Islet Remodeling in Female Mice with Spontaneous Autoimmune and Streptozotocin-Induced Diabetes

<div><p>Islet alpha- and delta-cells are spared autoimmune destruction directed at beta-cells in type 1 diabetes resulting in an apparent increase of non-beta endocrine cells in the islet core. We determined how islet remodeling in autoimmune diabetes compares to streptozotocin (STZ)-induced diabetes. Islet cell mass, proliferation, and immune cell infiltration in pancreas sections from diabetic NOD mice and mice with STZ-induced diabetes was assessed using quantitative image analysis. Serial sections were stained for various beta-cell markers and Ngn3, typically restricted to embryonic tissue, was only upregulated in diabetic NOD mouse islets. Serum levels of insulin, glucagon and GLP-1 were measured to compare hormone levels with respect to disease state. Total pancreatic alpha-cell mass did not change as autoimmune diabetes developed in NOD mice despite the proportion of islet area comprised of alpha- and delta-cells increased. By contrast, alpha- and delta-cell mass was increased in mice with STZ-induced diabetes. Serum levels of glucagon reflected these changes in alpha-cell mass: glucagon levels remained constant in NOD mice over time but increased significantly in STZ-induced diabetes. Increased serum GLP-1 levels were found in both models of diabetes, likely due to alpha-cell expression of prohormone convertase 1/3. Alpha- or delta-cell mass in STZ-diabetic mice did not normalize by replacement of insulin via osmotic mini-pumps or islet transplantation. Hence, the inflammatory milieu in NOD mouse islets may restrict alpha-cell expansion highlighting important differences between these two diabetes models and raising the possibility that increased alpha-cell mass might contribute to the hyperglycemia observed in the STZ model.</p></div

Proliferation of endocrine cells and leukocytes in NOD and Balb/c mouse islets.

<p>Co-immunostaining for the nuclear proliferation marker BrdU (green) and islet hormones (red) in 4, 12 and 20 wk old female NOD (A) and age-matched Balb/c mice (B). Sections are counterstained with DAPI to visualize nuclei (and infiltrating immune cells). White arrows indicate double-positive cells, and white arrowheads indicate single BrdU-positive cells within the islet mantel. Scale bar = 10 µm.</p

Quantification of proliferating endocrine cells in NOD and Balb/c mice with and without STZ-induced diabetes.

<p>Number of proliferating alpha, beta and delta-cells were quantified in relation to total respective endocrine cell number in 4-wk old diabetes-prone (black bars), 12-wk old insulitic (hatched bars) and 18–24 wk old diabetic (white bars) female NOD mice (A). Number of proliferating endocrine cells was quantified similarly in age-matched female Balb/c mice (B), and in 12 wk old Balb/c mice without (black bars) or with (white bars) STZ-induced diabetes (C). Significant changes among groups are indicated as: *<i>p<0.01</i>, **<i>p<0.001</i> and ***<i>p<0.0001</i>.</p

Changes in islet endocrine cell populations during progression of autoimmune diabetes.

<p>Alpha, beta and delta-cells were quantified as a percentage of total islet area (A) and total endocrine cell mass (B) in 4-wk old diabetes-prone (black bars), 12-wk old insulitic (hatched bars) and 18–24 wk old diabetic (white bars) female NOD mice. Endocrine cell area (C) and mass (D) was quantified similarly in age-matched female Balb/c mice. Significant changes among groups are indicated as: *<i>p<0.01</i>, **<i>p<0.001</i> and ***<i>p<0.0001</i>.</p

Changes in islet endocrine cell populations in mice with STZ-induced diabetes.

<p>Alpha, beta and delta-cells were quantified as a percentage of total islet area (A) and total endocrine cell mass (B) in 12-wk old Balb/c mice (black bars) and 12-wk Balb/c mice with STZ-induced diabetes (white bars) Significant changes among groups are indicated as: *<i>p<0.01</i>, **<i>p<0.001</i> and ***<i>p<0.0001</i>. Increased proportions of alpha (red) and delta (green) cells coincides with decreased insulin expression (data not shown, C) in 12 wk old female Balb/c mice with STZ-induced diabetes. This was observed irrespective of whether the mice were left untreated (middle panel) or received a syngeneic islet transplantation to restore euglycemia (right panel). Age- and gender-matched Balb/c control islets demonstrated normal endocrine cell distribution with peripherally located alpha and delta-cells and beta-cells throughout the core of the islet (left panel). Scale bar = 10 µm.</p

Body weight and blood glucose levels of non-obese diabetic (NOD) mice studied.

<p>Female NOD mice were analyzed every two weeks from age 4–24 weeks (n = 3–9 per group). Weights are presented as mean+/−SEM for each age group. Due to variability in onset of hyperglycemia blood glucose levels are listed as individual values.</p

Serum hormone levels reflect changes in endocrine cell mass.

<p>Levels of glucagon, insulin and GLP-1 in serum from 4-wk old diabetes-prone (black bars), 12-wk old insulitic (hatched bars) and 18–24 wk old diabetic (white bars) female NOD mice (A), and in 12 wk old Balb/c mice without (black bars) or with (white bars) STZ-induced diabetes (B). The ratios of the proglucagon gene products in percent is given for both the NOD and Balb/c mice (C). Significant changes among groups are indicated as: *<i>p<0.01</i>, **<i>p<0.001</i> and ***<i>p<0.0001</i>.</p

Correlation of insulitis progression with alpha-cell remodeling in individual islets in diabetic NOD mice.

<p>Composition of the insulitic lesion in 4-wk old diabetes-prone (islets from 5 mice), 12-wk old insulitic (islets from 7 mice) and 18–24 wk old diabetic (islets from 7 mice) female NOD mice (A, Insulitis score: white = 0 (pre-inflammation), lightest grey = 1 (less than 1/3 of the islet infiltrated), light grey = 2 (between 1/3 and 2/3 of the islet infiltrated), grey = 3 (more than 2/3 of the islet infiltrated), dark grey = 4 (islets with full insulitis) and black = 0 (post-inflammation)). Correlation between the severity of insulitis and degree of glucagon-positive alpha-cells in 4 wk old (diamond), 12 wk old (square) and 20 wk old diabetic NOD mice (triangle; B). Linear regression analysis revealed a negative correlation between the glucagon and insulitis score in the 12 wk old (dotted line R² = 0.79 and p<0.0005) and 20 wk old diabetic (solid line; R² = 0.85 and p<0.0001) groups. Note: islets with an insulitis score of zero may be either pre- or post-inflammation. Islets that were termed “post-inflammation” were easily identifiable; that is, the insulitis had dissipated following destruction of the beta-cells and largely been replaced by alpha and delta-cells.</p

Islet endocrine cell changes during progression of autoimmune diabetes as leukocyte infiltration increases and dissipates.

<p>Serial pancreas sections from 4(red) and somatostatin (green), or glucagon (red) and CD45-positive leukocytes (green). Scale bar = 10 µm.</p

Evaluation of an integrated care pathway for out-of-hospital treatment of older adults with an acute moderate-to-severe lower respiratory tract infection or pneumonia: protocol of a mixed methods study

Introduction Older adults with an acute moderate-to-severe lower respiratory tract infection (LRTI) or pneumonia are generally treated in hospitals causing risk of iatrogenic harm such as functional decline and delirium. These hospitalisations are often a consequence of poor collaboration between regional care partners, the lack of (acute) diagnostic and treatment possibilities in primary care, and the presence of financial barriers. We will evaluate the implementation of an integrated regional care pathway (‘The Hague RTI Care Bridge’) developed with the aim to treat and coordinate care for these patients outside the hospital.Methods and analysis This is a prospective mixed methods study. Participants will be older adults (age≥65 years) with an acute moderate-to-severe LRTI or pneumonia treated outside the hospital (care pathway group) versus those treated in the hospital (control group). In addition, patients, their informal caregivers and treating physicians will be asked about their experiences with the care pathway. The primary outcome of this study will be the feasibility of the care pathway, which is defined as the percentage of patients treated outside the hospital, according to the care pathway, whom fully complete their treatment without the need for hospitalisation within 30 days of follow-up. Secondary outcomes include the safety of the care pathway (30-day mortality and occurrence of complications (readmissions, delirium, falls) within 30 days); the satisfaction, usability and acceptance of the care pathway; the total number of days of bedridden status or hospitalisation; sleep quantity and quality; functional outcomes and quality of life.Ethics and dissemination The Medical Research Ethics Committee Leiden The Hague Delft (reference number N22.078) has confirmed that the Medical Research Involving Human Subjects Act does not apply to this study. The results will be published in international peer-reviewed journals.Trial registration number ISRCTN68786381