Pancreatic islet xenograft survival in mice is extended by a combination of alpha-1-antitrypsin and single-dose anti-CD4/CD8 therapy.

Clinical pancreatic islet transplantation is under evaluation for the treatment of autoimmune diabetes, yet several limitations preclude widespread use. For example, there is a critical shortage of human pancreas donors. Xenotransplantation may solve this problem, yet it evokes a rigorous immune response which can lead to graft rejection. Alpha-1-antitrypsin (AAT), a clinically available and safe circulating anti-inflammatory and tissue protective glycoprotein, facilitates islet alloimmune-tolerance and protects from inflammation in several models. Here, we examine whether human AAT (hAAT), alone or in combination with clinically relevant approaches, achieves long-term islet xenograft survival. Rat-to-mouse islet transplantation was examined in the following groups: untreated (n = 6), hAAT (n = 6, 60-240 mg/kg every 3 days from day -10), low-dose co-stimulation blockade (anti-CD154/LFA-1) and single-dose anti-CD4/CD8 (n = 5-7), either as mono- or combination therapies. Islet grafting was accompanied by blood glucose follow-up. In addition, skin xenografting was performed in order to depict responses that occur in draining lymph nodes. According to our results hAAT monotherapy and hAAT/anti-CD154/LFA-1 combined therapy, did not delay rejection day (11-24 days untreated vs. 10-22 day treated). However, host and donor intragraft inflammatory gene expression was diminished by hAAT therapy in both setups. Single dose T-cell depletion using anti-CD4/CD8 depleting antibodies, which provided 14-15 days of reduced circulating T-cells, significantly delayed rejection day (28-52 days) but did not achieve graft acceptance. In contrast, in combination with hAAT, the group displayed significantly extended rejection days and a high rate of graft acceptance (59, 61, >90, >90, >90). In examination of graft explants, marginal mononuclear-cell infiltration containing regulatory T-cells predominated surviving xenografts. We suggest that temporal T-cell depletion, as in the clinically practiced anti-thymocyte-globulin therapy, combined with hAAT, may promote islet xenograft acceptance. Further studies are required to elucidate the mechanism behind the observed synergy, as well as the applicability of the approach for pig-to-human islet xenotransplantation

Monitoring Attention in ADHD with an Easy-to-Use Electrophysiological Index

Attention deficit hyperactivity disorder (ADHD) involves characteristic electroencephalographic (EEG) activity. We developed a single-channel EEG marker for attention: the Brain Engagement Index (BEI’). In this study, we evaluated the use of BEI’ for distinguishing between ADHD patients and controls, and for monitoring the effect of pharmacological treatment on ADHD patients. The BEI’ values of 20 ADHD patients and 10 controls were measured using a 1-min auditory oddball paradigm and a continuous performance test (CPT) task. We showed that CPT BEI’ is trait-specific and separates controls from ADHD patients. At the same time, oddball BEI’ is state-specific and identifies differences in attention level within the two groups of ADHD participants and controls. The oddball BEI’ also associates with response to treatment, after distinguishing between treatment effect and learning/time effect. The combined use of this marker with common computerized tests holds promise for research and clinical use in ADHD. Further work is required to confirm the results of the present study

Human α1-Antitrypsin Binds to Heat-Shock Protein gp96 and Protects from Endogenous gp96-Mediated Injury In vivo

The extracellular form of the abundant heat shock protein, gp96, is involved in human autoimmune pathologies. In patients with type 1 diabetes, circulating gp96 is found to be elevated, and is bound to the acute-phase protein, α1-antitrypsin (AAT). The two molecules also engage intracellularly during the physiological folding of AAT. AAT therapy promotes pancreatic islet survival in both transplantation and autoimmune diabetes models, and several clinical trials are currently examining AAT therapy for individuals with type 1 diabetes. However, its mechanism of action is yet unknown. Here, we examine whether the protective activity of AAT is related to binding of extracellular gp96. Primary mouse islets, macrophages and dendritic cells were added recombinant gp96 in the presence of clinical-grade human AAT (hAAT, GlassiaTM, Kamada Ltd, Israel). Islet function was evaluated by insulin release. The effect of hAAT on IL-1β/IFNγ-induced gp96 cell surface levels was also evaluated. In vivo, skin transplants were performed for examination of robust immune responses, and systemic inflammation was induced by cecal puncture. Endogenous gp96 was inhibited by gp96-inhibitory peptide (gp96i, Compugen Ltd., Israel) in an allogeneic islet transplantation model. Our findings indicate that hAAT binds to gp96 and diminishes gp96-induced inflammatory responses; e.g., hAAT-treated gp96-stimulated islets released less pro-inflammatory cytokines (IL-1β by 6.16-fold and TNFα by 2.69-fold) and regained gp96-disrupted insulin release. hAAT reduced cell activation during both skin transplantation and systemic inflammation, as well as lowered inducible surface levels of gp96 on immune cells. Finally, inhibition of gp96 significantly improved immediate islet graft function. These results suggest that hAAT is a regulator of gp96-mediated inflammatory responses, an increasingly appreciated endogenous damage response with relevance to human pathologies that are exacerbated by tissue injury

AAT treatment combined with debulking therapy; graft survival.

<p><b>(A–C)</b> Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (<i>n</i> = 7) or with added AAT therapy (<i>n</i> = 5). (<b>A</b>) CD45⁺CD3⁺ cells from peripheral blood, as monitored by FACS analysis. Results presented as the percent out of initial amount prior to injection. Representative follow-up out of 10 mice. (<b>B</b>) Islet xenograft survival curve. ***p<0.001 between DB and BD/AAT. (<b>C</b>) Glucose follow-up. Representative mouse. Milestones indicated: <i>hAAT treatment stopped,</i> therapy withdrawn followed by glucose follow-up; <i>nephrectomy</i>, graft explantation followed by glucose follow-up; <i>second xenograft</i>, rat islets grafted into the right renal subcapsular space followed by glucose follow-up.</p

Human AAT monotherapy during pancreatic islet xenotransplantation.

<p>Rat pancreatic islets were grafted into the renal subcapsular space of hyperglycemic mice. Recipients were treated with saline (CT) or human AAT throughout the experiment. (<b>A</b>) Islet graft survival curve (<i>n</i> = 6/group). (<b>B</b>) Graft histology. Representative day-seven explanted grafts from CT and hATT-monotreated mice (<i>n</i> = 3/group). <i>Black arrows</i>, remains of rat pancreatic islets. (<b>C</b>) Mouse gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from <i>n</i> = 3 grafts/group; *p<0.05, **p<0.01, ***p<0.001. (<b>D</b>) Rat gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from <i>n</i> = 3 grafts/group; **p<0.01.</p

AAT treatment combined with debulking therapy; histology and gene expression.

<p>Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (<i>n</i> = 7) or with added AAT therapy (<i>n</i> = 5), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g003" target="_blank">Figure 3B</a>. (<b>A</b>) Graft site histology. <i>K</i>, kidney tissue; <i>G</i>, graft site. From <i>left</i> to <i>right</i>, representative syngeneic mouse islet graft (day 35), xenograft (debulking therapy alone, day 25), <i>black arrows</i> indicate immune cell mononuclear infiltration, xenograft (debulking therapy combined with AAT, day 11 after rejection) and xenograft (debulking therapy combined with AAT, day 90). (<b>B</b>) Treg cell content in xenograft sites. Immunofluorescent staining. DB, debulking therapy alone (rejected graft); DB/AAT, combined debulking and AAT therapy (rejected and accepted grafts). <i>Green</i>, foxp3; <i>blue</i>, DAPI nuclear counterstaining. Representative images. (<b>C</b>) Mouse (recipient) gene expression profiles. RT-PCR. CT vs. AAT monotherapy, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g001" target="_blank">Figure 1C</a>, shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; *p<0.05, **p<0.01, ***p<0.001. (<b>D</b>) Rat (donor) insulin expression profile. RT-PCR. CT vs. AAT monotherapy, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g001" target="_blank">Figure 1D</a>, are shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; **p<0.01.</p

DLN response to human AAT monotherapy after skin xenografting.

<p>Mice were either SHAM operated (CT) or recipients of rat skin (Tx) in the absence or presence of human AAT monotherapy. (<b>A</b>) 14-day DLN. FACS analysis. Results expressed as fold change from CT, mean ± SEM from n = 10/group; **p<0.01, ***p<0.001. (<b>B</b>) 72-h DLN. RT-PCR. Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; **p<0.01, ***p<0.001.</p