression was assessed in HeLa cells infected at vp MOIs of 2, 20 and 200 (lanes 2–4). Protein expression was compared to uninfected (lane 6) and RG vaccine-infected (lane 8) cells in a Western blot using HIV-1 Gag p24-specific mAb ab9071. ( b ) Using one cell passage from the VSS, material for the pre-clinical toxicity study in an animal model (ToxLot) was prepared and purified using AEX chromatography. A single virus peak was eluted at wavelengths of 260 nm (red) and 280 nm (blue). ( c ) Mouse immunogenicity. The VSS, ToxLot, VSS+5 and the RG vaccine were used to immunize BALB/cJ mice intramuscularly at a dose of 10 8 IU per animal, and the elicited T cells were tested for recognition of eight well-defined epitopes in tHIVconsv6, of which the AMQMLKETI is downstream of the mutated polycytidine region. Reactive splenocytes were enumerated as SFU in an IFN-γ ELISPOT assay. Mean ± SD ( n = 3) are shown. ( d ) DNA sequencing traces across the 11 cytidine regions of the starting BAC DNA, three SVI-3 clones and the VSS using one forward and two reverse primers indicated on the left. ( e ) tHIVconsv6 detection in Western blot by two HIV-1 Gag p24-specific antibodies given below in HeLa cells infected with the VSS (lanes 1 and 7), SVI-3 clone 3BG1.1AC11.1AE1 (lanes 3 and 8), SVI-2 clone 3BG1.1AC11 (lanes 4 and 9), uninfected (lane 5) and the RG vaccine (lane 10). ( f ) Schematic representation of the tHIVconsv6 protein indicating its regions, 6-5-4-3-2-1, originating from Pol (green) and Gag (navy blue); the regional junctions (yellow), including junction 2-1 generating 11 cytidines and two CD8 + T cells (red); and one mAb 91-5 (blue) epitopes used in the analyses. ( g ) Initially, the entire transgene in the VSS was sequenced directly from the purified genome (VSS). Then, the 11-C fragment was excised from the VSS genome and subcloned into pUC19. Next, 20 and, later, 100 bacterial colonies were sequenced. The table indicates the lengths of the cytidine run and their frequencies.

Article Snippet: An enzyme-linked immunospot (ELISPOT) assay was performed using a mouse IFN-γ ELISpot kit (Mabtech, Stockholm, Sweden) according to the manufacturer’s instructions as described previously [ ].

Techniques: Control, Suspension, Expressing, Infection, Western Blot, Animal Model, Purification, Chromatography, Virus, Immunopeptidomics, Enzyme-linked Immunospot, DNA Sequencing, Clone Assay