Journal: bioRxiv

Article Title: Characterization of bronchovascular-bundle mesenchymal stromal cells regulating antibody-secreting cell niche in rejecting lung allografts

doi: 10.1101/2025.02.16.638532

Figure Lengend Snippet: ( A ) FACS–sorted cell populations from adult mouse lung were analyzed by real-time PCR. Values are represented as means ± SEM. *p<0.05, ****p<0.0001. One-way ANOVA; post hoc test: Bonferroni’s. ( B ) Uniform manifold approximation and projection (UMAP) plot from scRNA-Seq on CD45 − CD31 − cell populations sorted from adult mouse lungs . ( C ) UMAP plots of Cxcl12 and ll6, with coordinates same as in B , but the color intensity indicates gene expression level within each cell. ( D ) Violin plots show expression intensity of Cxcl12 and Il6 in each of the 14 clusters. ( E ) Correlation plot showing Foxf1_MC cluster distinctly expressing Cxcl12 and Il6 gene expression. ( F ) Radar plot representing differentially expressed key genes marking the signature of each lung mesenchymal clusters. ( G ) Cxcl12 CreERT2 Rosa26 tdTomato adult mouse lung sections were immunolabeled for CXCL12 (red), mesenchymal αSMA (green) and epithelial EpCAM (white). Bronchovascular field shows presence of peri-bronchial and perivascular mesenchymal cells staining positive for CXCL12. n=3 per group. Original magnification: 200x; Scale bar = 50 µm. Abbreviations: AW, airway; V, vessel.

Article Snippet: CXCL12 levels were measured using the Human CXCL12/SDF-1⍺ Quantikine ELISA Kit (DSA00, R&D Systems) according to the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Expressing, Immunolabeling, Staining

Journal: bioRxiv

Article Title: Characterization of bronchovascular-bundle mesenchymal stromal cells regulating antibody-secreting cell niche in rejecting lung allografts

doi: 10.1101/2025.02.16.638532

Figure Lengend Snippet: ( A ) Flow cytometry to sort cell populations from Cxcl12 CreERT2-tdTomato mice that are CD45 − /CD31 − /EpCAM − /PDGFRα + MCs that are CXCL12(+) or CXCL12(−). ( B ) Cxcl12 and Il6 mRNA expression analyzed by real-time PCR of sorted populations from A. ( C ) mRNA expression of pro-fibrotic genes by real-time PCR of sorted populations from A. Values are represented as means ± SEM. *p<0.05, **p<0.01, ****p<0.0001 . One-way ANOVA; post hoc test: Bonferroni’s. ( C ) Immunofluorescent imaging of representative tissue section from Cxcl12 CreERT2-tdTomato mice in the pleural region showing CXCL12+ MCs (red). ( D ) Immunofluorescent imaging of representative tissue section from day 28 murine RAS allografts in the pleural region showing CD138 (red) overlapping with Ig deposition (green) and B220 (white). C-D. n=3. Original magnification = 200x. Scale bar = 50 μm.

Article Snippet: CXCL12 levels were measured using the Human CXCL12/SDF-1⍺ Quantikine ELISA Kit (DSA00, R&D Systems) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Imaging

Journal: bioRxiv

Article Title: Characterization of bronchovascular-bundle mesenchymal stromal cells regulating antibody-secreting cell niche in rejecting lung allografts

doi: 10.1101/2025.02.16.638532

Figure Lengend Snippet: ( A ) Cxcl12 and Il6 mRNA expression analyzed by real-time PCR in FACS-sorted cell populations from an isograft and day 28 RAS allografts. Values are represented as means ± SEM. *p<0.05, **p<0.01, ****p<0.0001 . One-way ANOVA; post hoc test: Bonferroni’s. ( B ) Schema for the generation of Cxcl12 CreERT2-tdTomato donor mice and lung transplant experiments. TAM, tamoxifen. ( C ) Representative immunofluorescence imaging of Cxcl12-tdTomato Red + MCs in n=3 RAS allografts. Scale bars: 100 μm (zoom, cropped: 50 μm).

Article Snippet: CXCL12 levels were measured using the Human CXCL12/SDF-1⍺ Quantikine ELISA Kit (DSA00, R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Characterization of bronchovascular-bundle mesenchymal stromal cells regulating antibody-secreting cell niche in rejecting lung allografts

doi: 10.1101/2025.02.16.638532

Figure Lengend Snippet: ( A, B ) CXCL12 mRNA expression analyzed by real-time PCR in MCs derived from lung transplant patients diagnosed with (CLAD) or without CLAD (non-CLAD) ( A ) and in non-CLAD MCs treated with TGF-β (2 ng/mL) and LPA (10 μM) ( B ). ( C ) non-CLAD MCs were treated with vehicle, IL-6 (50 ng/mL), sIL-6R (200 ng/mL) or the combination of IL-6 and sIL-6R for 30 minutes. Expression of CXCL12 mRNA (real-time PCR) and protein (ELISA) in MCs and conditioned media, respectively. ( D-F ) non-CLAD MCs were pre-treated with JAK1/2 pan-inhibitor, Ruxolitinib (RUX), or JAK3 inhibitor, WHI-P154 (WHI), and the transcription inhibitor, Actinomycin D (ActD) and then stimulated with IL-6 and sIL-6R. D. CXCL12 and IL6 mRNA expression in MCs (real-time PCR) E. CXCL12 protein expression analyzed by ELISA in the conditioned media. F. Representative western blot of STAT3-Tyr705 phosphorylation. ( G, H ) non-CLAD MCs were transfected with lentiviral vectors that allowed constitutional activation of STAT3 (CA- STAT3 ) or scrambled controls. G. STAT3 and IL6 mRNA expression analyzed by real-time PCR. H. CXCL12 mRNA and protein expression in MCs (real-time PCR) and conditioned media (ELISA), respectively. I. non-CLAD MCs were transfected with scrambled control siRNA or sequences specific to CXCL12 , and then stimulated with IL-6 and IL-6R for 48h. Conditioned media was used to stimulate 0.5 million splenocytes in the upper chamber to migrate through the membrane. The number of CD3 − CD19 + B lymphocytes in the lower chamber was measured by FACS analyses. Values are represented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 . One-way ANOVA; post hoc test: Bonferroni’s ( A-E , G-I ).

Article Snippet: CXCL12 levels were measured using the Human CXCL12/SDF-1⍺ Quantikine ELISA Kit (DSA00, R&D Systems) according to the manufacturer’s protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Transfection, Activation Assay, Control, Membrane

Journal: bioRxiv

Article Title: Characterization of bronchovascular-bundle mesenchymal stromal cells regulating antibody-secreting cell niche in rejecting lung allografts

doi: 10.1101/2025.02.16.638532

Figure Lengend Snippet: ( A ) Experimental schematic. B6D2F1/J donor lungs were transplanted into C57BL/6J recipient mice, followed by treatment with Olamkicept (2.5 mg/kg/week; intraperitoneal injections) between days 3 and 28. ( B ) CXCL12 protein concentration by ELISA in lung homogenates from A . ( C , D ) Isografts, or RAS allografts treated with or without Olamkicept or Bortezomib were sacrificed at day 28. Serum was analyzed for IgG ( C ) and IgM (C) levels by ELISA. ( E ) FACS analyses of antibody secreting cells (ASCs). (F) Immunofluorescent staining of representative tissue sections showing antibody secreting cells marked by B220/CD138/IgG and airway epithelial integrity marked by CCSP. Representative sections stained for Movat Pentachrome. n=3. Original magnification = 200x. Scale bar = 50 μm. ( G ) Quantitative assessment of fibrosis by measuring hydroxyproline content in graft lung homogenates. Values are represented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-way ANOVA; post hoc test: Bonferroni’s ( B-E , G ).

Article Snippet: CXCL12 levels were measured using the Human CXCL12/SDF-1⍺ Quantikine ELISA Kit (DSA00, R&D Systems) according to the manufacturer’s protocol.

Techniques: Protein Concentration, Enzyme-linked Immunosorbent Assay, Staining

Journal: The Prostate

Article Title: Pilot and feasibility study of serum chemokines as markers to distinguish prostatic disease in men with low total serum PSA.

doi: 10.1002/pros.20717

Figure Lengend Snippet: Fig. 1. A:LogserumCXCL5orCXCL12(ng/ml)differsdependingonprostatediseasestatus.ELISA-derivedvaluesforserumCXCL12formen withoutevidenceofprostaticdisease(whitetriangles),menwithoutcancerbutwithhistologicalprostatitis(gray triangles)andmenwithpros- tatecancer(blacktriangles),aswellasthelogserumCXCL5formenwithoutevidenceofprostaticdisease(whitediamonds),menwithoutcancer butwithhistologicalprostatitis (graydiamonds), andmenwithprostate cancer (blackdiamonds) are shown on a logarithmic scale. Significant differences (P < 0.050) are indicatedby *, trends (0.065 < P < 0.050) by #.B: Log serum CXCL5 (ng/ml) relevant to prostate volume.ELISA- derivedvalues for serumCXCL5 formenwithlow volume(37.5g)(circles)or high-volume(>37.5g)(diamonds) prostateswithoutcanceror histologicalprostatitis (white), withoutcancer butwithhistologicalprostatitis (gray), or with cancer (black) are shown on a logarithmic scale Differencesbetweengroupsthatachievedstatisticalsignificance(P < 0.05)areindicatedby*;trendsby#.Thedatarepresentedherearedrawn fromTable II.C: Log serum CXCL12 (ng/ml) relevant to prostate volume.ELISA-derived values for serum CXCL12 for men with low volume (37.5 g) (circles) or high-volume (>37.5 g) (diamonds) prostates without cancer or histological prostatitis (white), without cancer but with histological prostatitis (gray), or with cancer (black) are shown on a logarithmic scale. Differences between groups that achieved statistical significance (P < 0.05) is indicated by *; trends by #.The data represented here is drawn fromTable II.D: Log chemokine values in serum and plasma.ELISA-derivedvalues for CXCL12 (left) or CXCL5 (right) from serum (diamonds) orplasma (squares) are shown on alogarithmic scale for6of the51patientsexaminedinthisstudy.

Article Snippet: Circulating serum CXCL5 (ENA-78) or CXCL12 (SDF-1) chemokine levels were assessed using 50 ul frozen serum or plasma per direct sandwich ELISA in duplicate using the Human CXCL5/ENA-78 DuoSet kit DY254 or theHumanCXCL12/SDF-1 alpha capture antibody MAB350, detection antibody BAF310, and standard 350-NS ELISA reagents (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Clinical Proteomics