Journal: Journal of Fungi

Article Title: Vaginal Clinical Isolates of Candida albicans Differentially Modulate Complosome Activation in Vaginal Epithelial Cells

doi: 10.3390/jof11070501

Figure Lengend Snippet: Intracellular C3aR and C5aR1 analysis. A monolayer of VECs was infected or not (Mock) with the Colonizing strain (Col) or VVC strain (VVC) for 4 h. After incubation, intracellular C3aR ( a ) and C5aR1 ( b ) were analyzed by cytofluorimetric analysis. Data in the graphs show the mean ± SEM of C3aR- and C5aR1-positive cells (cell counts from 8000 live cells) obtained from 4 different experiments. ns—not significant; * p ≤ 0.05.

Article Snippet: The anti-C3aR antibody was obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Infection, Incubation

Journal: Journal of Fungi

Article Title: Vaginal Clinical Isolates of Candida albicans Differentially Modulate Complosome Activation in Vaginal Epithelial Cells

doi: 10.3390/jof11070501

Figure Lengend Snippet: Activation of complosome in VECs by C. albicans . Differential activation of C3, C3a and C3b ( a ) and C5 and C5a ( b ) and intracellular C3aR and C5aR1 involvement in VECs infected with the Colonizing or VVC-associated strain. Arrows indicate changes in the levels of complement factors and receptors after infection with either the Colonizing strain (left) or the VVC strain (right). Green indicates increased levels and red indicates lower levels as compared to resting levels. Created with BioRender (adapted from King B.C. and Blom A.M., 2024 ).

Article Snippet: The anti-C3aR antibody was obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Activation Assay, Infection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Botulinum Neurotoxin Induces Neurotoxic Microglia Mediated by Exogenous Inflammatory Responses.

doi: 10.1002/advs.202305326

Figure Lengend Snippet: Figure 5. Activation of Complement cascade. a) TGF𝛽is expressed substantially more in C5 treatment compared to DR5 or NAPs5 treated cells. b) C1q was not significantly expressed with either DR5 or NAP’s5 treatment. c–e) Immunofluorescent images show no significant microglial conversion to a proinflammatory phenotype with CD86 and C3aR upon treatment with DR5CM and NAPs5CM, hence not driving the microglia into a proinflammatory phenotype. f) Chemoattractant CCL2 was not significantly expressed with DR5CM and NAPs5CM treatment. g,h) No significant change in the number of synapses was observed with DR5MG and NAPs5MG. i) The schematic summarizes our mechanism study on BoNT/A-derived CNS neurodegeneration. Scale bar: 50 and 10 μm insets. All experiments were repeated ≥3 times; all parameters are presented as mean ± SD. p values were calculated using one-way ANOVA followed by Sidak’s multiple comparison test. ****p <0.0001, ***p <0.001, *p <0.1****.

Article Snippet: Antibodies Company Catalog # Dilution ratio pTau (AT8) Thermo Scientific MN1020 1:100 GFAP Sigma–Aldrich AB5541 1:200 MAP2 Cell Signaling Technology 4542 1:200 NeuN Abcam Ab177487 1:100 Cd86 Abcam AB196564 1:100 CD206 Fisher Scientific NB6001415 1:100 CD33 Stem cell 60096 1:200 C3aR Santacruz sc-133172 AF647 1:200 C5aR Biolegend 344306 1:200 TREM2 R&D systems AF1828 1:100 Synapsin Abcam Ab254349 1:100 ChAT Abcam ab223346 1:100 Cells were then fixed at RT, 20 min incubation in fresh 4% paraformaldehyde aqueous solution (157-4, Electron Microscopy Sciences) followed by rinsing twice with PBS.

Techniques: Activation Assay, Derivative Assay, Comparison

Journal: Cancers

Article Title: Deciphering the Role of the Anaphylatoxin C3a: A Key Function in Modulating the Tumor Microenvironment

doi: 10.3390/cancers15112986

Figure Lengend Snippet: Mouse Primers used for qRT-QPCR analyses.

Article Snippet: Serial FFPE sections were processed using the Leica Biosystems Bond-III automated staining system for immunohistochemical staining with the mouse anti-human C3a (clone 184 K13/16, Biolegend, San Diego, CA, USA),or the mouseanti-human C3aR (clone D12, ref SC133172, Santa Cruz, CA, USA), the anti-human CD163 (clone 10D6, Leica), the anti-human CD68 (clone 514H12, ready-to-use, Leica), the anti-human CD248 (clone E9Z7O, Cell Signaling, Danvers, MA, USA), and the anti-human SOX10 (clone EP268, ready-to-use, BIO SB).

Techniques: Sequencing

Journal: Cancers

Article Title: Deciphering the Role of the Anaphylatoxin C3a: A Key Function in Modulating the Tumor Microenvironment

doi: 10.3390/cancers15112986

Figure Lengend Snippet: Different models of inflammation regulate C3aR, C3a and PI3K expression (at the RNA level). 3T3-L1, B16/F0 and Raw 264.7 Blue cells were stimulated with IFN-γ (20 ng/mL), TGF-β (20 ng/mL) and LPS (1 µg/mL) for 24 h. ( A ) C3aR, ( B ) C3a, ( C ) PI3K, ( D ) IL-6, ( E ) TNF-α, ( F ) CCL-2 and ( G ) CCL-5 RNA expression was determined by qRT-PCR ( n = 3). Reported values are means ± SEM of three independent experiments. Two-way ANOVA followed by a multiple comparison test (Bonnferroni) was used to calculate the p value. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to unstimulated cells (Ct)).

Article Snippet: Serial FFPE sections were processed using the Leica Biosystems Bond-III automated staining system for immunohistochemical staining with the mouse anti-human C3a (clone 184 K13/16, Biolegend, San Diego, CA, USA),or the mouseanti-human C3aR (clone D12, ref SC133172, Santa Cruz, CA, USA), the anti-human CD163 (clone 10D6, Leica), the anti-human CD68 (clone 514H12, ready-to-use, Leica), the anti-human CD248 (clone E9Z7O, Cell Signaling, Danvers, MA, USA), and the anti-human SOX10 (clone EP268, ready-to-use, BIO SB).

Techniques: Expressing, RNA Expression, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Deciphering the Role of the Anaphylatoxin C3a: A Key Function in Modulating the Tumor Microenvironment

doi: 10.3390/cancers15112986

Figure Lengend Snippet: C3a modulates the expression of its own receptor and Pi3K. 3T3-L1, B16/F0 and Raw 264.7 Blue cells were stimulated with CHO F3 clone supernatants (2% and 20%) during 24 h. Ct cells are cells treated with conditioned medium from non-transfected CHO. ( A ) C3aR and ( B ) PI3K RNA expression were determined by qRT-PCR. Reported values are means ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared to non-stimulated cells (Ct)).

Article Snippet: Serial FFPE sections were processed using the Leica Biosystems Bond-III automated staining system for immunohistochemical staining with the mouse anti-human C3a (clone 184 K13/16, Biolegend, San Diego, CA, USA),or the mouseanti-human C3aR (clone D12, ref SC133172, Santa Cruz, CA, USA), the anti-human CD163 (clone 10D6, Leica), the anti-human CD68 (clone 514H12, ready-to-use, Leica), the anti-human CD248 (clone E9Z7O, Cell Signaling, Danvers, MA, USA), and the anti-human SOX10 (clone EP268, ready-to-use, BIO SB).

Techniques: Expressing, Transfection, RNA Expression, Quantitative RT-PCR

Journal: Cancers

Article Title: Deciphering the Role of the Anaphylatoxin C3a: A Key Function in Modulating the Tumor Microenvironment

doi: 10.3390/cancers15112986

Figure Lengend Snippet: Immunohistochemical assessment of C3a and C3aR expression in human melanoma and benign naevocelllular naevus. Serial formalin-fixed paraffin embedded (FFPE) sections of a human melanoma ( A – F ) and a benign case of human naevocelllular naevus ( G , H ) were processed via alkaline phosphatase-based immunostaining with either C3aR (clone D12) ( A , C , E , G ) and C3a (clone K13/16) ( B , D , F , H ). Using serial tissue sections, we were able to identify cells single or double stained for both markers. For example, the arrow points to a C3aR+/C3a− cell and the arrowheads to C3aR+/C3a+ perivascular cells (macrophage-like) in melanoma. Brown background staining was detected due to the presence of the melanin pigment in melanoma. The benign naevus revealed no C3a/C3aR expression. Images are shown at ×100 ( A , B ), ×200 ( C , D ) and ×400 ( E – H ) magnification. In addition, we confirmed that, in malignant melanoma, the majority of C3a and C3aR cells co-localized with CD68 and CD163 macrophages within the perivascular areas ( A–D). In addition, few CD248/endosialin + MSC were observed within the vascular walls ( E). To a lesser extent, C3a cells colocalized with SOX10 (nuclear staining) melanoma tumor cells ( F). Notably, double-staining experiments—for example, those using peroxidase-conjugated antibody and DAB brown color development—were not conclusive due to the intrinsic presence of brown melanin pigment within the melanoma cells and melanophages.

Article Snippet: Serial FFPE sections were processed using the Leica Biosystems Bond-III automated staining system for immunohistochemical staining with the mouse anti-human C3a (clone 184 K13/16, Biolegend, San Diego, CA, USA),or the mouseanti-human C3aR (clone D12, ref SC133172, Santa Cruz, CA, USA), the anti-human CD163 (clone 10D6, Leica), the anti-human CD68 (clone 514H12, ready-to-use, Leica), the anti-human CD248 (clone E9Z7O, Cell Signaling, Danvers, MA, USA), and the anti-human SOX10 (clone EP268, ready-to-use, BIO SB).

Techniques: Immunohistochemical staining, Expressing, Formalin-fixed Paraffin-Embedded, Immunostaining, Staining, Double Staining