Journal: medRxiv

Article Title: Identification of Plasma Growth Factors and Cytokines as Diagnostic Biomarkers for Lafora Disease

doi: 10.1101/2025.04.29.25326186

Figure Lengend Snippet: A) Heatmap showing the levels of different proteins detected using the Proteome Profiler Human Cytokine Array kit (R&D systems, see Methods). Plasma samples from eight healthy subjects and eleven LD patients were analyzed according to the manufacturer’s instructions. The intensity of the signals was analyzed using the Quick Spot software provided by the manufacturer. Heatmap includes only proteins with a fold change value between healthy and LD samples greater than 2 or lower than 0.5, along with their corresponding p-values. B) STRING analysis of the proteins identified in . Lines indicate possible protein-protein interactions, with a higher number of lines representing stronger predicted interactions.

Article Snippet: Plasma-EDTA samples from LD patients and healthy controls were analyzed using the Proteome Profiler Human Cytokine Array kit (R&D systems).

Techniques: Clinical Proteomics, Software, Protein-Protein interactions

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) levels were elevated in the synovial fluid (SF) of patients with periprosthetic joint infection (PJI), especially in the Gram-positive bacterial component-associated PJI (GPC-PJI) group. a) The expression level of CXCL5 in SF from patients with aseptic loosening (AL) and PJI was measured using protein-array assays. b) ImageJ software was used to quantify signal intensity of CXCL5 expression (two patients in the AL group and three patients in the PJI group). c) CXCL5 concentrations in SF of AL, GPC-PJI, and Gram-negative bacterial PJI (GNB-PJI) patients were measured by performing enzyme-linked immunosorbent assays. The expression of CXCL5 in the SF of PJI patients was significantly increased (four patients in the AL group, 12 patients in the GPC group, and three patients in the GNB group). Data are presented as the mean (standard error of the mean (SEM)) and analyzed using independent-samples t -test. *p < 0.05.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Infection, Expressing, Protein Array, Software

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Effects of lipoteichoic acid (LTA) treatment on chemokine (C-X-C motif) ligand 5 (CXCL5) expression in preosteoblasts and osteoblasts. a) Time-course analysis of CXCL5 expression following LTA stimulation. The preosteoblast cell line MC3T3-E1 was cultured in osteogenic induction medium (OIM) for 14 days to induce differentiation into osteoblasts. Cells were treated with LTA (0.1 μg/ml) for 24, 48, and 72 hours, and CXCL5 expression was measured at each timepoint. b) Dose-response analysis of CXCL5 expression in preosteoblasts and osteoblasts following treatment with LTA at concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μg/ml. CXCL5 expression levels positively correlated with increasing LTA concentrations in both cell types. Data are presented as mean (standard error of the mean (SEM)) and were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. PBS, phosphate-buffered saline.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Expressing, Cell Culture, Comparison, Saline

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) inhibits receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. a), c), and e) Immunofluorescence staining confirmed that CXCL5 (1 or 50 ng/ml) inhibits RANKL-induced differentiation of RAW264.7 cells into cathepsin K (CTSK)-positive osteoclast-like cells. b), d), and f) CXCL5 decreased the differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells. Purple indicates TRAP expression, and cyan indicates nuclei. g) and h) Western blot analysis showed that CXCL5 (1 ng/ml) inhibited RANKL-induced differentiation of RAW264.7 cells by suppressing the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), resulting in decreased CTSK expression at 72 hours of treatment. Data are presented as the mean (standard error of the mean (SEM)) and analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Comparison

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) attenuates receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation by inhibiting tumour necrosis factor receptor-associated factor 6 (TRAF6) downstream signalling through phospholipase Cγ2 (PLCγ2) and c-Fos. a) Schematic illustration depicting the experimental model, in which RAW264.7 cells were induced to undergo osteoclast differentiation by RANKL stimulation, with or without CXCL5 co-treatment, to investigate the regulatory role of CXCL5 in osteoclastogenesis and associated signalling pathways. b) and c) Western blot analysis was conducted to examine the time- and concentration-dependent effects of CXCL5 on RANKL-induced signalling in RAW264.7 cells. CXCR2 expression was positively correlated with CXCL5 concentration, and phosphorylation of PLCγ2 was reduced after 120 minutes of CXCL5 co-treatment, suggesting inhibition of TRAF6 downstream signalling. d) and e) CXCL5 co-treatment did not affect PLCγ1 signalling, indicating isoform specificity in the regulatory effect on PLCγ family proteins. f) and g) Western blot analysis of c-Fos and calcineurin A protein expression following CXCL5 treatment in RANKL-stimulated RAW264.7 cells. CXCL5 reduced c-Fos expression at 30 minutes post-stimulation, while having no significant impact at later timepoints. Calcineurin A levels remained unchanged across all conditions. Data are presented as mean (standard error of the mean (SEM)) and analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Western Blot, Concentration Assay, Expressing, Phospho-proteomics, Inhibition, Comparison

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) does not significantly alter mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), signal transducer and activator of transcription (STAT), or focal adhesion kinase (FAK) signalling during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. a) and b) To investigate whether CXCL5 modulates early intracellular signalling in osteoclastogenesis, RAW264.7 cells were stimulated with RANKL (50 ng/ml) to induce osteoclast differentiation, with or without co-treatment with recombinant CXCL5. Western blot analysis was conducted to examine the phosphorylation status of MAPK pathway components (p38, JNK, and ERK) at various timepoints. No significant changes in the p-p38/p38, p-JNK/JNK, or p-ERK/ERK ratios were observed following CXCL5 co-treatment. c) and d) Similarly, NF-κB signalling activity was assessed by analyzing phospho-p65 levels over time. CXCL5 had no significant effect on RANKL-induced p65 phosphorylation. e) to g) The effects of CXCL5 on other osteoclast-related signalling molecules, including STAT5, STAT6, and FAK, were also evaluated in both time- and dose-dependent settings. No significant changes were detected in STAT5/6 phosphorylation or FAK expression levels. Data are presented as mean (standard error of the mean (SEM)) and were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Recombinant, Western Blot, Phospho-proteomics, Activity Assay, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: CXCL5 suppresses osteoclastogenesis and protects against lipoteichoic acid-induced bone loss by modulating PLCγ2 and c-Fos signalling in gram-positive periprosthetic joint infection

doi: 10.1302/2046-3758.153.BJR-2025-0290.R1

Figure Lengend Snippet: Chemokine (C-X-C motif) ligand 5 (CXCL5) antibody treatment aggravates lipoteichoic acid (LTA)-induced trabecular bone loss in a mouse model. a) Representative cross-sectional and horizontal views of knee joint sections from LTA-treated mice with or without CXCL5 antibody treatment. Mice received intra-articular injections of phosphate-buffered saline (PBS; vehicle control) or LTA (10 mg/kg) on day 0 and daily intraperitoneal injections of PBS or CXCL5 antibody (2 μg/mouse) from day 0 to day 6 for a total of seven injections and were killed on day 7. The results of micro-CT revealed that CXCL5 treatment enhanced the bone loss caused by LTA. b) Quantitative results of micro-CT analysis. The medial and lateral subchondral bone plate and epiphyseal trabecular bone were analyzed separately. Data are presented as mean (standard error of the mean (SEM)). Statistical analysis was performed using two-way analysis of variance followed by Tukey’s multiple comparisons test. *p < 0.05. BV, bone volume; BS, bone surface; PI.Th, plate thickness; Po, porosity; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness; TV, tissue volume.

Article Snippet: SF specimens from two AL and three PJI patients were analyzed using a Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems, USA), which simultaneously measures the expression levels of 31 chemokines.

Techniques: Saline, Control, Micro-CT