CXCL16-dependent scavenging of oxidized lipids by islet macrophages promotes differentiation of pathogenic CD8+ T cells in diabetic autoimmunity.

The pancreatic islet microenvironment is highly oxidative, rendering ß cells vulnerable to autoinflammatory insults. Here, we examined the role of islet resident macrophages in the autoimmune attack that initiates type 1 diabetes. Islet macrophages highly expressed CXCL16, a chemokine and scavenger receptor for oxidized low-density lipoproteins (OxLDLs), regardless of autoimmune predisposition. Deletion of Cxcl16 in nonobese diabetic (NOD) mice suppressed the development of autoimmune diabetes. Mechanistically, Cxcl16 deficiency impaired clearance of OxLDL by islet macrophages, leading to OxLDL accumulation in pancreatic islets and a substantial reduction in intra-islet transitory (Texint) CD8+ T cells displaying proliferative and effector signatures. Texint cells were vulnerable to oxidative stress and diminished by ferroptosis; PD-1 blockade rescued this population and reversed diabetes resistance in NOD.Cxcl16-/- mice. Thus, OxLDL scavenging in pancreatic islets inadvertently promotes differentiation of pathogenic CD8+ T cells, presenting a paradigm wherein tissue homeostasis processes can facilitate autoimmune pathogenesis in predisposed individuals.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Pancreatic islets communicate with lymphoid tissues via exocytosis of insulin peptides.

Tissue-specific autoimmunity occurs when selected antigens presented by susceptible alleles of the major histocompatibility complex are recognized by T cells. However, the reason why certain specific self-antigens dominate the response and are indispensable for triggering autoreactivity is unclear. Spontaneous presentation of insulin is essential for initiating autoimmune type 1 diabetes in non-obese diabetic mice1,2. A major set of pathogenic CD4 T cells specifically recognizes the 12-20 segment of the insulin B-chain (B:12-20), an epitope that is generated from direct presentation of insulin peptides by antigen-presenting cells3,4. These T cells do not respond to antigen-presenting cells that have taken up insulin that, after processing, leads to presentation of a different segment representing a one-residue shift, B:13-214. CD4 T cells that recognize B:12-20 escape negative selection in the thymus and cause diabetes, whereas those that recognize B:13-21 have only a minor role in autoimmunity3-5. Although presentation of B:12-20 is evident in the islets3,6, insulin-specific germinal centres can be formed in various lymphoid tissues, suggesting that insulin presentation is widespread7,8. Here we use live imaging to document the distribution of insulin recognition by CD4 T cells throughout various lymph nodes. Furthermore, we identify catabolized insulin peptide fragments containing defined pathogenic epitopes in ß-cell granules from mice and humans. Upon glucose challenge, these fragments are released into the circulation and are recognized by CD4 T cells, leading to an activation state that results in transcriptional reprogramming and enhanced diabetogenicity. Therefore, a tissue such as pancreatic islets, by releasing catabolized products, imposes a constant threat to self-tolerance. These findings reveal a self-recognition pathway underlying a primary autoantigen and provide a foundation for assessing antigenic targets that precipitate pathogenic outcomes by systemically sensitizing lymphoid tissues.

Cytocidal macrophages in symbiosis with CD4 and CD8 T cells cause acute diabetes following checkpoint blockade of PD-1 in NOD mice.

Autoimmune diabetes is one of the complications resulting from checkpoint blockade immunotherapy in cancer patients, yet the underlying mechanisms for such an adverse effect are not well understood. Leveraging the diabetes-susceptible nonobese diabetic (NOD) mouse model, we phenocopy the diabetes progression induced by programmed death 1 (PD-1)/PD-L1 blockade and identify a cascade of highly interdependent cellular interactions involving diabetogenic CD4 and CD8 T cells and macrophages. We demonstrate that exhausted CD8 T cells are the major cells that respond to PD-1 blockade producing high levels of IFN-γ. Most importantly, the activated T cells lead to the recruitment of monocyte-derived macrophages that become highly activated when responding to IFN-γ. These macrophages acquire cytocidal activity against ß-cells via nitric oxide and induce autoimmune diabetes. Collectively, the data in this study reveal a critical role of macrophages in the PD-1 blockade-induced diabetogenesis, providing new insights for the understanding of checkpoint blockade immunotherapy in cancer and infectious diseases.

Echo speckle imaging of dynamic processes in soft materials.

We present a laser-speckle imaging technique, termed Echo speckle imaging (ESI), that quantifies the local dynamics in biological tissue and soft materials with a noise level around or below 10% of the measured signal without affecting the spatial resolution. We achieve this through an unconventional speckle beam illumination that creates changing, statistically independent illumination conditions and substantially increases the measurement accuracy. Control experiments for dynamically homogeneous and heterogeneous soft materials and tissue phantoms illustrate the performance of the method. We show that this approach enables us to precision-monitor purely dynamic heterogeneities in turbid soft media with a lateral resolution of 100 µm and better.

Ergodic and non-ergodic regimes in temporal laser speckle imaging.

The non-ergodicity problem of temporal averaging in the laser speckle contrast imaging (LSCI) is discussed both theoretically and numerically. We demonstrate that temporal speckle statistics assessed within a finite time window might differ from the statistics of the speckle ensemble. The dependence of temporal speckle contrast on sample dynamics is non-monotonic and demonstrates regimes of negative (ergodic), as well as positive correlations with dynamics (non-ergodic). The ergodic regime is similar to an ensemble (spatial) averaging case and is typically assumed for interpretation of LSCI measurements. The positive relation in the non-ergodic regime is an artifact of temporal statistics which we further quantify and describe the transition between two regimes.

Molecular and Mechanical Causes of Microtubule Catastrophe and Aging.

Tubulin polymers, microtubules, can switch abruptly from the assembly to shortening. These infrequent transitions, termed "catastrophes", affect numerous cellular processes but the underlying mechanisms are elusive. We approached this complex stochastic system using advanced coarse-grained molecular dynamics modeling of tubulin-tubulin interactions. Unlike in previous simplified models of dynamic microtubules, the catastrophes in this model arise owing to fluctuations in the composition and conformation of a growing microtubule tip, most notably in the number of protofilament curls. In our model, dynamic evolution of the stochastic microtubule tip configurations over a long timescale, known as the system's "aging", gives rise to the nonexponential distribution of microtubule lifetimes, consistent with experiment. We show that aging takes place in the absence of visible changes in the microtubule wall or tip, as this complex molecular-mechanical system evolves slowly and asymptotically toward the steady-state level of the catastrophe-promoting configurations. This new, to our knowledge, theoretical basis will assist detailed mechanistic investigations of the mechanisms of action of different microtubule-binding proteins and drugs, thereby enabling accurate control over the microtubule dynamics to treat various pathologies.

Mechanical Properties of the Pt-CNT Composite under Uniaxial Deformation: Tension and Compression.

Composite materials are gaining increasing attention from researchers worldwide due to their ability to offer tailored properties for various technical challenges. One of these promising fields is metal matrix composites, including carbon-reinforced metals and alloys. These materials allow for the reduction of density while simultaneously enhancing their functional properties. This study is focused on the Pt-CNT composite, its mechanical characteristics, and structural features under uniaxial deformation depending on temperature and mass fractions of carbon nanotube (CNT). The mechanical behavior of platinum reinforced with carbon nanotubes of diameters varying in the interval 6.62-16.55 Å under uniaxial tension and compression deformation has been studied by the molecular dynamics method. Simulations for tensile and compression deformations have been done for all specimens at different temperatures (viz. 300 K, 500 K, 700 K, 900 K, 1100 K, and 1500 K). The calculated mechanical characteristics allow us to conclude that, compared to pure platinum, the Young's modulus increased by about 60%. The results indicate that yield and tensile strength values decreases with increase in temperature for all simulation blocks. This increase was due to the inherent high axial rigidity of CNTs. In this work, these characteristics are calculated for the first time for Pt-CNT. It can be concluded that CNTs can be an effective reinforcing material for composites based on a metal matrix under tensile strain.

Single-cell RNA sequencing of murine islets shows high cellular complexity at all stages of autoimmune diabetes.

Tissue-specific autoimmune diseases are driven by activation of diverse immune cells in the target organs. However, the molecular signatures of immune cell populations over time in an autoimmune process remain poorly defined. Using single-cell RNA sequencing, we performed an unbiased examination of diverse islet-infiltrating cells during autoimmune diabetes in the nonobese diabetic mouse. The data revealed a landscape of transcriptional heterogeneity across the lymphoid and myeloid compartments. Memory CD4 and cytotoxic CD8 T cells appeared early in islets, accompanied by regulatory cells with distinct phenotypes. Surprisingly, we observed a dramatic remodeling in the islet microenvironment, in which the resident macrophages underwent a stepwise activation program. This process resulted in polarization of the macrophage subpopulations into a terminal proinflammatory state. This study provides a single-cell atlas defining the staging of autoimmune diabetes and reveals that diabetic autoimmunity is driven by transcriptionally distinct cell populations specialized in divergent biological functions.

First Experiences With a Wearable Multisensor in an Outpatient Glucose Monitoring Study, Part I: The Users' View.

BACKGROUND: Extensive past work showed that noninvasive continuous glucose monitoring with a wearable Multisensor device worn on the upper arm provides useful information about glucose trends to improve diabetes therapy in controlled and semicontrolled conditions. METHODS: To test previous findings also in uncontrolled in-clinic and outpatient conditions, a long-term study has been conducted to collect Multisensor and reference glucose data in a population of 20 type 1 diabetes subjects. A total of 1072 study days were collected and a fully on-line compatible algorithmic routine linking Multisensor data to glucose applied to estimate glucose trends noninvasively. The operation of a digital log book, daily semiautomated data transfer and at least 10 daily SMBG values were requested from the patient. RESULTS: Results showed that the Multisensor is capable of indicating glucose trends. It can do so in 9 out of 10 cases either correctly or with one level of discrepancy. This means that in 90% of all cases the Multisensor shows the glucose dynamic to rapidly increase or at least increase. CONCLUSIONS: The Multisensor and the algorithmic routine used in controlled conditions can track glucose trends in all patients, also in uncontrolled conditions. Training of the patient proved to be essential. The workload imposed on patients was significant and should be reduced in the next step with further automation. The feature of glucose trend indication was welcomed and very much appreciated by patients; this value creation makes a strong case for the justification of wearing a wearable.

First Experiences With a Wearable Multisensor Device in a Noninvasive Continuous Glucose Monitoring Study at Home, Part II: The Investigators' View.

BACKGROUND: Extensive past work showed that noninvasive continuous glucose monitoring with a wearable multisensor device worn on the upper arm provides useful information about glucose trends to improve diabetes therapy in controlled and semicontrolled conditions. METHOD: To test previous findings also in uncontrolled conditions, a long term at home study has been organized to collect multisensor and reference glucose data in a population of 20 type 1 diabetes subjects. A total of 1072 study days were collected and a fully on-line compatible algorithmic routine linking multisensor data to glucose applied to estimate glucose levels noninvasively. RESULTS: The algorithm used here calculates glucose values from sensor data and adds a constant obtained by a daily calibration. It provides point inaccuracy measured by a MARD of 35.4 mg/dL on test data. This is higher than current state-of-the-art minimally invasive devices, but still 86.9% of glucose rate points fall within the zone AR+BR. CONCLUSIONS: The multisensor device and the algorithmic routine used earlier in controlled conditions tracks glucose changes also in uncontrolled conditions, although with lower accuracy. The examination of learning curves suggests that obtaining more data would not improve the results. Therefore, further efforts would focus on the development of more complex algorithmic routines able to compensate for environmental and physiological confounders better.

The islet-resident macrophage is in an inflammatory state and senses microbial products in blood.

We examined the transcriptional profiles of macrophages that reside in the islets of Langerhans of 3-wk-old non-obese diabetic (NOD), NOD.Rag1-/-, and B6.g7 mice. Islet macrophages expressed an activation signature with high expression of Tnf, Il1b, and MHC-II at both the transcript and protein levels. These features are common with barrier macrophages of the lung and gastrointestinal tract. Moreover, injection of lipopolysaccharide induced rapid inflammatory gene expression, indicating that blood stimulants are accessible to the macrophages and that these macrophages can sense them. In NOD mice, the autoimmune process imparted an increased inflammatory signature, including elevated expression of chemokines and chemokine receptors and an oxidative response. The elevated inflammatory signature indicates that the autoimmune program was active at the time of weaning. Thus, the macrophages of the islets of Langerhans are poised to mount an immune response even at steady state, while the presence of the adaptive immune system elevates their activation state.

The Effect of a Global, Subject, and Device-Specific Model on a Noninvasive Glucose Monitoring Multisensor System.

BACKGROUND: We study here the influence of different patients and the influence of different devices with the same patients on the signals and modeling of data from measurements from a noninvasive Multisensor glucose monitoring system in patients with type 1 diabetes. The Multisensor includes several sensors for biophysical monitoring of skin and underlying tissue integrated on a single substrate. METHOD: Two Multisensors were worn simultaneously, 1 on the upper left and 1 on the upper right arm by 4 patients during 16 study visits. Glucose was administered orally to induce 2 consecutive hyperglycemic excursions. For the analysis, global (valid for a population of patients), personal (tailored to a specific patient), and device-specific multiple linear regression models were derived. RESULTS: We find that adjustments of the model to the patients improves the performance of the glucose estimation with an MARD of 17.8% for personalized model versus a MARD of 21.1% for the global model. At the same time the effect of the measurement side is negligible. The device can equally well measure on the left or right arm. We also see that devices are equal in the linear modeling. Thus hardware calibration of the sensors is seen to be sufficient to eliminate interdevice differences in the measured signals. CONCLUSIONS: We demonstrate that the hardware of the 2 devices worn on the left and right arms are consistent yielding similar measured signals and thus glucose estimation results with a global model. The 2 devices also return similar values of glucose errors. These errors are mainly due to nonstationarities in the measured signals that are not solved by the linear model, thus suggesting for more sophisticated modeling approaches.

Follow-up of cortical activity and structure after lesion with laser speckle imaging and magnetic resonance imaging in nonhuman primates.

The nonhuman primate model is suitable to study mechanisms of functional recovery following lesion of the cerebral cortex (motor cortex), on which therapeutic strategies can be tested. To interpret behavioral data (time course and extent of functional recovery), it is crucial to monitor the properties of the experimental cortical lesion, induced by infusion of the excitotoxin ibotenic acid. In two adult macaque monkeys, ibotenic acid infusions produced a restricted, permanent lesion of the motor cortex. In one monkey, the lesion was monitored over 3.5 weeks, combining laser speckle imaging (LSI) as metabolic readout (cerebral blood flow) and anatomical assessment with magnetic resonance imaging (T2-weighted MRI). The cerebral blood flow, measured online during subsequent injections of the ibotenic acid in the motor cortex, exhibited a dramatic increase, still present after one week, in parallel to a MRI hypersignal. After 3.5 weeks, the cerebral blood flow was strongly reduced (below reference level) and the hypersignal disappeared from the MRI scan, although the lesion was permanent as histologically assessed post-mortem. The MRI data were similar in the second monkey. Our experiments suggest that LSI and MRI, although they reflect different features, vary in parallel during a few weeks following an excitotoxic cortical lesion.

Characteristics of a multisensor system for non invasive glucose monitoring with external validation and prospective evaluation.

The Multisensor Glucose Monitoring System (MGMS) features non invasive sensors for dielectric characterisation of the skin and underlying tissue in a wide frequency range (1 kHz-100 MHz, 1 and 2 GHz) as well as optical characterisation. In this paper we describe the results of using an MGMS in a miniaturised housing with fully integrated sensors and battery. Six patients with Type I Diabetes Mellitus (age 44±16 y; BMI 24.1±1.3 kg/m(2), duration of diabetes 27±12 y; HbA1c 7.3±1.0%) wore a single Multisensor at the upper arm position and performed a total of 45 in-clinic study days with 7 study days per patient on average (min. 5 and max. 10). Glucose changes were induced either orally or by i.v. glucose administration and the blood glucose was measured routinely. Several prospective data evaluation routines were applied to evaluate the data. The results are shown using one of the restrictive data evaluation routines, where measurements from the first 22 study days were used to train a linear regression model. The global model was then prospectively applied to the data of the remaining 23 study days to allow for an external validation of glucose prediction. The model application yielded a Mean Absolute Relative Difference of 40.8%, a Mean Absolute Difference of 51.9 mg dL(-1), and a correlation of 0.84 on average per study day. The Clarke error grid analyses showed 89.0% in A+B, 4.5% in C, 4.6% in D and 1.9% in the E region. Prospective application of a global, purely statistical model, demonstrates that glucose variations can be tracked non invasively by the MGMS in most cases under these conditions.

Cutaneous blood perfusion as a perturbing factor for noninvasive glucose monitoring.

It is widely accepted that noninvasive glucose monitoring (NIGM) has the potential to revolutionize diabetes therapy. However, current approaches to NIGM studied to date have not yet demonstrated a level of acceptable functionality to allow real-time use, beyond restricted fields of application. A number of reviews have been devoted to the subject of NIGM with different focuses related to challenges and a description of the respective underlying problems. This review is aimed at addressing a fundamental topic in the application of NIGM that seems to have received less attention, by describing the perturbations that result in a reduced functionality of NIGM in daily use. Here we provide a short general introduction to glucose monitoring and a basic illustration of the electromagnetic spectrum with a description of the respective physical mechanisms underlying the measurement techniques. This allows for a better understanding of how these perturbing factors affect the measured properties. Cutaneous blood perfusion is one of the major perturbing factors to NIGM, along with variations in temperature, migration of water, and the effect of attachment of the sensor to the skin. An understanding of the mechanisms underlying perfusion variation over time and within the measured human skin tissue matrix is required to enable a discrimination between glucose-induced effects within the tissue and various biophysical impacts to be made. It is suggested that a plurality of probing frequencies is required to discriminate glucose-related changes from the perturbations. A system designed to perform the measurements in different regions of the electromagnetic spectrum with dedicated sensors (multisensor approach) has the potential to more efficiently and reliably discriminate glucose-related information from perturbations. This can be achieved by combining signals related to measurements with different physical underlying mechanisms of the interaction between the probing field propagation and the tissue to help account for the different sources of perturbations.

Quantitative modeling of laser speckle imaging.

We have analyzed the image formation and dynamic properties in laser speckle imaging (LSI) both experimentally and with Monte Carlo simulation. We show for the case of a liquid inclusion that the spatial resolution and the signal itself are both significantly affected by scattering from the turbid environment. Multiple scattering leads to blurring of the dynamic inhomogeneity as detected by LSI. The presence of a nonfluctuating component of scattered light results in the significant increase in the measured image contrast and complicates the estimation of the relaxation time. We present a refined processing scheme that allows a correct estimation of the relaxation time from LSI data.

Multiple-scattering suppression in dynamic light scattering based on a digital camera detection scheme.

We introduce a charge-coupled device (CCD) camera-based detection scheme in dynamic light scattering that provides information on the single-scattered autocorrelation function even for fairly turbid samples. It is based on the single focused laser beam geometry combined with the selective cross-correlation analysis of the scattered light intensity. Using a CCD camera as a multispeckle detector, we show how spatial correlations in the intensity pattern can be linked to single- and multiple-scattering processes. Multiple-scattering suppression is then achieved by an efficient cross-correlation algorithm working in real time with a temporal resolution down to 0.02 s. Our approach allows access to the extensive range of systems that show low-order scattering by selective detection of the singly scattered light. Model experiments on slowly relaxing suspensions of titanium dioxide in glycerol were carried out to establish the validity range of our approach. Successful application of the method is demonstrated up to a scattering coefficient of more than micro(S) = 5 cm(-1) for the sample size of L = 1 cm.