Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) and B) Development of T1D in the spontaneous (A) and cell transfer (B) models of WT and CD44-deficient mice was monitored by measuring blood glucose. In the transfer model, irradiated CD44-deficient and WT young (6–8 weeks-old) male recipients were respectively transplanted with splenocytes from CD44-deficient and WT diabetic females. Percentage of diabetes-free mice (showing <250 mg/dL blood glucose) was recorded versus time. Statistical analysis by Breslow; A , P < 0.05; B , P < 0.005. C) The invasion capacity of infiltrating cells derived from WT (n = 6) and CD44-deficient (n = 6) NOD females (spontaneous model) was measured as indicated in Materials and Methods . A total of 754 pancreatic islets in each mouse group were scored by an uninformed observer. The percentage of islets showing each one of the infiltrating scores was calculated for each mouse, and the average values are presented. Islets from CD44-deficient mice display higher percentage of infiltrating scores (2, 3 and 4) than islets from wild type mice. P < 0.0001 by Pearson's χ2 test comparing the distribution of scores in the two mouse groups. D) Wild type or CD44-deficient pre-diabetic spleen cells were added to the top compartment of transwell migration chamber, separated by HA-coated filter (10 μg/filter) from the bottom compartment, and the % of cells migrating through the filter toward the chemoatractant in the bottom compartment was calculated by flow cytometry. Statistical analysis by Student’s- t- test. Accumulated data of 4 experiments (n = 8; error bars, SEM).

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Irradiation, Derivative Assay, Migration, Flow Cytometry

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) CD44-deficient non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44 +/+ →CD44 -/- ; marked #1) or CD44-deficient (CD44 -/- →CD44 -/- ; marked #2) diabetic NOD female donors. In addition, WT non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44 +/+ →CD44 +/+ ; marked #3) or CD44-deficient (CD44 -/- →CD44 +/+ ; marked #4) diabetic NOD female donors. Inflammatory cells are marked by arrows and local islet cells by arrowhead. The pancreases of all 4 combinations were removed after detection of diabetes or 200 days after cell transfer. Doing so, pancreases that were harvested approximately at the same time, were stained with anti-CD44 mAb. B) The % of diabetes-free mice (exhibiting less than 250 mg/dL urine glucose) was determined in corresponding four mouse groups, described in A . Statistical analysis by Breslow: #2 versus #3, P < 0.01; #1 versus #2 (follow up 75 to 200 days), P < 0.03; #3 versus #4 (follow up 20 to 75 days), P < 0.03.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Derivative Assay, Staining

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) Enhanced caspase-3 activity is detected in cell extracts of WT than in those of CD44-deficient pancreatic islet cells. Pancreatic islet cells were freshly removed from WT and CD44-deficient pre-diabetic NOD females. Cell extracts from islets were subjected to Western blot analysis, using anti-cleaved caspase-3 antibody. A representative Fig of three experiments. (B and C) Pancreata were harvested from 10-weeks-old normoglycemic WT and CD44-deficient NOD females (n = 5 in each group). β-cell apoptosis was assessed using TUNEL assay (green) and co-staining for insulin (red) and DNA (blue). B) Apoptosis was calculated as percent of TUNEL positive nuclei (+/- SEM) in insulin-positive cells out of the total number of insulin-positive cells per mouse. WT (white bar, ~17,560 total β-cells); CD44-deficient (black bar, ~14,800 total β-cells). C) Pictures are representative images of islets from WT (left panel) and CD44-deficient NOD females (right panel). Original images were taken at a magnification of x40. Inset images were digitally increased x5. White arrowheads point to apoptotic β-cells.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Activity Assay, Western Blot, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: (A and B) In vitro pro-inflammatory conditions. A) Griess assay. Wild type and CD44-deficient pancreatic islet cells were incubated with medium (-) or Cytomix for 24 and 48 h. Nitrite release was assessed by Griess assay (n = 3). Inset: Cell extracts from pancreatic islets described in ( A ) were subjected to Western blot analysis, using anti-iNOS antibody. A representative Fig of three experiments. B) Glucose-stimulated insulin secretion (GSIS). Pancreatic islet cells from WT and CD44-deficient NOD mice were incubated with medium (-) or Cytomix for 48 h and then stimulated with 3.3 or 16.7 mM glucose for additional one hour and their ability to secrete insulin was measured by ELISA. Inset: Insulin content in each one of the samples (n = 4–5) (error bars, SEM). Statistical analysis for A and B by standard two-tailed Student’s t -test. A , one representative experiment of five. B , one representative experiment of two. (C and D) Ex vivo pro-inflammatory conditions. C) Freshly isolated pancreatic islet cells from WT and CD44-deficient pre-diabetic NOD females were processed as described in ( B ) (n = 6–8) (error bars, SEM). D) Content of insulin in pancreatic islets of WT and CD44-deficient pre-diabetic NOD females was analyzed by ELISA. Statistical analysis in C and D by standard two-tailed Student’s t -test. The statistical analysis in C shows that the ex vivo insulin release of CD44-deficient pancreatic islets at 12 and 14 weeks is significantly higher (p<0.05) than the insulin release of the corresponding WT cells.The statistical analysis in D shows that the insulin content in pancreatic islets of 14 wks-old pre-diabetic WT mice is significantly different ( P < 0.05) from all other samples. (n = 6–8). In C—D , at least one representative experiment of two.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: In Vitro, Griess Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Ex Vivo, Isolation