Journal: Frontiers in Immunology

Article Title: Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation

doi: 10.3389/fimmu.2022.1018774

Figure Lengend Snippet: Detection of antibodies against endogenous Pgp3 in C. muridarum -infected cell samples. Sera collected from each group were used to detect endogenous Pgp3 by an immunofluorescence assay. C. muridarum -infected cells were costained with mice antisera (red) as indicated at the top of the figure, rabbit anti-chlamydial EB polyclone antibody (green), and DNA dye (blue). Only Pgp3 trimer-immunized mice produced antibodies that recognized endogenous Pgp3.

Article Snippet: A genus-specific rabbit antibody against chlamydial EBs and Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)-labeled goat anti-rabbit IgG secondary antibody were used to visualize the chlamydial inclusions.

Techniques: Infection, Immunofluorescence, Produced

Journal: Frontiers in Immunology

Article Title: Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation

doi: 10.3389/fimmu.2022.1018774

Figure Lengend Snippet: Live chlamydial organism shedding from mice vaginas following challenge. (A) For the experiment, 5-6-week-old BALB/c mice were immunized intranasally with Pgp3 trimer + CpG, Pgp3 monomer + CpG, or CpG alone on days 0, 20, and 30. In total, five mice/group were sacrificed before the challenge to monitor specific humoral and cellular immune responses. The remaining mice (n = 10) were challenged intravaginally with ( C ) muridarum , and vaginal live chlamydial organism shedding was monitored every 4 days until 28 days after infection. Day 60 post-challenge, all mice were sacrificed for pathological evaluation. (B) Vaginal swabs were collected for measuring IFU numbers, and each swab IFU number was converted into log 10 . The log 10 IFUs were compared between the three groups of mice at each time point using the ANOVA Test and a Wilcoxon rank sum test was used to further analyze the speed of clearance. Significant differences were observed between the Pgp3 trimer versus CpG alone groups. ** p < 0.01, * p < 0.05, analyzed by ANOVA and a Wilcoxon rank sum test.

Article Snippet: A genus-specific rabbit antibody against chlamydial EBs and Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)-labeled goat anti-rabbit IgG secondary antibody were used to visualize the chlamydial inclusions.

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Efficacy of Pgp3 vaccination for Chlamydia urogenital tract infection depends on its native conformation

doi: 10.3389/fimmu.2022.1018774

Figure Lengend Snippet: Percentage of positive chlamydial shedding in diverse immunization groups.

Article Snippet: A genus-specific rabbit antibody against chlamydial EBs and Cy2 (green; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)-labeled goat anti-rabbit IgG secondary antibody were used to visualize the chlamydial inclusions.

Techniques:

Journal: BMC Microbiology

Article Title: CCL5 regulation of mucosal chlamydial immunity and infection

doi: 10.1186/1471-2180-8-136

Figure Lengend Snippet: CCL5, CCR5, and IFN-γ mRNA expression during Chlamydia infections . Groups of female BALB/c mice challenged with C. muridarum and total RNA was isolated from lymphocytes isolated from the spleen, ILNs, fallopian tubes, uterus, and cervix of each mouse, under sterile conditions before or 7 and 14, 21 and 42 days after challenge. The levels of CCL5, CCR5 and IFN-γ mRNA expression were ascertained after RT-PCR analysis. Log 10 copies of transcripts were expressed relative to the actual copies of 18S rRNA ± SEM. Experiments were repeated 3 times to yield 15 mice per group. Asterisks (*) indicate significant differences ( p < 0.01) between tissues from naïve mice.

Article Snippet: Briefly, Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti- Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence [ ].

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Microbiology

Article Title: CCL5 regulation of mucosal chlamydial immunity and infection

doi: 10.1186/1471-2180-8-136

Figure Lengend Snippet: Chlamydia-specific CD4 + T cell proliferation . Groups of naïve or female BALB/c mice challenged with C. muridarum and received 100 μl of control Ab or anti-CCL5 Ab solution every 3 days. Following sacrifice 42 days after challenge, spleen-, ILN-, fallopian tubes-, uterus-, and cervix-derived CD4 + T cells were purified and cultured at a density of 5 × 10 6 cells/ml with 10 6 cells/ml of γ-irradiated feeder splenocytes for 3 days. Proliferation of CD4 + T cells was measured by BrdU incorporation. The data presented are the mean OD 450 for proliferative ± SEM of quadruplicate cultures. Experiments were repeated 3 times to yield 15 mice per group. Asterisks (*) indicate statistically significant differences ( p < 0.01) between untreated, control Ab-treated mice and mice treated with anti-CCL5 Ab.

Article Snippet: Briefly, Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti- Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence [ ].

Techniques: Derivative Assay, Purification, Cell Culture, Irradiation, BrdU Incorporation Assay

Journal: BMC Microbiology

Article Title: CCL5 regulation of mucosal chlamydial immunity and infection

doi: 10.1186/1471-2180-8-136

Figure Lengend Snippet: Serum and vaginal Ab responses of mice infected with genital Chlamydia . Groups of naïve or female BALB/c mice challenged with C. muridarum and received 100 μl of control Ab or anti-CCL5 Ab solution every 3 days. Following sacrifice 42 days after challenge, Chlamydia -specific serum and vaginal Ab responses of mice were determined by ELISA that was capable of detecting > 20 pg/ml of IgM, IgG, IgA, and IgG subclass Abs. The data presented are the mean concentration of IgG1, IgG2a, IgG2b, IgG3, IgM, IgG, or IgA ± SEM of three separate experiments. Experiments were repeated 3 times to yield 15 mice per group. Asterisks (*) indicate statistically significant differences ( p < 0.01) between untreated and control Ab- or anti-CCL5 Ab-treated mice.

Article Snippet: Briefly, Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti- Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence [ ].

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: BMC Microbiology

Article Title: CCL5 regulation of mucosal chlamydial immunity and infection

doi: 10.1186/1471-2180-8-136

Figure Lengend Snippet: T helper cytokine secretion by CD4 + T cells from Chlamydia -infected mice . Groups of naïve or female BALB/c mice challenged with C. muridarum and received 100 μl of control Ab or anti-CCL5 Ab solution every 3 days. Following sacrifice 42 days after challenge, spleen- and ILN-derived CD4 + T cells from these mice were purified and cultured at a density of 5 × 10 6 cells/ml with 10 6 cells/ml of γ-irradiated feeder splenocytes for 3 days. Cytokines present in cultured supernatants were determined by ELISA that was capable of detecting < 10 pg of IL-2, IFN-γ, IL-4, IL-6, IL-10, or GM-CSF. The data presented are the mean cytokine (pg/ml) ± SEM of quadruplicate cultures. Asterisks (*) indicate statistically significant differences ( p < 0.01) between untreated and control Ab- or anti-CCL5 Ab-treated mice.

Article Snippet: Briefly, Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti- Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence [ ].

Techniques: Infection, Derivative Assay, Purification, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: BMC Microbiology

Article Title: CCL5 regulation of mucosal chlamydial immunity and infection

doi: 10.1186/1471-2180-8-136

Figure Lengend Snippet: Changes in Chlamydia -shedding . Mice treated with control Ab or anti-CCL5 Ab were infected with C. muridarum . The status of infection was monitored for live organism shedding by culturing periodic cervico-vaginal swabs from individual mice every week for 42 days after challenge. Chlamydia present in swabs were detected by infecting McCoy cells with vaginal swab rinses and staining infected monolayer with FITC-labeled, genus-specific, anti- Chlamydia Abs to count inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence. BD indicates the presence of Chlamydiae was below this level of detection. Experiments were repeated 3 times to yield 15 mice per group. Asterisks (*) indicate statistically significant differences ( p < 0.01) between untreated and control Ab- or anti-CCL5 Ab-treated mice.

Article Snippet: Briefly, Chlamydia present in swabs were detected by infecting McCoy cells vaginal/swab rinses and staining infected monolayer with fluorescein isothiocyanate (FITC)-labeled, genus-specific, anti- Chlamydia Abs (Kallestad Diagnostics) to verify inclusion bodies (> 10 IFUs/ml) by direct immuno-fluorescence [ ].

Techniques: Infection, Staining, Labeling, Fluorescence