Amelioration of type 1 diabetes following treatment of non-obese diabetic mice with INGAP and lisofylline

Type 1 diabetes mellitus results from the autoimmune and inflammatory destruction of insulin-producing islet β cells, rendering individuals devoid of insulin production. Recent studies suggest that combination therapies consisting of anti-inflammatory agents and islet growth-promoting factors have the potential to cause sustained recovery of β cell mass, leading to amelioration or reversal of type 1 diabetes in mouse models. In this study, we hypothesized that the combination of the anti-inflammatory agent lisofylline (LSF) with an active peptide fragment of islet neogenesis associated protein (INGAP peptide) would lead to remission of type 1 diabetes in the non-obese diabetic (NOD) mouse. We treated groups of spontaneously diabetic NOD mice with combinations of LSF, INGAP peptide, or control saline parenterally for up to 6 weeks. Our results demonstrate that the mice receiving combined treatment with LSF and INGAP peptide exhibited partial remission of diabetes with increased plasma insulin levels. Histologic assessment of pancreata in mice receiving combined therapy revealed the presence of islet insulin staining, increased β cell replication, and evidence of Pdx1-positivity in ductal cells. By contrast, diabetic animals showed severe insulitis with no detectible insulin or Pdx1 staining. We conclude that the novel combination treatment with LSF and INGAP peptide has the potential to ameliorate hyperglycemia in the setting of established type 1 diabetes via the recovery of endogenous β cells and warrant further studies

K21-Antigen: A Molecule Shared by the Microenvironments of the Human Thymus and Germinal Centers

The mouse IgG1 monoclonal antibody (mAb) K21 recognizes a 230-kD molecule (K21-Ag) on Hassall's corpuscles in the human thymus. This mAb also stains cultured thymic epithelial cells as well as other epithelial cell lines, revealing a predominant intracellular localization. Further analysis with mAb K21 on other lymphoid tissues showed that it also stains cells within the germinal centers of human tonsils, both lymphoid (B) cells and some with the appearance of follicular dendritic cells. Double immunostaining of tonsil sections shows that K21-Ag is not expressed by T cells, whereas staining with anti-CD22 and -CD23 mAb revealed some doublepositive cells. A subpopulation of the lymphoid cells express the K21-Ag much more strongly. This K21++/CD23++ subpopulation of cells is localized in the apical light zone of germinal centers, suggesting that K21-Ag may be an important marker for the selected centrocytes within germinal centers and may play a role in B-cell selection and/or development of B-cell memory. Flow cytometric analysis showed that K21-Ag is expressed on the surface of a very low percentage of thymocytes, tonsillar lymphocytes, and peripheral blood mononuclear cells. Analysis of purified/separated tonsillar T and B lymphocytes showed that T cells do not express the K21-Ag; in contrast, B cells express low levels of the K21-Ag, and this together with CD23 is upregulated after mitogenic stimulation. Our data therefore raise the possibility that the K2l- Ag may play a role in B-lymphocyte activation/selection

12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets

Context: The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D). Objectives: We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function. Design: Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1β, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355. Setting: In vitro study. Participants: Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%. Intervention: Glucose stimulation. Main Outcome Measures: Static and dynamic insulin secretion and oxygen consumption rate (OCR). Results: ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors. Conclusions: ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo

Interleukin-12 (IL-12)/STAT4 Axis Is an Important Element for β-Cell Dysfunction Induced by Inflammatory Cytokines

<div><p>Pathology driving β-cell loss in diabetes is poorly defined. Chronic subclinical inflammation is associated with β-cell dysfunction. Acute <i>in vitro</i> exposure of islets and β-cells to an inflammatory cytokine cocktail (IL-1β/TNF-α/IFN-γ) results in loss of cell function and viability. The contribution of each cytokine alone or in combination has been evaluated in homogeneous mouse β-cell lines and primary mouse islets. Cytokine cooperation is required for β-cell apoptosis with the most potent combinations including IL-1β. Single cytokine exposure did not induce β-cell apoptosis. Expression of endogenous interleukin-12 in β-cells correlated with inflammatory cytokine combinations that induced β-cell apoptosis. Uncoupling of the IL-12 axis by a block of IL-12 production, inhibition of IL-12 receptor/ligand interaction or disruption of IL-12 receptor signaling conferred protection to β-cells from apoptosis induced by inflammatory cytokine stimulation. Signaling through STAT4 is indicated since disruption of IL-12 concomitantly reduced inflammatory cytokine stimulation of endogenous IFN-γ expression. Primary mouse islets isolated from mice deficient in STAT4 show resistance to inflammatory-cytokine-induced cell death when compared to islets isolated from wild type mice. Collectively, the data identify IL-12 as an important mediator of inflammation induced β-cell apoptosis. Modulation of IL-12/STAT4 signaling may be a valuable therapeutic strategy to preserve islet/β-cell viability in established diabetes.</p></div

Lisofylline protects β-cells from PIC-induced apoptosis.

<p>(A) IL-12 p40 and (B) IL-12 p35 gene expression in PIC-treated βTC-3 cells without or with 20μM Lisofylline (LSF). (C) PIC-induced caspase-3 activation in the absence or presence of 20μM LSF or 50μM c47 in βTC-3 cells. Graph shows pro-caspase-3 cleavage relative fluorescent units (RFU). (D) Apoptosis was measured in PIC-treated βTC-3 cells without or with 20μM LSF or 50μM c47. Cells were examined microscopically following labeling with the viability dye, YO-PRO-1 (green). Representative images are shown for untreated (Ctl), PIC-treated, PIC with 50μM c47, or PIC with 20μM LSF respectively. (E) Graph shows quantified apoptotic index. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 relative to ctl, ## <i>p</i> < 0.01 relative to PIC and n ≥ 3.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Cytokine treatment affects IL-12 gene expression in mouse islets.

<p>(A) IL-12 p40 and (B) IL-12 p35 gene expression was measured in islets treated with PICs or the dual cytokine combinations of IL-1β/TNF-α, IL-1β/IFN-γ, or TNF-α/IFN-γ. # <i>p</i> < 0.05 relative to PIC and n = 3.</p

c47 confers protection to mouse islets from PIC-induced apoptosis.

<p>(A) PIC-induced caspase-3 activation in the absence or presence of 50μM c47 in mouse islets. Graph shows pro-caspase-3 cleavage. (B) Apoptosis was measured in PIC-treated islets without or with 50μM c47. Cells were examined microscopically following labeling with YO-PRO-1 (green) and PI (red). Representative images are shown for untreated (Ctl), PIC-treated, or PIC with 50μM c47 respectively. (C) Graph shows quantified apoptotic index. * <i>p</i> < 0.05, *** <i>p</i> < 0.001 relative to ctl, # <i>p</i> < 0.05, ### <i>p</i> < 0.001 relative to PIC and n > 3.</p

A neutralizing antibody to IL-12 p40 protects β-cells from PIC-induced apoptosis.

<p>(A) MCP-1 gene expression in PIC-treated βTC-3 cells without or with IL-12 p40 neutralizing antibody. (B) Caspase-3 activity in PIC-treated βTC-3 cells without or with IL-12 p40 neutralizing antibody. Graph (B) shows pro-caspase-3 cleavage (RFU). # <i>p</i> < 0.05, ## <i>p</i> < 0.01 relative to PIC and n = 3.</p