Mast Cell Deficiency in Mice Attenuates Insulin Phenolic Preservative-Induced Inflammation.

One major obstacle that limits the lifespan of insulin infusion pumps is surmounting the tissue site reaction at the device implantation site. All commercial insulin formulations contain insulin phenolic preservatives (IPPs) designed to ensure insulin protein stability and prolong shelf-life. However, our laboratory demonstrated that these preservatives are cytotoxic and induce inflammation. Mature mast cells (MCs) reside in cutaneous tissue and are one of the first responders to an epidermal breach. Upon activation, MCs release proinflammatory and immunomodulatory prepacked mediators that exacerbate these inflammatory reactions. Thus, we hypothesized that once the epidermis is breached, cutaneous MCs are triggered inciting the inflammatory response to IPP-induced inflammation. This hypothesis was pursued utilizing our modified in vivo mouse air pouch model, including a c-kit dependent (C57BL/6J-kitW-sh/W-sh) and a c-kit independent (Cpa3-Cre; Mcl-1fl/fl) MC-deficient mouse model. Leukocytes were quantified in the mouse air pouch lavage fluid following flow cytometry analysis for IPP infusion under three different states, insulin-containing phenolic preservatives (Humalog®), insulin preservatives alone, and normal saline as a control. The air pouch wall was assessed using histopathological evaluations. Flow cytometry analysis demonstrated a statistically significant difference in inflammatory cell recruitment for both MC-deficient mouse models when compared to the control strain including infused control saline. Significantly less inflammation was observed at the site of infusion for the MC-deficient strains compared to the control strain. Overall, concordant results were obtained in both mouse types, C57Bl6-kitW-sh/W-sh and Cpa3-Cre; Mcl-1fl/fl. These findings in multiple model systems support the conclusion that MCs have important or possible unique roles in IPP-induced inflammation.

Insulin Derived Fibrils Induce Cytotoxicity in vitro and Trigger Inflammation in Murine Models.

BACKGROUND: Effective exogenous insulin delivery is the cornerstone of insulin dependent diabetes mellitus management. Recent literature indicates that commercial insulin-induced tissue reaction and cellular cytotoxicity may contribute to variability in blood glucose as well as permanent loss of injection or infusion site architecture and function. It is well accepted that insulin formulations are susceptible to mechanical and chemical stresses that lead to insulin fibril formation. This study aims to characterize in vitro and in vivo toxicity, as well as pro-inflammatory activity of insulin fibrils. METHOD: In vitro cell culture evaluated cytotoxicity and fibril uptake by macrophages and our modified murine air-pouch model quantified inflammatory activity. The latter employed FLOW cytometry and histopathology to characterize fibril-induced inflammation in vivo, which included fibril uptake by inflammatory phagocytes. RESULTS: These studies demonstrated that insulin derived fibrils are cytotoxic to cells in vitro. Furthermore, inflammation is induced in the murine air-pouch model in vivo and in response, macrophages uptake fibrils both in vitro and in vivo. CONCLUSIONS: Administration of insulin fibrils can lead to cytotoxicity in macrophages. In vivo data demonstrate insulin fibrils to be pro-inflammatory which over time can lead to cumulative cell/tissue toxicity, inflammation, and destructive wound healing. Long term, these tissue reactions could contribute to loss of insulin injection site architecture and function.

Inflammation at Site of Insulin Infusion Diminishes Glycemic Control.

The approximation of euglycemia is the most effective means of preventing diabetic complications, which is achieved through effective insulin delivery. Recent reports indicate that insulin phenolic preservatives, which are found in all commercial insulin formulations, are cytotoxic, pro-inflammatory and induce secondary fibrosis. Therefore, we hypothesize that these preservatives induce an inflammatory response at the site of insulin infusion leading to diminished glycemic control and adverse pharmacokinetic outcomes. Insulin degradation by inflammatory cell proteases was quantitated following protease treatment in vitro. A modified murine air pouch model was utilized to evaluate the relative inflammatory responses following infusions of saline, insulin preservatives, and insulin, utilizing the adjuvant irritant thioglycolate. Blood glucose levels were monitored in diabetic mice with and without air pouch irritation. A pharmacokinetic analysis evaluated insulin effectiveness for diabetic mice between these two conditions. Inflammatory cells are significantly present in insulin preservative-induced inflammation, which effects diminished blood glucose control by both insulin uptake and degradation. Insulin containing these preservatives resulted in similar degrees of inflammation as observed with the irritant thioglycolate. These studies imply that the preservative agents found in commercial insulin formulations induce an intense localized inflammatory reaction. This inflammatory reaction may be responsible for the premature failure of insulin infusion devices. Future studies directed at reducing this inflammatory reaction may prove to be an important step in extending the lifespan of insulin infusion devices.

A pharmacological approach assessing the role of mast cells in insulin infusion site inflammation.

Background Extending the lifespan of subcutaneous insulin administration sets and infusion pumps requires overcoming unreliable insulin delivery induced by dermal reactions. All commercially available insulin formulations contain insulin phenolic preservatives (IPP), which stabilize the insulin molecule but result in unwanted cell and tissue toxicity. Mast cells, which are the first line of defense once the epithelium is breached, are particularly abundant beneath the skin surface. Thus, we hypothesize a sequence of events initiated by device insertion that activates skin mast cells (MC) that subsequently trigger neutrophil and monocyte/macrophage recruitment. The ensuing inflammatory response compromises effective insulin infusion therapy. Methods We employed a non-genetic, pharmacological approach to MC membrane stabilization using Cromolyn sodium (CS), which inhibits MC degranulation. These studies were conducted in our modified air pouch mouse model using non-diabetic and streptozotocin induced diabetic mice. We evaluated the impact of systemic CS through intraperitoneal injections, as well as the impact of local CS through co-infusion, on infusion catheter insertion and IPP-induced inflammation. Results CS at a concentration of 50 mg/kg minimized inflammation triggered in response to insulin phenolic preservatives present in standard insulin formulations. The resultant degree of tissue inflammation was comparable to that observed with saline injections. Conclusion Targeting MC has the potential to extend the longevity of insulin infusion sets by mitigating the inflammatory response. Future studies should be directed at employing other MC models, such as newer Cre/loxP mouse strains, to confirm the sentinel role of MC in insulin infusion therapy.

Phenolic Preservative Removal from Commercial Insulin Formulations Reduces Tissue Inflammation while Maintaining Euglycemia.

Background: Exogenous insulin therapy requires stabilization of the insulin molecule, which is achieved through the use of excipients (e.g., phenolic preservatives (PP)) that provide protein stability, sterility and prolong insulin shelf life. However, our laboratory recently reported that PP, (e.g., m-creosol and phenol) are also cytotoxic, inducing inflammation and fibrosis. Optimizing PP levels through filtration would balance the need for insulin preservation with PP-induced inflammation. Method: Zeolite Y (Z-Y), a size-exclusion-based resin, was employed to remove PP from commercial insulin formulations (Humalog) before infusion. Results: PP removal significantly decreased cell toxicity in vitro and inflammation in vivo. Infusion site histological analysis after a 3 day study demonstrated that leukocyte accumulation increased with nonfiltered preparations but decreased after filtration. Additional studies demonstrated that a Z-Y fabricated filter effectively removed excess PP such that the filtered insulin solution achieved equivalent glycemic control in diabetic mice when compared to nonfiltered insulin. Conclusion: This approach represents the proof of concept that using Z-Y for in-line PP removal assists in lowering inflammation at the site of insulin infusion and thus could lead to extending the functional lifespan of insulin infusion sets in vivo.

Nourin-Dependent miR-137 and miR-106b: Novel Early Inflammatory Diagnostic Biomarkers for Unstable Angina Patients.

BACKGROUND: Currently, no blood biomarkers exist that can diagnose unstable angina (UA) patients. Nourin is an early inflammatory mediator rapidly released within 5 min by reversible ischemic myocardium, and if ischemia persists, it is also released by necrosis. Nourin is elevated in acute coronary syndrome (ACS) patients but not in symptomatic noncardiac and healthy subjects. Recently, circulating microRNAs (miRNAs) have been established as markers of disease, including cardiac injury and inflammation. OBJECTIVES: To profile and validate the potential diagnostic value of Nourin-dependent miR-137 (marker of cell damage) and miR-106b-5p (marker of inflammation) as early biomarkers in suspected UA patients and to investigate the association of their target and regulating genes. METHODS: Using Nourin amino acid sequence, an integrated bioinformatics analysis was conducted. Analysis indicated that Nourin is a direct target for miR-137 and miR-106b-5p in myocardial ischemic injury. Two linked molecular networks of lncRNA/miRNAs/mRNAs were also retrieved, including CTB89H12.4/miR-137/FTHL-17 and CTB89H12.4/miR-106b-5p/ANAPC11. Gene expression profiling was assessed in serum samples collected at presentation to an emergency department (ED) from: (1) UA patients (n = 30) (confirmed by invasive coronary angiography with stenosis greater than 50% and troponin level below the clinical decision limit); (2) patients with acute ST elevation myocardial infarction (STEMI) (n = 16) (confirmed by persistent ST-segment changes and elevated troponin level); and 3) healthy subjects (n = 16). RESULTS: Gene expression profiles showed that miR-137 and miR-106b-5p were significantly upregulated by 1382-fold and 192-fold in UA compared to healthy, and by 2.5-fold and 4.6-fold in STEMI compared to UA, respectively. Healthy subjects showed minimal expression profile. Receiver operator characteristics (ROC) analysis revealed that the two miRNAs were sensitive and specific biomarkers for assessment of UA and STEMI patients. Additionally, Spearman's correlation analysis revealed a significant association of miRNAs with the associated mRNA targets and the regulating lncRNA. CONCLUSIONS: Nourin-dependent gene expression of miR-137 and miR-106b-5p are novel blood-based biomarkers that can diagnose UA and STEMI patients at presentation and stratify severity of myocardial ischemia, with higher expression in STEMI compared to UA. Early diagnosis of suspected UA patients using the novel Nourin biomarkers is key for initiating guideline-based therapy that improves patients' health outcomes.

Noninflammatory Stress-Induced Remodeling of Mandibular Bone: Impact of Age and Pregnancy.

PURPOSE: The impact of noninflammatory stress, such as aging and pregnancy, on human long bone remodeling is well-established, but little is known about the impact of these stressors on oral bones, including the mandibular bone. To begin to fill this gap in our knowledge, we utilized a mouse mandibular model to evaluate the impact of noninflammatory simple stressors, ie, aging and pregnancy, on bone mandibular architecture and bone density in the mandible using micro-CT. MATERIALS AND METHODS: For the present study, mandibles were obtained from both aged and pregnant C57BL/6 mice and analyzed using micro-CT technology. Micro-CT metrics included bone volume fraction (BVF), total volume (TV), tissue density, and apparent density in the mandible on the mandibular area of compact and trabecular bone, in which the teeth are embedded. All bone-related metrics data from aged and pregnant mice were analyzed using ANOVA analysis and visualized in boxplots. RESULTS: Age-dependent bone remodeling occurred over 4 to 18 weeks of age, ie, increases in BVF, TV, BV/TV, as well as tissue and bone density. Evaluation of bone remodeling in breeder mice (repeated pregnancy model) and virgin mice (age-matched controls) at 37 weeks of age demonstrated that breeder mice had a dramatic decline in all bone metrics measured. CONCLUSIONS: This study underscores the need for more research on noninflammatory stress-related mandibular bone remodeling (eg, age and pregnancy), which compromises bone strength and tooth anchoring. The data also underscores loss of alveolar bone height, as in periodontitis, is an important metric for a more complete assessment of bone loss. This report on mice provides essential data that can be applied for oral-maxillofacial surgeons and periodontists when planning for dental implants in patients with such stressors. Periodontitis related bone loss occurs independent of skeletal homeostasis, although osteoporosis may adversely affect alveolar bone height in humans.

Advancing continuous subcutaneous insulin infusion in vivo: New insights into tissue challenges.

Continuous Subcutaneous Insulin Infusion (CSII) is superior to conventional insulin therapy as it improves glycemic control thus reducing the probability of diabetic complications. Notwithstanding CSII's benefits, insulin dependent diabetic patients rarely achieve optimal glucose control. Moreover, CSII is only FDA approved for 3 days and often fails prematurely for reasons that have not been fully elucidated. We hypothesize that phenolic compounds, such as m-cresol and phenol, which are present in all commercial insulin formulations are responsible for the tissue reaction occurring at the insulin infusion site. This hypothesis was examined with in vitro cell cultures and a mouse air-pouch model to determine cellular and tissue reactions following infusions with saline, phenolic compounds, (i.e., commercial diluent), and insulin. We demonstrated that diluent and insulin were cytotoxic to cells in culture at sub-clinical concentrations (e.g., >1:10 of commercial insulin). Air pouch studies demonstrated that infusion of either diluted insulin or diluent itself induced three to five-fold level of recruited leukocytes as compared to saline. At both 3- and 7-days post infusion, these were predominantly neutrophils and macrophages. We conclude that phenolic compounds in commercial insulin preparations are cell and tissue toxic, which contributes to the failure of effective insulin infusion therapy.

Continuous Glucose Monitoring Devices: Past, Present, and Future Focus on the History and Evolution of Technological Innovation.

The concept of implantable glucose sensors has been promulgated for more than 40 years. It is now accepted that continuous glucose monitoring (CGM) increases quality of life by allowing informed diabetes management decisions as a result of more optimized glucose control. The focus of this article is to provide a brief overview of the CGM market history, emerging technologies, and the foreseeable challenges for the next CGM generations as well as proposing possible solutions in an effort to advance the next generation of implantable sensor.

Crosslinked basement membrane-based coatings enhance glucose sensor function and continuous glucose monitoring in vivo.

Overcoming sensor-induced tissue reactions is an essential element of achieving successful continuous glucose monitoring (CGM) in the management of diabetes, particularly when used in closed loop technology. Recently, we demonstrated that basement membrane (BM)-based glucose sensor coatings significantly reduced tissue reactions at sites of device implantation. However, the biocompatible BM-based biohydrogel sensor coating rapidly degraded over a less than a 3-week period, which effectively eliminated the protective sensor coating. In an effort to increase the stability and effectiveness of the BM coating, we evaluated the impact of crosslinking BM utilizing glutaraldehyde as a crosslinking agent, designated as X-Cultrex. Sensor performance (nonrecalibrated) was evaluated for the impact of these X-Cultrex coatings in vitro and in vivo. Sensor performance was assessed over a 28-day time period in a murine CGM model and expressed as mean absolute relative difference (MARD) values. Tissue reactivity of Cultrex-coated, X-Cultrex-coated, and uncoated glucose sensors was evaluated over a 28-day time period in vivo using standard histological techniques. These studies demonstrated that X-Cultrex-based sensor coatings had no effect on glucose sensor function in vitro. In vivo, glucose sensor performance was significantly enhanced following X-Cultrex coating throughout the 28-day study. Histological evaluations of X-Cultrex-treated sensors demonstrated significantly less tissue reactivity when compared to uncoated sensors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 7-16, 2018.

Impact of CCL2 and CCR2 chemokine/receptor deficiencies on macrophage recruitment and continuous glucose monitoring in vivo.

The accumulation of macrophages (MΦ) at the sensor-tissue interface is thought to be a major player in controlling tissue reactions and sensor performance in vivo. Nevertheless until recently no direct demonstration of the causal relationship between MΦ aggregation and loss of sensor function existed. Using a Continuous Glucose Monitoring (CGM) murine model we previously demonstrated that genetic deficiencies of MΦ or depletion of MΦ decreased MΦ accumulation at sensor implantation sites, which led to significantly enhanced CGM performance, when compared to normal mice. Additional studies in our laboratories have also demonstrated that MΦ can act as "metabolic sinks" by depleting glucose levels at the implanted sensors in vitro and in vivo. In the present study we extended these observations by demonstrating that MΦ chemokine (CCL2) and receptor (CCR2) knockout mice displayed a decrease in inflammation and MΦ recruitment at sensor implantation sites, when compared to normal mice. This decreased MΦ recruitment significantly enhanced CGM performance when compared to control mice. These studies demonstrated the importance of the CCL2 family of chemokines and related receptors in MΦ recruitment and sensor performance and suggest chemokine targets for enhancing CGM in vivo.

Basement Membrane-Based Glucose Sensor Coatings Enhance Continuous Glucose Monitoring in Vivo.

BACKGROUND: Implantable glucose sensors demonstrate a rapid decline in function that is likely due to biofouling of the sensor. Previous efforts directed at overcoming this issue has generally focused on the use of synthetic polymer coatings, with little apparent effect in vivo, clearly a novel approach is required. We believe that the key to extending sensor life span in vivo is the development of biocompatible basement membrane (BM) based bio-hydrogels as coatings for glucose sensors. METHOD: BM based bio-hydrogel sensor coatings were developed using purified BM preparations (ie, Cultrex from Trevigen Inc). Modified Abbott sensors were coated with Cultrex BM extracts. Sensor performance was evaluated for the impact of these coatings in vitro and in vivo in a continuous glucose monitoring (CGM) mouse model. In vivo sensor function was assessed over a 28-day time period expressed as mean absolute relative difference (MARD) values. Tissue reactivity of both Cultrex coated and uncoated glucose sensors was evaluated at 7, 14, 21 and 28 days post-sensor implantation with standard histological techniques. RESULTS: The data demonstrate that Cultrex-based sensor coatings had no effect on glucose sensor function in vitro. In vivo glucose sensor performance was enhanced following BM coating as determined by MARD analysis, particularly in weeks 2 and 3. In vivo studies also demonstrated that Cultrex coatings significantly decreased sensor-induced tissue reactions at the sensor implantation sites. CONCLUSION: Basement-membrane-based sensor coatings enhance glucose sensor function in vivo, by minimizing or preventing sensor-induced tissues reactions.

Cell based metabolic barriers to glucose diffusion: macrophages and continuous glucose monitoring.

It is assumed that MQ are central to glucose sensor bio-fouling and therefore have a major negative impact on continuous glucose monitoring (CGM) performance in vivo. However to our knowledge there is no data in the literature to directly support or refute this assumption. Since glucose and oxygen (O2) are key to glucose sensor function in vivo, understanding and controlling glucose and O2 metabolic activity of MQ is likely key to successful glucose sensor performance. We hypothesized that the accumulation of MQ at the glucose sensor-tissue interface will act as "Cell Based Metabolic Barriers" (CBMB) to glucose diffusing from the interstitial tissue compartment to the implanted glucose sensor and as such creating an artificially low sensor output, thereby compromising sensor function and CGM. Our studies demonstrated that 1) direct injections of MQ at in vivo sensor implantation sites dramatically decreased sensor output (measured in nA), 2) addition of MQ to glucose sensors in vitro resulted in a rapid and dramatic fall in sensor output and 3) lymphocytes did not affect sensor function in vitro or in vivo. These data support our hypothesis that MQ can act as metabolic barriers to glucose and O2 diffusion in vivo and in vitro.

Impact of macrophage deficiency and depletion on continuous glucose monitoring in vivo.

Although it is assumed that macrophages (MQ) have a major negative impact on continuous glucose monitoring (CGM), surprisingly there is no data in the literature to directly support or refute the role of MQ or related foreign body giant cells in the bio-fouling of glucose sensors in vivo. As such, we developed the hypothesis that MQ are key in controlling glucose sensor performance and CGM in vivo and MQ deficiencies or depletion would enhance CGM. To test this hypothesis we determined the presence/distribution of MQ at the sensor tissue interface over a 28-day time period using F4/80 antibody and immunohistochemical analysis. We also evaluated the impact of spontaneous MQ deficiency (op/op mice) and induced-transgenic MQ depletions (Diphtheria Toxin Receptor (DTR) mice) on sensor function and CGM utilizing our murine CGM system. The results of these studies demonstrated: 1) a time dependent increase in MQ accumulation (F4/80 positive cells) at the sensor tissue interface; and 2) MQ deficient mice and MQ depleted C57BL/6 mice demonstrated improved sensor performance (MARD) when compared to normal mice (C57BL/6). These studies directly demonstrate the importance of MQ in sensor function and CGM in vivo.

Role of vascular networks in extending glucose sensor function: Impact of angiogenesis and lymphangiogenesis on continuous glucose monitoring in vivo.

The concept of increased blood vessel (BV) density proximal to glucose sensors implanted in the interstitial tissue increases the accuracy and lifespan of sensors is accepted, despite limited existing experimental data. Interestingly, there is no previous data or even conjecture in the literature on the role of lymphatic vessels (LV) alone, or in combination with BV, in enhancing continuous glucose monitoring (CGM) in vivo. To investigate the impact of inducing vascular networks (BV and LV) at sites of glucose sensor implantation, we utilized adenovirus based local gene therapy of vascular endothelial cell growth factor-A (VEGF-A) to induce vessels at sensor implantation sites. The results of these studies demonstrated that (1) VEGF-A based local gene therapy increases vascular networks (blood vessels and lymphatic vessels) at sites of glucose sensor implantation; and (2) this local increase of vascular networks enhances glucose sensor function in vivo from 7 days to greater than 28 days postsensor implantation. This data provides "proof of concept" for the effective usage of local angiogenic factor (AF) gene therapy in mammalian models in an effort to extend CGM in vivo. It also supports the practice of a variety of viral and nonviral vectors as well as gene products (e.g. anti-inflammatory and anti-fibrosis genes) to engineer "implant friendly tissues" for the usage with implantable glucose sensors as well as other implantable devices.

Role of interleukin-1/interleukin-1 receptor antagonist family of cytokines in long-term continuous glucose monitoring in vivo.

BACKGROUND: Glucose-sensor-induced tissue reactions (e.g., inflammation and wound healing) are known to negatively impact sensor function in vivo. The roles of cytokine networks in controlling these tissue reactions (i.e., sensor biofouling) is not understood. In the present study, we investigated the role of interleukin-1 receptor antagonist (IL-1Ra), a key anti-inflammatory antagonist of the proinflammatory interleukin-1 cytokines [i.e. interleukin-1 (IL-1) alpha and IL-1 beta] in controlling continuous glucose monitoring (CGM). METHODS: To investigate the role of IL-1Ra in long-term CGM in vivo, we compared CGM in transgenic mice that overexpress IL-1Ra [interleukin-1 receptor antagonist overexpresser (IL-1Ra~OE), B6.Cg-Tg(IL1rn)1Dih/J] or are deficient in IL-1Ra [interleukin-1 receptor antagonist knockout (IL-1Ra~KO), B6.129S-IL1rn(tm1Dih)/J] with mice that have normal levels of IL-1Ra (C57BL/6) over a 28-day time period. RESULTS: Mean absolute relative difference (MARD) analysis of CGM results among the mice of varying IL-1Ra levels demonstrated that during the first 21 days, IL-1~KO mice had the greatest tissue inflammation and the poorest sensor performance (i.e., higher MARD values) when compared with normal or IL-1Ra~OE mice. By 28 days post-sensor implantation, the inflammatory reactions had subsided and were replaced by varying degrees of fibrosis. CONCLUSIONS: These data support our hypothesis on the importance of the IL-1 family of agonists and antagonists in controlling tissue reactions and sensor function in vivo. These data also suggest that local delivery of IL-1Ra genes or recombinant proteins (anakinra) or other IL-1 antagonists such as antibodies or soluble IL-1 receptors would suppress sensor-induced tissue reactions and likely enhance glucose sensor function by inhibiting inflammation and wound healing at sensor implantation sites.

Analysis: on the path to overcoming glucose-sensor-induced foreign body reactions.

It is generally accepted that unreliable in vivo performance of implantable glucose sensors originates, in large part, from tissue reactions to the implanted sensor, including foreign body reactions (i.e., inflammation, fibrosis, and vessel regression). Development of glucose sensor coatings with increased biocompatibility would contribute to the development of a reliable long-term glucose sensor. In this issue of Journal of Diabetes Science and Technology, Van den Bosch and coauthors report on their initial in vitro results on a candidate biocompatibility coating for sensors (silica nanoparticle- polyethylene-glycol-based coating). Although the initial standard testing is encouraging, it is important that sensor-specific testing protocol be utilized to more accurately predict sensor performance in vivo. The development and application of sensor-specific testing standards will likely speed the development of biocompatible coatings that will increase sensor accuracy and lifespan in the future.

Metabolic biofouling of glucose sensors in vivo: role of tissue microhemorrhages.

OBJECTIVE: Based on our in vitro study that demonstrated the adverse effects of blood clots on glucose sensor function, we hypothesized that in vivo local tissue hemorrhages, induced as a consequence of sensor implantation or sensor movement post-implantation, are responsible for unreliable readings or an unexplained loss of functionality shortly after implantation. RESEARCH DESIGN AND METHODS: To investigate this issue, we utilized real-time continuous monitoring of blood glucose levels in a mouse model. Direct injection of blood at the tissue site of sensor implantation was utilized to mimic sensor-induced local tissue hemorrhages. RESULTS: It was found that blood injections, proximal to the sensor, consistently caused lowered sensor glucose readings, designated temporary signal reduction, in vivo in our mouse model, while injections of plasma or saline did not have this effect. CONCLUSION: These results support our hypothesis that tissue hemorrhage and resulting blood clots near the sensor can result in lowered local blood glucose concentrations due to metabolism of glucose by the clot. The lowered local blood glucose concentration led to low glucose readings from the still functioning sensor that did not reflect the systemic glucose level.

Importance of interleukin-1 and interleukin-1 receptor antagonist in short-term glucose sensor function in vivo.

BACKGROUND: The importance of the interleukin (IL)-1 cytokine family in inflammation and immunity is well established as a result of extensive in vitro and in vivo studies. In fact, much of our understanding of the in vivo importance of interleukin-1beta (IL-1B) is the result of research utilizing transgenic mice, such as overexpression or deficiencies of the naturally occurring inhibitor of IL-1 known as interleukin-1 receptor antagonist (IL-1RA). For the present studies, we utilized these transgenic mice to determine the role of IL-1B in glucose sensor function in vivo. METHODS: To investigate the role of IL-1B in glucose sensor function in vivo, we compared glucose sensor function in trans-genic mice that (1) overexpressed IL-1RA [B6.Cg-Tg(II1rn)1Dih/J] and (2) are deficient in IL-1RA (B6.129S-Il1rn(tm1Dih)/J), with mice that have normal levels of IL-1RA (C57BL/6). RESULTS: Our studies demonstrated that, during the first 7 days post-sensor implantation (PSI), mice deficient in IL-1RA had extensive inflammation and decreased sensor function when compared to normal or IL-1RA-overexpressing mice. CONCLUSION: These data directly support our hypothesis that the IL-1 family of cytokines and antagonists play a critical role in controlling tissue reactions and thereby sensor function in vivo during the first 7 days PSI.

Critical role of tissue mast cells in controlling long-term glucose sensor function in vivo.

Little is known about the specific cells, mediators and mechanisms involved in the loss of glucose sensor function (GSF) in vivo. Since mast cells (MC) are known to be key effector cells in inflammation and wound healing, we hypothesized that MC and their products are major contributors to the skin inflammation and wound healing that controls GSF at sites of sensor implantation. To test this hypothesis we utilized a murine model of continuous glucose monitoring (CGM) in vivo in both normal C57BL/6 mice (mast cell sufficient), as well as mast cell deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (Sash) mice over a 28 day CGM period. As expected, both strains of mice displayed excellent CGM for the first 7 days post sensor implantation (PSI). CGM in the mast cell sufficient C57BL/6 mice was erratic over the remaining 21 days PSI. CGM in the mast cell deficient Sash mice displayed excellent sensor function for the entire 28 day of CGM. Histopathologic evaluation of implantation sites demonstrated that tissue reactions in Sash mice were dramatically less compared to the reactions in normal C57BL/6 mice. Additionally, mast cells were also seen to be consistently associated with the margins of sensor tissue reactions in normal C57BL/6 mice. Finally, direct injection of bone marrow derived mast cells at sites of sensor implantation induced an acute and dramatic loss of sensor function in both C57BL/6 and Sash mice. These results demonstrate the key role of mast cells in controlling glucose sensor function in vivo.