Journal: International Journal of Molecular Sciences

Article Title: Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model

doi: 10.3390/ijms20092134

Figure Lengend Snippet: Altered gut morphology in Shank3αβ knock-out (KO) mice. ( A – D ) Histological evaluation of GI tract from wild type and Shank3αβ KO mice. ( A ) Longitudinal cross sections of Shank3αβ KO mice and wild type (WT) mice were stained with hematoxylin/eosin (HE) (upper panels) and periodic acid schiff (PAS) reaction (lower panels). Exemplary images are shown. ( B – D ) Morphological analysis of ( B ) villi length and ( C ) width, and ( D ) crypt depth reveals a significantly decreased villi length (Mann-Whitney U -test, p = 0.009; n = 5 animals per group) but not width ( p = 0.534), and normal crypt depth ( p = 0.983) in Shank3αβ KO mice. ( E , F ) Immunohistochemistry was performed on 5 mice per group and 5 optic fields of view each from 3 sections per mouse were analyzed. ( E ) A slight but non-significant decrease in FABP2 signal intensity was observed in Shank3αβ KO mice compared to wild types (left panel). Significantly higher ZONULIN-1 levels were found in Shank3αβ KO mice (right panel) ( t -test, p = 0.0413). ( F ) The levels of CLAUDIN3 and lipopolysaccharide (LPS) were not significantly different between Shank3αβ KO mice and wild types in gut epithelium. ( G ) Significantly higher ZONULIN-1 levels in Shank3αβ KO mice were confirmed by western blotting using gut epithelium protein lysate ( t -test, p = 0.0434, n = 3 per group). ( H ) Protein lysate from liver tissue from WT and Shank3αβ KO mice ( n = 3 per group) were analyzed for E. coli LPS levels using Western Blotting. The results show significantly higher LPS levels in the liver of Shank3αβ β KO mice ( t -test, p = 0.0452). * p < 0.05, ** p < 0.01.

Article Snippet: Zonulin 1 antibody was purchased from Thermo Fisher Scientific (Invitrogen) (Waltham, MA, USA); Claudin3 antibody from Abcam (Berlin, Germany); FABP2 antibody from Thermo Fisher Scientific (Invitrogen); LPS antibody from Origene (Rockville, MD, USA); and IL6 antibody was purchased from Cell signaling Technologies (Danvers, MA, USA); GFAP antibody was purchased from Sigma Aldrich (St. Louis, MO, USA); Cytokeratin and Vimentin antibody from Abcam.

Techniques: Knock-Out, Staining, MANN-WHITNEY, Immunohistochemistry, Western Blot

Journal: Cellular immunology

Article Title: Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation.

doi: 10.1016/j.cellimm.2019.103962

Figure Lengend Snippet: Fig. 1. Stimulation with mAb-iFt enhances BMDC maturation. WT BMDCs were stimulated with LPS (2.25 ng/mL), iFt (MOI 50), or mAb-iFt (MOI 50) for 24 h. Supernatants were collected for cytokine analysis (D) and cells were stained for CD80, CD86, and MHC II (A–C). (A) After gating out debris and doublets, unstained cells were used to draw gates on CD80+, CD86+, or MHC II+ cell populations. Histograms representative of one experiment are shown. (B) Geometric mean fluorescent intensities (MFI) for CD80, CD86, or MHC II positive cells from the histograms in (A) were combined from three separate experiments and plotted as mean ± SEM. Further characterization of the MHC II+ cells was then conducted by plotting CD80 vs. CD86 as shown in (C). Contour Plots are representative of one experiment whereas combined data from three separate experiments is shown in the bar graphs as mean ± SEM. (D) Cytokine analysis of BMDC supernatants was completed via Bio-Rad multiplex kit. Concentrations of IL-6, IL-12p70, and TNF-α from three different experiments are shown as mean ± SEM. All bar graphs were analyzed with an ordinary one-way ANOVA with a Tukey’s post-test after Log10 transforming the data to account for heteroskedasticity. *p ≤0.05, **p ≤0.01, ***p ≤0.001 compared to media unless noted with a bar.

Article Snippet: Anti-F. tularensis LPS monoclonal antibody (mouse IgG2a) was purchased from Fitzgerald Industries (Acton, MA).

Techniques: Staining, Multiplex Assay

Journal: Cellular immunology

Article Title: Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation.

doi: 10.1016/j.cellimm.2019.103962

Figure Lengend Snippet: Fig. 6. MyD88 deficient BMDCs do not mature when stimulated with mAb-iFt. (A) WT (white bars) or MyD88−/−(gray bars) BMDCs were stimulated with iFt (50 iFt/ cell), mAb-iFt (50 iFt/cell), LPS (2.25 ng/mL), or Poly(I:C) (10 µg/mL) for 24 h before being stained for maturation markers. (B) Cytokines analysis was completed on the BMDC supernatants via multiplex. Data are combined from three experiments where bars represent mean ± SEM and Log10 transformed to account for het- eroskedasticity prior to analysis via two-way ANOVA with Sidak’s post-test (WT vs. knockout). *p ≤0.05, ***p ≤0.001, & ****p ≤0.0001.

Article Snippet: Anti-F. tularensis LPS monoclonal antibody (mouse IgG2a) was purchased from Fitzgerald Industries (Acton, MA).

Techniques: Staining, Multiplex Assay, Transformation Assay, Knock-Out

Journal: Cellular immunology

Article Title: Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation.

doi: 10.1016/j.cellimm.2019.103962

Figure Lengend Snippet: Fig. 7. mAb-iFt-stimulated BMDCs have reduced maturation marker expression when TLR9 signaling is blocked. (A) WT BMDCs were pretreated with either media (white bars) or the TLR9 antagonist ODN 2088 (10 µM, gray bars) for 1 h before being stimulated with iFt (50 iFt/cell), mAb-iFt (50 iFt/cell), LPS (2.25 ng/mL), or the TLR9 ligand ODN 1826 (5 µM) for 24 h. BMDCs were subsequently stained for maturation markers (A) and supernatants analyzed by multiplex for cytokines (B). Data are combined from three experiments where bars represent mean ± SEM. Data were Log10 transformed to account for heteroskedasticity and subsequently analyzed via two-way ANOVA with Sidak’s post-test (media vs. antagonist). *p ≤0.05, **p ≤0.01, ***p ≤0.001, & ****p ≤0.0001.

Article Snippet: Anti-F. tularensis LPS monoclonal antibody (mouse IgG2a) was purchased from Fitzgerald Industries (Acton, MA).

Techniques: Marker, Expressing, Staining, Multiplex Assay, Transformation Assay

Journal:

Article Title: Chlamydia trachomatis infection modulates trophoblast cytokine/chemokine production 1

doi: 10.4049/jimmunol.0800764

Figure Lengend Snippet: Inclusion formation and infection rates in trophoblast cells infected with C. trachomatis. (A) Trophoblast cells (Sw.71) were exposed to either No infection (NI), Ct serovar D (Ct-D) or Ct serovar L1 (Ct-L1) at a MOI of 1, by rocking/resting at room temperature for 2 hour. Inclusion formation was evaluated by light microscopy at 24 and 36 hours post-infection for Ct-L1 and Ct-D, respectively. Inclusions are highlighted by arrow heads (Mag. ×40). (B) After 36 hours of infection with or without Ct-L1 or Ct-D, inclusion formation in the Sw.71 and H8 cells was evaluated by by staining cells intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. Infection rates were then determined by both immmunofluorescent microscopy and flow cytometry. The flow cytometry histograms show two distinct populations: the left hand peak being the uninfected cells, and the right hand peak with the marker representing the infected trophoblast population.

Article Snippet: After this, cells were centrifuged and then resuspended in 100ml of Perm/Wash and 3μl of a FITC-conjugated anti-Ct-LPS monoclonal antibody (mAb) was added, (#1649; ViroStat Inc, Portland, ME) and incubated for 25 minutes on ice in the dark.

Techniques: Infection, Light Microscopy, Staining, Microscopy, Flow Cytometry, Marker

Journal:

Article Title: Chlamydia trachomatis infection modulates trophoblast cytokine/chemokine production 1

doi: 10.4049/jimmunol.0800764

Figure Lengend Snippet: Infection rates of trophoblast cells infected with C. trachomatis by different techniques. (A) H8 and Sw.71 cells were infected with Ct (serovar D) at an MOI of 1 by either rocking or by centrifugation. After 36 hours, the cells were collected and stained intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. Centrifugation resulted in a higher rate of infection in both cell lines (B) H8 and Sw.71 cells were infected with or without Ct (serovar D) at an MOI of 1 by centrifugation. After 48 and 72 hours inclusion formation was visualized by light microscopy. Inclusions contained within the cells are highlighted by arrow heads, while extruded inclusions are highlighted by asterisks (Mag. ×40).

Article Snippet: After this, cells were centrifuged and then resuspended in 100ml of Perm/Wash and 3μl of a FITC-conjugated anti-Ct-LPS monoclonal antibody (mAb) was added, (#1649; ViroStat Inc, Portland, ME) and incubated for 25 minutes on ice in the dark.

Techniques: Infection, Centrifugation, Staining, Light Microscopy

Journal:

Article Title: Chlamydia trachomatis infection modulates trophoblast cytokine/chemokine production 1

doi: 10.4049/jimmunol.0800764

Figure Lengend Snippet: Chlamydia-infected trophoblast cells produce viable EBs. H8 and Sw.71 cells were infected with Ct (serovar D) at an MOI of 1 by centrifugation. After 36 hours, the cells were collected and stained intracellularly with a FITC-conjugated mouse anti-Ct LPS mAb. The infection levels were then determined by flow cytometry (i & iv). In parallel, lysates were prepared from Ct-infected H8 and Sw.71 cells and these were then immediately applied to a culture of uninfected HeLa cells. After 48 hours, the HeLa cells exposed to infected Sw.71 or H8 lysates were collected and the infection levels determined by flow cytometry. Histograms (ii & v) show the levels of HeLa cell Ct infection (solid line), when compared to the uninfected HeLa cells (dotted line). The HeLa cells exposed to infected Sw.71 or H8 lysates were also evaluated for inclusion formation by light microcopy (iii & vi). Inclusions contained within the cells are highlighted by arrow heads, while extruded inclusions are highlighted by asterisks (Mag. ×40) (iii & iv).

Article Snippet: After this, cells were centrifuged and then resuspended in 100ml of Perm/Wash and 3μl of a FITC-conjugated anti-Ct-LPS monoclonal antibody (mAb) was added, (#1649; ViroStat Inc, Portland, ME) and incubated for 25 minutes on ice in the dark.

Techniques: Infection, Centrifugation, Staining, Flow Cytometry