im7 8 1  (Miltenyi Biotec)

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 1 CD44 expression in renal specimens from LN patients. (A) Left panel: representative images of CD44 staining in normal kidney specimens (Control, n = 6) and patients with active proliferative LN (n=15). Black arrows depict recruitment of CD44+ immune cells into the kidney and yellow arrows indicate CD44 staining in tubular cells. Original magnification x400. Right panel: violin plot showing CD44-positive staining as a percentage of the whole image area using ImageJ software. Each dot represents CD44 staining from an individual control or LN patient. Data analysed using Mann- Whitney test. Box and Whiskers plots comparing CD44 gene expression in micro-dissected (B) glomeruli from 37 healthy controls and 73 LN patients and (C) tubulo-interstitial compartments from 36 healthy controls and 95 LN patients. Data obtained from bulk RNA-Seq datasets GSE127797, GSE32591, GSE69438 and GSE37463. (D) tSNE plots showing CD44 expression in resident renal cells using public scRNA-Seq dataset obtained from 21 LN patients and 3 healthy controls (33). Each dot represents a single cell and grey to blue colour represents low to high CD44 expression. (E) tSNE plots showing CD44 expression in renal tubular cells (n = 1223 cells) after sub-clustering according to their canonical markers: ALDOB and MIOX for PTEC (n = 177 cells), CALB1 for distal tubular cells (DC, n = 400 cells) and UMOD and SLC12A1 for Loop of Henle cells (LoH, n = 646 cells). Data obtained from public scRNA-Seq dataset from 21 LN patients and 3 healthy controls (33). (F) Histogram showing CD44 expression in renal tubular cells after sub-clustering in healthy controls and LN patients.

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Expressing, Staining, Control, Software, MANN-WHITNEY, Gene Expression, RNA Sequencing

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 3 CD44 expression in NZB/W F1 mice with progressive LN. Left panel: Representative images showing CD44 expression in NZB/W F1 mice with progressive disease, where 8, 16, 25 and 32 weeks of age represent different stages of disease, namely pre-nephritis, emergence of anti-dsDNA antibodies, active nephritis and severe disease respectively (n = 6 mice per timepoint). Original magnification x200. Right panel: scatterplot showing CD44-positive staining as a percentage of the whole image area using ImageJ software. Each dot represents an individual mouse. Horizontal line represents the mean for each group. ***P<0.001, compared to 8 weeks of age, ##P<0.001, 16 weeks vs 32 weeks. Data analysed using Kruskal- Wallis test followed by Dunn’s multiple comparison post-test.

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Expressing, Staining, Software, Comparison

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 2 CD44 expression in immune cells in LN patients. (A) tSNE plots showing CD44 gene expression in immune cells using public scRNA-Seq dataset obtained from kidney specimens from 24 LN patients and 10 healthy controls (34). (B) Violin plots showing CD44 gene expression in different subsets of macrophages, dendritic cells, T cells and B cells in LN patients. Violin plots showing CD44 expression in (C) tissue resident macrophages (D) CD56dimCD16+ NK cells, (E) effector memory CD4+ T cells and (F) resident memory CD8+ T cells in 10 healthy controls and 24 LN patients. Each dot represents a single cell. Horizontal line represents mean value for each group.

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Expressing, Gene Expression

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 4 Effect of anti-CD44 antibody on clinical, serological and histological parameters of disease in NZB/W F1 Mice. Serum (A) creatinine and (B) urea levels in NZB/W F1 mice treated with either Control IgG (Cont, n = 4) or anti-CD44 antibody (CD44 Ab, n = 5) for 4 weeks. (C) Urine albumin-to- creatinine ratio (ACR) determined weekly from commencement of study to study’s end for mice treated with Control IgG (n = 4) or anti-CD44 antibody (n = 5). *P<0.05, Control IgG vs anti-CD44 antibody for the same time-point. (D) Gene expression of CD44, VCAM-1, ICAM-1, TGF-b1, a- SMA and FN in the renal cortex from NZB/W F1 mice treated with either Control IgG (Control, n = 4) or CD44 Ab (n = 5) for 4 weeks. Each sample was assessed in triplicate by qPCR, normalized to GAPDH and each dot represents the mean value for each mouse. Data analysed using Mann- Whitney test for each gene. (E) Upper panel: representative images showing H & E, CD3, CD4, CD19, F4/80, CD44, HA, collagen (determined by Masson’s trichrome), VCAM-1 and NGAL staining in mice treated with either Control IgG (Control, n = 6) or CD44 Ab (n = 6) for 4 weeks. In H & E image, asterisks depict tubular atrophy, hashtag depicts protein cast formation, arrowhead depicts immune cell infiltration and arrow depicts areas of glomerulosclerosis. Original magnification x200. Lower panels: Glomerular and tubulo-interstitial lesion scores as determined by H & E staining was graded for each mouse as described in the Methods and Materials. Scatterplots showing staining of CD3+ T cells, CD4+ T cells, CD19+ B cells and macrophages as determined by F4/80 staining, and expression of CD44, HA, collagen, VCAM-1 and NGAL as a percentage of the whole image area as assessed by ImageJ software. Horizontal line represents the mean for each group. Data analysed using Mann-Whitney test. (F) Serum CD44 and (G) Serum VCAM-1 levels in NZB/W F1 mice treated with Control IgG (n = 4) or CD44 Ab (n = 5) for 4 weeks. In a parallel study, serum CD44 and VCAM-1 levels were measured in mice treated with vehicle (n = 5) or MMF (n = 6) for 4 weeks. Serum from 8-week old pre-disease mice (n = 6) served as baseline CD44 and VCAM-1 levels. **P<0.01, compared to pre-disease mice, #P<0.05, ##P<0.01, Control vs CD44 Ab, or Vehicle vs MMF. Each dot represents an individual mouse. Data analysed using Kruskal-Wallis test followed by Dunn’s multiple comparison post-test. Mice were 29- 32 weeks of age at the time of sacrifice for panels (A–G). Although 6 mice were assigned to Control IgG- and anti-CD44 antibody-treated groups, frozen biological samples from 2 Control IgG-treated mice and 1 anti-CD44 antibody-treated mouse were compromised, and these 3 samples were not used for subsequent clinical (C), serological (A, B, F, G) or mRNA analyses (D). (H) PPI network for CD44, HA, FN (FN1), TGF-b1, VCAM-1 and NGAL (LCN2) constructed using STRING database, with a minimum required interaction score of 0.7 (high confidence). Eleven nodes were identified with 27 edges (PPI enrichment P value 5.56 x10-10). Line thickness indicates confidence level of protein-protein interaction. Coloured nodes show their interaction in various biological processes, molecular functions, KEGG pathways and association with disease. False discovery rates (FDR) are shown. ACAN, aggrecan; HAS2, hyaluronan synthase 2; HYAL2, hyaluronidase-2; ITGAD, integrin aD (receptor for VCAM-1); ITGAV, integrin aV; SPP1, osteopontin (ligand for CD44).

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Control, Gene Expression, MANN-WHITNEY, Staining, Expressing, Software, Comparison, Construct

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 5 Serum CD44 level in LN patients and control groups. (A) Scatterplot showing serum CD44 level in healthy subjects (n = 46), LN patients with active disease (LN A, n = 41) or remission (LN R, n = 39), non-renal SLE patients with active disease (SLE A, n = 18), non-renal SLE patients in remission (SLE R, n = 35) and non-lupus CKD patients (n = 37). ***P<0.001, compared to LN A; ###P<0.001, compared to LN R. Data analysed using Kruskal-Wallis test followed by Dunn’s multiple comparison post-test. (B) Serum CD44 level was determined in paired serum samples whereby one sample was obtained during active disease and another during remission (n = 39). Horizontal line represents mean for each group. Data analysed using Wilcoxon signed-ranked test. (C) Heatmap showing correlation of serum CD44 level with serological and clinical parameters of disease (n = 80). Graphs showing the temporal relationship between serial serum CD44 level and (D) anti-dsDNA antibody level, (E) serum C3 level, (F) SLEDAI-2K score, (G) renal SLEDAI-2K score (H) urine albumin-to-creatinine ratio, (I) serum creatinine level, (J) eGFR and (K) serum urea level in LN patients. Results are expressed as mean ± SEM (n = 39). Dashed line represents mean CD44 level in healthy subjects. (L) ROC AUC analysis of serum CD44 level in distinguishing patients with active LN (n = 41) from patients with quiescent LN (blue line, n = 39), active non-renal SLE (green line, n = 18), CKD (red line, n = 37) or healthy subjects (black line, n = 46).

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Control, Comparison

Journal: Frontiers in immunology

Article Title: Cluster of differentiation-44 as a novel biomarker of lupus nephritis and its role in kidney inflammation and fibrosis.

doi: 10.3389/fimmu.2024.1443153

Figure Lengend Snippet: FIGURE 6 Schematic illustration showing the potential of CD44 as a novel biomarker of active LN, and its role in LN pathogenesis. During active LN, serum CD44 level is increased and correlates with serological and clinical parameters of disease. Its level is increased approximately 4.5 months before clinical symptoms of active LN is detected suggesting both predictive and prognostic potential. Serum CD44 level can distinguish between patients with active LN and quiescent LN, active non-renal SLE and non-lupus CKD suggesting specificity for renal manifestation in SLE patients. It is expressed by both immune and non-immune cells, and its location in the kidney parenchyma is dependent on disease progression, being present in glomerular and tubular cells and immune cells during increased immunological activity, and in crescents and interstitial space during onset of nephritis and kidney fibrosis. CD44 contributes to the recruitment of myeloid cells and lymphocytes into the tubulo-interstitium and increases tubulo-interstitial deposition of HA, a-SMA, collagen and FN resulting in tubular atrophy and proteinuria. Increased a-SMA expression may indicate epithelial-to-mesenchymal transition in PTEC. Treatment with anti-CD44 antibody attenuates histopathological changes in the kidney and reduces lymphocyte recruitment into the kidney parenchyma and this is associated with improvement in kidney function. CD44 may be a novel target of therapeutic intervention. Created with BioRender.com.

Article Snippet: Rat B cell hybridomas that produce monoclonal antibody against mouse CD44 (clone IM7.8.1) were purchased from American Type Culture Collection (ATCC, Tin Hang Technology Limited, Hong Kong).

Techniques: Biomarker Discovery, Activity Assay, Expressing