Marked expansion of exocrine and endocrine pancreas with incretin therapy in humans with increased exocrine pancreas dysplasia and the potential for glucagon-producing neuroendocrine tumors.

Controversy exists regarding the potential regenerative influences of incretin therapy on pancreatic β-cells versus possible adverse pancreatic proliferative effects. Examination of pancreata from age-matched organ donors with type 2 diabetes mellitus (DM) treated by incretin therapy (n = 8) or other therapy (n = 12) and nondiabetic control subjects (n = 14) reveals an ∼40% increased pancreatic mass in DM treated with incretin therapy, with both increased exocrine cell proliferation (P < 0.0001) and dysplasia (increased pancreatic intraepithelial neoplasia, P < 0.01). Pancreata in DM treated with incretin therapy were notable for α-cell hyperplasia and glucagon-expressing microadenomas (3 of 8) and a neuroendocrine tumor. β-Cell mass was reduced by ∼60% in those with DM, yet a sixfold increase was observed in incretin-treated subjects, although DM persisted. Endocrine cells costaining for insulin and glucagon were increased in DM compared with non-DM control subjects (P < 0.05) and markedly further increased by incretin therapy (P < 0.05). In conclusion, incretin therapy in humans resulted in a marked expansion of the exocrine and endocrine pancreatic compartments, the former being accompanied by increased proliferation and dysplasia and the latter by α-cell hyperplasia with the potential for evolution into neuroendocrine tumors

Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model

<p>Abstract</p> <p>Background</p> <p>Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA).</p> <p>Methods</p> <p>DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA.</p> <p>Results</p> <p>Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially.</p> <p>Conclusion</p> <p>These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.</p

Sustained Expression with Partial Correction of Neutrophil Defects 5 Years After Intramuscular rAAV1 Gene Therapy for Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin (AAT) deficiency is a common monogenic disorder resulting in emphysema, which is currently treated with weekly infusions of protein replacement. We previously reported achieving plasma wild-type (M) AAT concentrations at 2.5-3.8% of the therapeutic level at 1 year after intramuscular (IM) administration of 6×1012vg/kg of a recombinant adeno-associated virus serotype 1 (rAAV1)-AAT vector in AAT-deficient patients, with an associated regulatory T cell (Treg) response to AAV1 capsid epitopes in the absence of any exogenous immune suppression. Here, we report sustained expression at greater than 2% of the therapeutic level for 5 years after one-time treatment with rAAV1-AAT in an AAT-deficient patient from that study, with partial correction of neutrophil defects previously reported in AAT-deficient patients. There was also evidence of an active Treg response (FoxP3+, Helios+) and an exhausted cytotoxic T cell response (PD-1+, LAG-3+) to AAV1 capsid. These findings suggest that muscle-based AAT gene replacement is toleragenic and that very stable levels of M AAT may exert beneficial effects at lower concentrations than previously anticipated

Large-scale electron microscopy database for human type 1 diabetes

Autoimmune β-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of 'intermediate cells' containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity

Salivary PYY: A Putative Bypass to Satiety

Peptide YY3-36 is a satiation hormone released postprandially into the bloodstream from L-endocrine cells in the gut epithelia. In the current report, we demonstrate PYY3-36 is also present in murine as well as in human saliva. In mice, salivary PYY3-36 derives from plasma and is also synthesized in the taste cells in taste buds of the tongue. Moreover, the cognate receptor Y2R is abundantly expressed in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland. The acute augmentation of salivary PYY3-36 induced stronger satiation as demonstrated in feeding behavioral studies. The effect is mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. In a long-term study involving diet-induced obese (DIO) mice, a sustained increase in PYY3-36 was achieved using viral vector-mediated gene delivery targeting salivary glands. The chronic increase in salivary PYY3-36 resulted in a significant long-term reduction in food intake (FI) and body weight (BW). Thus this study provides evidence for new functions of the previously characterized gut peptide PYY3-36 suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity

Glycogen Storage Disease Type Ia in Canines: A Model for Human Metabolic and Genetic Liver Disease

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including “lactic acidosis”, larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases

5 Year Expression and Neutrophil Defect Repair after Gene Therapy in Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated

CD226 Deletion Reduces Type 1 Diabetes in the NOD Mouse by Impairing Thymocyte Development and Peripheral T Cell Activation.

The costimulatory molecule CD226 is highly expressed on effector/memory T cells and natural killer cells. Costimulatory signals received by T cells can impact both central and peripheral tolerance mechanisms. Genetic polymorphisms in CD226 have been associated with susceptibility to type 1 diabetes and other autoimmune diseases. We hypothesized that genetic deletion of Cd226 in the non-obese diabetic (NOD) mouse would impact type 1 diabetes incidence by altering T cell activation. CD226 knockout (KO) NOD mice displayed decreased disease incidence and insulitis in comparison to wild-type (WT) controls. Although female CD226 KO mice had similar levels of sialoadenitis as WT controls, male CD226 KO mice showed protection from dacryoadenitis. Moreover, CD226 KO T cells were less capable of adoptively transferring disease compared to WT NOD T cells. Of note, CD226 KO mice demonstrated increased CD8+ single positive (SP) thymocytes, leading to increased numbers of CD8+ T cells in the spleen. Decreased percentages of memory CD8+CD44+CD62L- T cells were observed in the pancreatic lymph nodes of CD226 KO mice. Intriguingly, CD8+ T cells in CD226 KO mice showed decreased islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-tetramer and CD5 staining, suggesting reduced T cell receptor affinity for this immunodominant antigen. These data support an important role for CD226 in type 1 diabetes development by modulating thymic T cell selection as well as impacting peripheral memory/effector CD8+ T cell activation and function

Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for \u3e1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT \u3e20 μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations