Dose responsiveness of autoimmune CD4 T cells to anti-CD3 mediated stimulation.

<p>The number of mitoses per 10,000 CD4 T cells was determined for cultures stimulated with a range (0–2 µg/ml) of anti-CD3 concentrations; each culture received 4 µg/ml anti-CD28. The percent of the maximal mitosis is shown plotted against the dose of anti-CD3. The autoimmune strain CD4 T cells exhibit dose-response curves that are less responsive than the normal control B6. Significant differences in response are noted at the lower concentrations of anti-CD3 (*p<0.01, B6 vs all other strains, ANOVA followed by Bonferroni post-test). It is important to note that 100% represents the maximal number of mitoses achieved by that strain; the maximum for the normal strain B6 is greater than that for the autoimmune strains. Data are from three separate experiments (n>6 for all strains).</p

Susceptibility to activation induced cell death is governed by cell divisions.

<p>The percentage of cell death as detected by 7-AAD staining in the divided CD4 T cell gate is plotted against the fold reduction in median fluorescence intensity as an indicator of the extent of cell division. Dots from twelve stimulation conditions in four separate experiments are plotted. Stimulated cells from C57BL/6 animals achieve nearly twice the maximal division seen for the autoimmune strains as indicated by the maximum reduction in CFSE intensity and, therefore, they undergo more AICD. However, at shared points along the ordinate, all four strains demonstrate similar levels of cell death.</p

Plate bound stimulation normalizes the responsiveness of autoimmune CD4 T cells.

<p>Splenocytes were harvested from each strain, CFSE labeled, and stimulated in culture plates that had been previously coated with anti-CD3 and anti-CD28. The probability that a stimulated precursor will produce a daughter cell in any division is calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106347#pone-0106347-g001" target="_blank">Figure 1</a>. Following plate bound stimulation, this measure is shared among all strains. No significant differences were detected. Data are obtained from three independent experiments (n>6 for all strains).</p

B lymphocytes are required for proliferation of T cells in autoimmune prone lineages.

<p>Splenocytes harvested from B6, NOD, MRL, or NZBW were depleted of B cells by MACS with anti-B220 antibodies. The resultant populations were stimulated with 2 µg/ml anti-CD3 and 2 µg/ml anti-CD28 for 65 hours (black histogram) or left unstimulated (red histogram). At that time, minimal proliferation was detected in B cell depleted cultures in autoimmune-prone mice whereas B6 splenocytes maintained normal proliferation in the absence of B cells. Data are representative of three to four separate experiments.</p

CD8 T cells from all strains are able to reach advanced cell divisions.

<p>A) The probability that a dividing precursor generates a daughter cell in division N was calculated under maximally stimulating conditions (2 µg/ml anti-CD3/28, 65 hours) and represented as a bar graph (n = 6 for all strains). Differences in response were most evident in later divisions (*p<0.05, B6 vs all other strains, ANOVA followed by Bonferroni post-test). B) Dose Responsiveness of autoimmune CD8 T cells to anti-CD3 mediated stimulation. The number of mitoses per 10,000 CD4 T cells was determined for cultures stimulated with a range (0–2 µg/ml) of anti-CD3 concentrations; each culture received 4 µg/ml anti-CD28. The percent of the maximal mitosis is shown plotted against the dose of anti-CD3. The NOD CD8 T cells exhibit a dose-response curve that diminishes more rapidly than the other strains. Significant differences in response are noted at the lower concentrations of anti-CD3 (n>6 for all strains, *p<0.01, NOD vs all other strains, ANOVA followed by Bonferroni post-test). Presented data are obtained from two separate experiments.</p

CD4 T cells isolated from autoimmune strains exhibit truncated cell division profiles.

<p>A) Splenocytes from NOD, MRL, NZBW, or C57BL/6 female mice at 8–11 weeks of age were labeled homogeneously with CFSE and incubated with 2 µg/ml anti-CD3 and 4 µg/ml anti-CD28 for 65 hours (dotted histogram); unstimulated cells are shown for comparison (solid line). The dotted line demarcates the third division in each graph. B) The probability of reaching any division is identical among autoimmune strains. The probability that a dividing precursor generates a daughter cell in division N was calculated under maximally stimulating conditions (2 µg/ml anti-CD3/28, 65 hours) and represented as a bar graph. An activated CD4 T cell from any autoimmune strain is unlikely to produce a daughter cell in divisions 4–6 (probability<10%) while there is a significant chance for this event in the normal strain B6 (probability>40%). The distribution of B6 daughter cells was significantly different from all other strains in each division except division 3 (*p<0.01, ANOVA followed by post-test, B6 was compared to each other strain, n>6). All data are representative of at least three separate experiments in each group.</p

Use of the Electronic Medical Record to Assess Pancreas Size in Type 1 Diabetes

<div><p>Aims</p><p>This study harnessed the electronic medical record to assess pancreas volume in patients with type 1 diabetes (T1D) and matched controls to determine whether pancreas volume is altered in T1D and identify covariates that influence pancreas volume.</p><p>Methods</p><p>This study included 25 patients with T1D and 25 age-, sex-, and weight-matched controls from the Vanderbilt University Medical Center enterprise data warehouse. Measurements of pancreas volume were made from medical imaging studies using magnetic resonance imaging (MRI) or computed tomography (CT).</p><p>Results</p><p>Patients with T1D had a pancreas volume 47% smaller than matched controls (41.16 ml vs. 77.77 ml, P < 0.0001) as well as pancreas volume normalized by subject body weight, body mass index, or body surface area (all P < 0.0001). Pancreatic volume was smaller with a longer duration of T1D across the patient population (N = 25, P = 0.04). Additionally, four individual patients receiving multiple imaging scans displayed progressive declines in pancreas volume over time (~ 6% of volume/year), whereas five controls scanned a year apart did not exhibit a decline in pancreas size (P = 0.03). The pancreas was uniformly smaller on the right and left side of the abdomen.</p><p>Conclusions</p><p>Pancreas volume declines with disease duration in patients with T1D, suggesting a protracted pathological process that may include the exocrine pancreas.</p></div

Pancreas volume is smaller with longer duration of type 1 diabetes.

<p>A) Pancreatic volume index is similar across the age range analyzed in control subjects (P = 0.56), but is smaller with age in patients with type 1 diabetes (P = 0.02). B) Patient age at the imaging scan correlates linearly with the duration of type 1 diabetes (P < 0.0001). C) Pancreatic volume index is smaller with increasing duration of type 1 diabetes (P = 0.04). D) In individual patients receiving multiple longitudinal imaging scans (N = 4) pancreatic volume index declines monotonically, whereas controls with repeated imaging scans do not display a decline in pancreas volume (P = 0.03).</p

Demographic and Imaging Characteristics for Patients and Controls in This Study.

<p>Demographic and Imaging Characteristics for Patients and Controls in This Study.</p

Flowchart demonstrating criteria used to identify patient records for inclusion in the study.

<p>Flowchart demonstrating criteria used to identify patient records for inclusion in the study.</p