Serum from pregnant donors induces human beta cell proliferation.

Pancreatic beta cells are among the slowest replicating cells in the human body and have not been observed to increase in number except during the fetal and neonatal period, in cases of obesity, during puberty, as well as during pregnancy. Pregnancy is associated with increased beta cell mass to meet heightened insulin demands. This phenomenon raises the intriguing possibility that factors present in the serum of pregnant individuals may stimulate beta cell proliferation and offer insights into expansion of the beta cell mass for treatment and prevention of diabetes. The primary objective of this study was to test the hypothesis that serum from pregnant donors contains bioactive factors capable of inducing human beta cell proliferation. An immortalized human beta cell line with protracted replication (EndoC-ßH1) was cultured in media supplemented with serum from pregnant and non-pregnant female and male donors and assessed for differences in proliferation. This experiment was followed by assessment of proliferation of primary human beta cells. Sera from five out of six pregnant donors induced a significant increase in the proliferation rate of EndoC-ßH1 cells. Pooled serum from the cohort of pregnant donors also increased the rate of proliferation in primary human beta cells. This study demonstrates that serum from pregnant donors stimulates human beta cell proliferation. These findings suggest the existence of pregnancy-associated factors that can offer novel avenues for beta cell regeneration and diabetes prevention strategies. Further research is warranted to elucidate the specific factors responsible for this effect.

Immune engineered extracellular vesicles to modulate T cell activation in the context of type 1 diabetes.

Extracellular vesicles (EVs) can affect immune responses through antigen presentation and costimulation or coinhibition. We generated designer EVs to modulate T cells in the context of type 1 diabetes, a T cell-mediated autoimmune disease, by engineering a lymphoblast cell line, K562, to express HLA-A*02 (HLA-A2) alongside costimulatory CD80 and/or coinhibitory programmed death ligand 1 (PD-L1). EVs presenting HLA-A2 and CD80 activated CD8+ T cells in a dose, antigen, and HLA-specific manner. Adding PD-L1 to these EVs produced an immunoregulatory response, reducing CD8+ T cell activation and cytotoxicity in vitro. EVs alone could not stimulate T cells without antigen-presenting cells. EVs lacking CD80 were ineffective at modulating CD8+ T cell activation, suggesting that both peptide-HLA complex and costimulation are required for EV-mediated immune modulation. These results provide mechanistic insight into the rational design of EVs as a cell-free approach to immunotherapy that can be tailored to promote inflammatory or tolerogenic immune responses.

Serum from pregnant donors induces human beta cell proliferation and insulin secretion.

Pancreatic beta cells are among the slowest replicating cells in the human body. Human beta cells usually do not increase in number with exceptions being during the neonatal period, in cases of obesity, and during pregnancy. This project explored maternal serum for stimulatory potential on human beta cell proliferation and insulin output. Gravid, full-term women who were scheduled to undergo cesarean delivery were recruited for this study. A human beta cell line was cultured in media supplemented with serum from pregnant and non-pregnant donors and assessed for differences in proliferation and insulin secretion. A subset of pregnant donor sera induced significant increases in beta cell proliferation and insulin secretion. Pooled serum from pregnant donors also increased proliferation in primary human beta cells but not primary human hepatocytes indicating a cell-type specific effect. This study suggests stimulatory factors in human serum during pregnancy could provide a novel approach for human beta cell expansion.

Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

Extracellular vesicles in ß cell biology: Role of lipids in vesicle biogenesis, cargo, and intercellular signaling.

BACKGROUND: Type 1 diabetes (T1D) is a complex autoimmune disorder whose pathogenesis involves an intricate interplay between ß cells of the pancreatic islet, other islet cells, and cells of the immune system. Direct intercellular communication within the islet occurs via cell surface proteins and indirect intercellular communication has traditionally been seen as occurring via secreted proteins (e.g., endocrine hormones and cytokines). However, recent literature suggests that extracellular vesicles (EVs) secreted by ß cells constitute an additional and biologically important mechanism for transmitting signals to within the islet. SCOPE OF REVIEW: This review summarizes the general mechanisms of EV formation, with a particular focus on how lipids and lipid signaling pathways influence their formation and cargo. We review the implications of EV release from ß cells for T1D pathogenesis, how EVs and their cargo might be leveraged as biomarkers of this process, and how EVs might be engineered as a therapeutic candidate to counter T1D outcomes. MAJOR CONCLUSIONS: Islet ß cells have been viewed as initiators and propagators of the cellular circuit giving rise to autoimmunity in T1D. In this context, emerging literature suggests that EVs may represent a conduit for communication that holds more comprehensive messaging about the ß cells from which they arise. As the field of EV biology advances, it opens the possibility that intervening with EV formation and cargo loading could be a novel disease-modifying approach in T1D.

Physical tuning of galectin-3 signaling.

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Distributed Temperature Sensing to Measure Infiltration Rates Across a Groundwater Recharge Basin.

Managed aquifer recharge is used to augment groundwater resources and provide resiliency to water supplies threatened by prolonged droughts. It is important that recharge facilities operate at their maximum efficiency to increase the volume of water stored for future use. In this study, we evaluate the use of distributed temperature sensing (DTS) technology as a tool to measure high-resolution infiltration rates at a large-scale recharge facility. Fiber optic cable was laid out inside a spreading basin in a spiral pattern, at two different depths. The cables measured the propagation of diurnal surface water temperature oscillations into the basin depth. The rate of heat propagation is proportional to the velocity of the water, making it possible to estimate the infiltration rate from the temperature measurements. Our results showed that the infiltration rate calculated from DTS, averaged over the entire basin, was within 5% of the infiltration rate calculated using a conventional metering method. The high-resolution data obtained from DTS, both spatially and temporally, revealed heterogeneous infiltration rates throughout the basin; furthermore, tracking the evolution of infiltration rates over time revealed regions with consistently high infiltration rates, regions with consistently low infiltration rates, and regions that evolved from high to low rates, which suggested clogging within that region. Water utilities can take advantage of the high-resolution information obtained from DTS to better manage recharge basins and make decisions about cleaning schedule, frequency, and extent, leading to improved basin management strategies, reduced O&M costs, and increased groundwater recharge.

Distributed Acoustic Sensing of Strain at Earth Tide Frequencies.

The solid Earth strains in response to the gravitational pull from the Moon, Sun, and other planetary bodies. Measuring the flexure of geologic material in response to these Earth tides provides information about the geomechanical properties of rock and sediment. Such measurements are particularly useful for understanding dilation of faults and fractures in competent rock. A new approach to measuring earth tides using fiber optic distributed acoustic sensing (DAS) is presented here. DAS was originally designed to record acoustic vibration through the measurement of dynamic strain on a fiber optic cable. Here, laboratory experiments demonstrate that oscillating strain can be measured with DAS in the microHertz frequency range, corresponding to half-day (M2) lunar tidal cycles. Although the magnitude of strain measured in the laboratory is larger than what would be expected due to earth tides, a clear signal at half-day period was extracted from the data. With the increased signal-to-noise expected from quiet field applications and improvements to DAS using engineered fiber, earth tides could potentially be measured in deep boreholes with DAS. Because of the distributed nature of the sensor (0.25 m measurement interval over kilometres), fractures could be simultaneously located and evaluated. Such measurements would provide valuable information regarding the placement and stiffness of open fractures in bedrock. Characterization of bedrock fractures is an important goal for multiple subsurface operations such as petroleum extraction, geothermal energy recovery, and geologic carbon sequestration.

Mechanism and effects of pulsatile GABA secretion from cytosolic pools in the human beta cell.

Pancreatic beta cells synthesize and secrete the neurotransmitter γ-aminobutyric acid (GABA) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel (VRAC), functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from type 1 and type 2 diabetic patients, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

Engineered microenvironments and microdevices for modeling the pathophysiology of type 1 diabetes.

The pathophysiology of type 1 diabetes is a complex process involving tightly controlled microenvironments, a number of highly specific immune cell - islet cell interactions, and the eventual breaking of immune tolerance leading to beta cell death. Modeling this process can provide researchers with powerful insights into how and when to best provide treatment, but has proven difficult to accurately model due to its complex nature and differences between animal models and humans. Much progress has been made in determining the genetic, molecular, and cellular mechanisms of type 1 diabetes, yet translating that knowledge to clinical treatments remains challenging. Thus, there exists a capabilities gap between understanding the disease pathophysiology and engineering effective clinical treatment strategies. Biomimetic modeling of human type 1 diabetes is a valuable tool to study and manipulate islet function and can be employed to address immunological aspects of type 1 diabetes. This article will review recent advances in this field, and will suggest ways to synergize systems to model and observe the pathophysiology of autoimmune diabetes with bioengineered therapeutic strategies.

Measuring artificial recharge with fiber optic distributed temperature sensing.

Heat was used as a tracer to measure infiltration rates from a recharge basin. The propagation of diurnal oscillation of surface water temperature into the basin bed was monitored along a transect using Fiber Optic Distributed Temperature Sensing (FODTS). The propagation rate was related to downward specific discharge using standard theory of heat advection and dispersion in saturated porous media. An estimate of the temporal variation of heat propagation was achieved using a wavelet transform to find the phase lag between the surface temperature diurnal oscillation and the correlated oscillation at 0.33 and 0.98 m below the bed surface. The wavelet results compared well to a constant velocity model of thermal advection and dispersion during periods of relatively constant discharge rates. The apparent dispersion of heat was found to be due primarily to hydrodynamic mechanisms rather than thermal diffusion. Specific discharge estimates using the FODTS technique also compared well to water balance estimates over a four month period, although there were occasional deviations that have yet to be adequately explained. The FODTS technique is superior to water balance in that it produces estimates of infiltration rate every meter along the cable transect, every half hour. These high resolution measurements highlighted areas of low infiltration and demonstrated the degradation of basin efficiency due to source waters of high suspended solids. FODTS monitoring promises to be a useful tool for diagnosing basin performance in an era of increasing groundwater demand.

Potential for satellite remote sensing of ground water.

Predicting hydrologic behavior at regional scales requires heterogeneous data that are often prohibitively expensive to acquire on the ground. As a result, satellite-based remote sensing has become a powerful tool for surface hydrology. Subsurface hydrology has yet to realize the benefits of remote sensing, even though surface expressions of ground water can be monitored from space. Remotely sensed indicators of ground water may provide important data where practical alternatives are not available. The potential for remote sensing of ground water is explored here in the context of active and planned satellite-based sensors. Satellite technology is reviewed with respect to its ability to measure ground water potential, storage, and fluxes. It is argued here that satellite data can be used if ancillary analysis is used to infer ground water behavior from surface expressions. Remotely sensed data are most useful where they are combined with numerical modeling, geographic information systems, and ground-based information.

Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater.

The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates.

Bacterial transport experiments in fractured crystalline bedrock.

The efficiency of contaminant biodegradation in ground water depends, in part, on the transport properties of the degrading bacteria. Few data exist concerning the transport of bacteria in saturated bedrock, particularly at the field scale. Bacteria and microsphere tracer experiments were conducted in a fractured crystalline bedrock under forced-gradient conditions over a distance of 36 m. Bacteria isolated from the local ground water were chosen on the basis of physicochemical and physiological differences (shape, cell-wall type, motility), and were differentially stained so that their transport behavior could be compared. No two bacterial strains transported in an identical manner, and microspheres produced distinctly different breakthrough curves than bacteria. Although there was insufficient control in this field experiment to completely separate the effects of bacteria shape, reaction to Gram staining, cell size, and motility on transport efficiency, it was observed that (1) the nonmotile, mutant strain exhibited better fractional recovery than the motile parent strain; (2) Gram-negative rod-shaped bacteria exhibited higher fractional recovery relative to the Gram-positive rod-shaped strain of similar size; and (3) coccoidal (spherical-shaped) bacteria transported better than all but one strain of the rod-shaped bacteria. The field experiment must be interpreted in the context of the specific bacterial strains and ground water environment in which they were conducted, but experimental results suggest that minor differences in the physical properties of bacteria can lead to major differences in transport behavior at the field scale.