Biology subjects

Ramirez, D. G.

Publications and source records attributed to Ramirez, D. G..

5 recordsLinked to original sources

bioRxiv2020

Detecting Insulitis in Type 1 Diabetes with Ultrasound Phase-change Contrast Agents

Type 1 diabetes (T1D) results from immune infiltration and destruction of insulin-producing {beta}-cells within the pancreatic islets of Langerhans (insulitis), resulting in loss of glucose homeostasis. Early diagnosis during pre-symptomatic T1D would allow for therapeutic intervention prior to substantial loss of {beta}-cell mass at T1D onset. There are limited methods to track the progression of insulitis and {beta}-cell mass decline in pre-symptomatic T1D. During insulitis, the islet microvasculature increases permeability, such that sub-micron sized particles can extravasate and accumulate within the islet microenvironment. Ultrasound is a widely deployable and cost-effective clinical imaging modality. However, conventional microbubble contrast agents are restricted to the vasculature. Sub-micron sized nanodroplet (ND) phasechange agents can be vaporized into micron-sized bubbles; serving as a circulating microbubble precursor. We tested if NDs extravasate into the immune-infiltrated islet microenvironment. We performed ultrasound contrast-imaging following ND infusion in NOD mice and NOD;Rag1ko controls, and tracked diabetes development. We measured the biodistribution of fluorescently labeled NDs, with histological analysis of insulitis. Ultrasound contrast signal was elevated in the pancreas of 10w NOD mice following ND infusion and vaporization, but was absent in both the non-infiltrated kidney of NOD mice and pancreas of Rag1ko controls. High contrast elevation also correlated with rapid diabetes onset. In pancreata of NOD mice, infiltrated islets and nearby exocrine tissue were selectively labeled with fluorescent NDs. Thus, contrast ultrasound imaging with ND phase-change agents can detect insulitis prior to diabetes onset. This will be important for monitoring disease progression to guide and assess preventative therapeutic interventions for T1D. SignificanceThere is a need for imaging methods to detect type1 diabetes (T1D) progression prior to clinical diagnosis. T1D is a chronic disease that results from autoreactive T cells infiltrating the islet of Langerhans and destroying insulin-producing {beta}-cells. Overt disease takes years to present and is only diagnosed after significant {beta}-cells loss. As such, the possibility of therapeutic intervention to preserve {beta}-cell mass is hampered by an inability to follow pre-symptomatic T1D progression. There are immunotherapies that can delay T1D development. However identifying at risk individuals, and tracking whether therapeutic interventions are impacting disease progression, prior to T1D onset, is lacking. A method to detect insulitis and {beta}-cell mass decline would present an opportunity to guide therapeutic treatments to prevent T1D.

bioengineering↗

bioRxiv2020

Dynamic changes in β-cell electrical activity and regulates NFATc3 activation and downstream gene transcription

Diabetes results from insufficient insulin secretion as a result of dysfunction to {beta}-cells within the islet of Langerhans. Elevated glucose causes {beta}-cell membrane depolarization and action potential generation, voltage gated Ca2+ channel activation and oscillations in free-Ca2+ activity ([Ca2+]), triggering insulin release. Nuclear Factor of Activated T-cell (NFAT) is a transcription factor that is regulated by increases in [Ca2+] and calceineurin (CaN) activation. NFAT regulation links cell activity with gene transcription in many systems, and within the {beta}-cell regulates proliferation and insulin granule biogenesis. However the link between the regulation of {beta}-cell electrical activity and oscillatory [Ca2+], with NFAT activation and downstream transcription is poorly understood. In this study we tested whether dynamic changes to {beta}-cell electrical activity and [Ca2+] regulates NFAT activation and downstream transcription. In cell lines, mouse islets and human islets, including those from donors with type2 diabetes, we applied both agonists/antagonists of ion channels together with optogenetics to modulate {beta}-cell electrical activity. Both glucose-induced membrane depolarization and optogenetic-stimulation triggered NFAT activation, and increased transcription of NFAT targets and intermediate early genes (IEGs). Importantly only conditions in which slow sustained [Ca2+] oscillations were generated led to NFAT activation and downstream transcription. In contrast in human islets from donors with type2 diabetes NFAT activation by glucose was diminished, but rescued upon pharmacological stimulation of electrical activity. Thus, we gain insight into the specific patterns of electrical activity that regulate NFAT activation and gene transcription and how this is disrupted in diabetes.

biophysics↗

bioRxiv2020

Contrast-enhanced ultrasound measurement of pancreatic blood flow dynamics predicts type1 diabetes therapeutic reversal in preclinical models

In type 1 diabetes (T1D) immune-cell infiltration into the islets of Langerhans (insulitis) and {beta}-cell decline occurs many years before diabetes presents. Non-invasively detecting insulitis and {beta}-cell decline would allow diagnosis of eventual diabetes and provide a means to monitor the efficacy of therapeutic intervention. However, there is a lack of validated clinical approaches for non-invasively imaging disease progression leading to T1D. Islets have a dense microvasculature that reorganizes during diabetes. We previously demonstrated contrast-enhanced ultrasound measurements of pancreatic blood-flow dynamics could predict disease progression in T1D pre-clinical models. Here we test whether these measurements can predict successful therapeutic prevention of T1D. We performed destruction-reperfusion measurements using a small-animal ultrasound machine and size-isolated microbubbles, in NOD-scid mice receiving an adoptive transfer of diabetogenic splenocytes (AT mice). Mice received vehicle control or either of the following treatments: 1) antiCD4 to deplete CD4+ T cells; 2) antiCD3 to block T cell activation, 3) Verapamil to reduce {beta}-cell apoptosis and 4) TUDCA to reduce ER stress. We compared measurements of pancreas blood-flow dynamics with subsequent progression to diabetes. In AT mice blood-flow dynamics were altered >2 weeks after splenocyte transfer. AntiCD4, antiCD3 and verapamil provided a significant delay in diabetes development. Treated AT mice with delayed or absent diabetes development showed significantly altered blood flow dynamics compared to untreated AT mice. Conversely, treated AT mice that developed diabetes, despite therapy, showed similar blood-flow dynamics to untreated AT mice. Thus, contrast-enhanced ultrasound measurement of pancreas blood-flow dynamics can predict the successful or unsuccessful delay or prevention of diabetes upon therapeutic treatments that target both immune activity or {beta}-cell protection. This strategy may provide a clinically deployable predictive marker for disease progression and therapeutic reversal in asymptomatic T1D.

bioengineering↗

bioRxiv2019

Contrast-enhanced ultrasound with sub-micron sized contrast agents detects insulitis and beta-cell mass decline in mouse models of type 1 diabetes

Type 1 diabetes (T1D) is characterized by the infiltration of autoreactive T-cells into the islet of Langerhans, and depletion of insulin-secreting {beta}-cells. This immune cell infiltration (insulitis) first occurs during an asymptomatic phase of T1D that can take place many years prior to clinical diagnosis. Methods to diagnose insulitis and changes in {beta}-cell mass during this asymptomatic phase are limited, thus precluding early therapeutic intervention. While therapeutic treatments can delay T1D progression, treatment efficacy is limited and widely varying, and a method to track this efficacy is also lacking. During T1D progression, the islet microvasculature increases permeability as a result of insulitis, in both mouse models of T1D and humans with T1D. This increased permeability can allow nanoparticles, such as contrast agents for diagnostic imaging, to access the islet microenvironment. Contrast enhanced ultrasound (CEUS) uses shell-stabilized gas bubbles to provide high acoustic backscatter in vasculature and tissue and is clinically approved. A novel, sub-micron sized nanobubble (NB) ultrasound contrast agent has been developed and shown to extravasate and accumulate in tumors, where microvascular permeability is high. To test whether CEUS can be used to measure increased islet microvasculature permeability and indicate the asymptomatic phase of T1D, we applied CEUS measurements with NBs in pre-clinical T1D models. NOD mice and mice receiving an adoptive-transfer of diabetogenic splenocytes showed accumulation of NBs specifically within the pancreatic islets, and only in the presence of insulitis. This accumulation was measured by both ultrasound contrast and histological analysis, and accumulation only occurred for sub-micron sized bubbles. Importantly, accumulation was detected as early as 4w in NOD mice. Thus, CEUS with sub-micron sized NB contrast agent may provide a predicative marker for disease progression early in asymptomatic T1D, as well as monitoring of disease prevention or reversal.

bioengineering↗

bioRxiv2019

Optogenetic Stimulation of Pancreatic Function via Vagal Cholinergic Axons

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.

bioengineering↗