Distinct gene expression pathways in islets from individuals with short- and long-duration type 1 diabetes.

AIMS: Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non-obese diabetic (NOD) mouse model. In the present study, we chose a human-focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. MATERIALS AND METHODS: We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high-depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non-diabetic donors. RESULTS: The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up-regulated in short-duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL-23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL-23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. CONCLUSION: While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications.

Adiponectin receptor fragmentation in mouse models of type 1 and type 2 diabetes.

The protein hormone adiponectin regulates glucose and fatty acid metabolism by binding to two PAQR-family receptors (AdipoR1 and AdipoR2). Both receptors feature a C-terminal segment which is released by proteolysis to form a freely circulating C-terminal fragment (CTF) found in the plasma of normal individuals but not in some undefined diabetes patients. The AdipoR1-CTF344-376 is a competitive inhibitor of tumor necrosis factor α cleavage enzyme (TACE) but it contains a shorter peptide domain (AdipoR1 CTF351-362) that is a strong non-competitive inhibitor of insulin-degrading enzyme (IDE). The link between adiponectin receptor fragmentation and diabetes pathology is unclear but could lead to new therapeutic strategies. We therefore investigated physiological variations in the concentrations of CTF in non-obese diabetic (NOD/ShiLtJ) mice and C57BL/6 mice with diet-induced obesity (DIO) as models of diabetes types 1 and 2, respectively. We tested for changes in adiponectin receptor signaling, immune responses, disease progression, and the abundance of neutralizing autoantibodies. Finally, we administered exogenous AdipoR1-CTF peptides either containing or lacking the IDE-binding domain. We observed the more pronounced CTF shedding in the TACE-active NOD mice, which represents an inflammatory autoimmune phenotype, but fragmentation was also observed to a lesser extent in the DIO model. Autoantibodies to CTF were detected in both models. Neither exogenous CTF peptide affected IgG-CTF plasma levels, body weight or the conversion of NOD mice to diabetes. The pattern of AdipoR1 fragmentation and autoantibody production under physiological conditions of aging, DIO, and autoimmune diabetes therefore provides insight into the association adiponectin biology and diabetes.

Utilization of electronic health records for the assessment of adiponectin receptor autoantibodies during the progression of cardio-metabolic comorbidities.

BACKGROUND: Diabetes is a complex, multi-symptomatic disease whose complications drives increases in healthcare costs as the diabetes prevalence grows rapidly world-wide. Real-world electronic health records (EHRs) coupled with patient biospecimens, biological understanding, and technologies can characterize emerging diagnostic autoimmune markers resulting from proteomic discoveries. METHODS: Circulating autoantibodies for C-terminal fragments of adiponectin receptor 1 (IgG-CTF) were measured by immunoassay to establish the reference range using midpoint samples from 1862 participants in a 20-year observational study of type 2 diabetes and cardiovascular arterial disease (CVAD) conducted by the Fairbanks Institute. The White Blood Cell elastase activity in these patients was assessed using immunoassays for Bikunin and Uristatin. Participants were assigned to four cohorts (healthy, T2D, CV, CV+T2D) based on analysis of their EHRs and the diagnostic biomarkers values and patient status were assessed ten-years post-sample. RESULTS: The IgG-CTF reference range was determined to be 75-821 ng/mL and IgG-CTF out-of-range values did not predict cohort or comorbidity as determined from the EHRs at 10 years after sample collection nor did IgG-CTF demonstrate a significant risk for comorbidity or death. Many patients at sample collection time had other conditions (hypertension, hyperlipidemia, or other risk factors) of which only hypertension, Uristatin and Bikunin values correlated with increased risk of developing additional comorbidities (odds ratio 2.58-13.11, P<0.05). CONCLUSIONS: This study confirms that retrospective analysis of biorepositories coupled with EHRs can establish reference ranges for novel autoimmune diagnostic markers and provide insights into prediction of specific health outcomes and correlations to other markers.

Analysis of ligand binding by bioaffinity mass spectrometry.

BACKGROUND: Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. METHODS: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. RESULTS: A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. CONCLUSIONS: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.

Clinical utility of a rapid test for uristatin.

OBJECTIVES: Uristatin is a trypsin inhibitor present in urine that is increased in most patients with bacterial or viral infections and in many with inflammatory disorders. We included the assay of uristatin as part of a screening program carried out by pediatricians on 4207 Japanese schoolchildren to judge the ability of uristatin to identify those with an infection and (or) inflammation of any cause. We used urine dipsticks for the assay of uristatin, creatinine, albumin, blood, leukocyte esterase, and protein. We also performed quantitative assays for uristatin and creatinine. Another aim was to estimate the reference range for uristatin in schoolchildren, ages 5 to 14 yr. METHODS: We prepared dipstick pads that were impregnated with a chromogenic substrate for trypsin and measured the uristatin-caused inhibition of trypsin in urine. We measured creatinine so that the ratio of uristatin to creatinine could be calculated to correct for urine concentration. RESULTS: We obtained quantitative uristatin and creatinine results for 4207 children. Of these, 177 had an abnormal urine dipstick for albumin or blood or protein or leukocyte esterase or a combination of these. We used data from 3622 children to establish the reference range for the uristatin dipsticks. The 3622 were diagnosed by their pediatricians as free from an infection or inflammation of any cause and with normal urine dipstick tests. We recommend an upper reference limit for uristatin by dipstick of < or = 7.5 mg uristatin/g creatinine. The leftover 408 children ( [4207-3622-177] = 408) fell into two groups: 205 with diagnoses of no infection, possible infection, or possible inflammatory disorders. The remaining 203 children were renal disease follow-up cases. The diagnoses were based on a physical examination, microscopic urinalysis plus urine dipstick tests for albumin, blood, creatinine, protein, leukocyte esterase and a complete blood count. In the 205 children, 46 had an abnormal uristatin dipstick test, 39 had an abnormal uristatin by immunoassay, 41 had an abnormal erythrocyte sedimentation rate (ESR), 27 had an abnormal serum C-reactive protein (CRP), and one had an abnormal urine microscopic exam. For the first 938 children in the study, the agreement was 93% of negative dipstick uristatin results and immunoassays. The agreement of positive uristatin dipsticks with immunoassays was 85%. We assumed that the immunoassay results were correct. In the evaluation of 189 children with fever, 62 also had an abnormal uristatin by dipstick. DISCUSSION: A rapid dipstick test for uristatin read on a reflectance photometer gave values that compared well with a quantitative immunoassay method. The uristatin test is sensitive but not specific for any cause of infection or inflammation. Uristatin is easy to determine and appears to be a better indicator than fever, ESR, or CRP for the diagnosis of an infection or inflammation.

The uristatin dipstick is useful in distinguishing upper respiratory from urinary tract infections.

BACKGROUND: We determined the diagnostic value of the trypsin inhibitor, uristatin, that is commonly found in urine and plasma in patients with infections or inflammations of any kind. METHODS: We collected urine specimens from patients with infections of the urinary or upper respiratory tract and from healthy controls. We also collected blood from patients with a likely upper respiratory tract infection and healthy controls. A bacterial count of >10(5) organisms/ml in urine was considered to represent infection rather than contamination. RESULTS: The uristatin dipstick test in urine showed acceptable negative predictive values (NPV of up to 93%) for patients without infection or inflammation. Here, the dipsticks could eliminate some urine cultures. For those with infection or inflammation, the positive predictive values (PPV) of the dipsticks were lower (up to 57%). Including the leukocyte esterase and nitrite values increased the PPV of the dipsticks for those with disease. CONCLUSIONS: The uristatin strip was more accurate than the leukocyte and nitrite dipsticks for predicting upper respiratory infections (URI) and C-reactive protein for those with infection or inflammation. The uristatin dipstick was able to detect both the bikunin and uristatin inhibitors.

Bikunin (urinary trypsin inhibitor): structure, biological relevance, and measurement.

Inflammatory processes, such as phagocytosis, coagulation, and vascular dilation, promote the release of serine proteases by neutrophils, macrophages, mast cells, lymphocytes, and the epithelial or endothelial cells. These proteases further facilitate the release of inflammatory cytokines and growth factors as well as take part in signal-cell proliferation through protease-activated receptors (PARs). Controlling the action of this cascade is necessary to prevent further damage to the normal tissues. One of the main anti-inflammatory response mediators is bikunin (Bik) that is responsible for inhibiting the activity of many serine proteases such as trypsin, thrombin, chymotrypsin, kallikrein, plasmin, elastase, cathepsin, Factors IXa, Xa, XIa, and XlIa. During the acute-phase response, Bik is released into plasma from proinhibitors primarily due to increased elastase activity. Bik is a glycoprotein, also referred to as urinary trypsin inhibitor, which in plasma inhibits the trypsin family of serine proteases by binding to either of the two Kunitz-binding domains. Bik also accumulates in urine. In conditions such as infection, cancer, tissue injury during surgery, kidney disease, vascular disease, coagulation, and diabetes, the concentrations of Bik in plasma and urine are increased. Several trypsin inhibitory assays for urine and immunoassays for both blood and urine have been described for measuring Bik. In addition to presenting the synthesis, structure, and pathophysiology of Bik, we will summarize various diagnostic approaches for measuring Bik. Analysis of Bik may provide a rapid approach in assessing various conditions involving the inflammatory processes.

Immunological evaluation of urinary trypsin inhibitors in blood and urine: role of N- & O-linked glycoproteins.

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-alpha-I, and I-alpha-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm-Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.

Au nanoparticle conjugation for impedance and capacitance signal amplification in biosensors.

Amplification of the electrochemical impedance and capacitance signals in a biosensor is demonstrated for the model fluorescein/anti-fluorescein system. Following immobilization of fluorescein onto Au through formation of a self-assembled monolayer, goat anti-fluorescein conjugated with 10-nm Au nanoparticles is introduced into the system. This results in an increase in the capacitance of approximately 400 nF/cm(2), whereas no change can be observed for goat anti-fluorescein without the Au nanoparticle conjugate. An even greater sensitivity is obtained by introduction of a redox probe, [Fe(CN)6]3-/4-, whereby the charge-transfer resistance (R(ct)) is reduced to approximately 25% of its original value. This allows construction of high-sensitivity electrochemical impedance biosensors at a single low frequency, where the signal is sensitive to the interfacial R(ct). This change in the electrochemical impedance signal upon binding to goat anti-fluorescein conjugated with Au nanoparticles can be attributed to the much higher electrochemical activity of Au surfaces relative to the underlying organic layer.

Pathophysiology and diagnostic value of urinary trypsin inhibitors.

Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down.

Microfluidic tool box as technology platform for hand-held diagnostics.

BACKGROUND: Use of microfluidics in point-of-care testing (POCT) will require on-board fluidics, self-contained reagents, and multistep reactions, all at a low cost. Disposable microchips were studied as a potential POCT platform. METHODS: Micron-sized structures and capillaries were embedded in disposable plastics with mechanisms for fluidic control, metering, specimen application, separation, and mixing of nanoliter to microliter volumes. Designs allowed dry reagents to be on separate substrates and liquid reagents to be added. Control of surface energy to +/-5 dyne/cm2 and mechanical tolerances to < or = 1 microm were used to control flow propulsion into adsorptive, chromatographic, and capillary zones. Fluidic mechanisms were combined into working examples for urinalysis, blood glucose, and hemoglobin A(1c) testing using indicators (substances that react with analyte, such as dyes, enzyme substrates, and diazonium salts), catalytic reactions, and antibodies as recognition components. Optical signal generation characterized fluid flow and allowed detection. RESULTS: We produced chips that included capillary geometries from 10 to 200 microm with geometries for stopping and starting the flow of blood, urine, or buffer; vented chambers for metering and splitting 100 nL to 30 microL; specimen inlets for bubble-free specimen entry and containment; capillary manifolds for mixing; microstructure interfaces for homogeneous transfer into separation membranes; miniaturized containers for liquid storage and release; and moisture vapor barrier seals for easy use. Serum was separated from whole blood in <10 s. Miniaturization benefits were obtained at 10-200 microm. CONCLUSION: Disposable microchip technology is compatible with conventional dry-reagent technology and allows a highly compact system for complex assay sequences with minimum manual manipulations and simple operation.

Near-patient testing for infection using urinalysis and immuno-chromatography strips.

Urinary tract infections require costly confirmatory tests such as a urine culture to establish the diagnosis. Elimination of the culture step would save resources; diagnosis and treatment could begin in hours rather than days. We tested a new dip-and-read strip that uses immuno-chromatography (IC) to detect infectious agents in urine. We used a goat-derived polyclonal antibody with reactivity to the cell-wall proteins of Escherichia coli (E. coli). Fluorescein linked to the anti-E. coli antibody served to trap the bacteria on a strip coated with an anti-fluorescein mouse antibody. Blue latex particles were linked to anti-E. coli antibodies by standard methods and were used for detection of E. coli. We found that the combination of leukocyte esterase and nitrite dipsticks gave negative predictive values of 93% for culture-negative urines, i.e., there were very few false-negative results. Using the same dipsticks on culture-positive specimens, the positive predictive values were unacceptably low; we obtained too many false-positive values. By contrast, the IC strips gave negative predictive values of 89%. The major advantage of the IC strips is that the positive predictive values were higher, i.e., there were fewer false-positive results. The combined use of both IC strips and urinalysis dipsticks offers the best strategy for diagnosing infection with dipsticks. The IC strip test could reduce the necessity of a urine culture in patients with suspected infections and provide rapid point-of-care testing.

Sensitive noninvasive marker for the diagnosis of probable bacterial or viral infection.

Urinary trypsin inhibitor (uTi) is a product of elastase-mediated degradation of interleukin-alpha-inhibitor (I-alpha-I). Its activity increases in the urine of patients with a malignancy, inflammation, or infection, or in late pregnancy. The objective of this study was to compare the sensitivity of uTi in urine with that of serum quantitative C-reactive protein (CRP) for diagnosing infection, as indicated by white cell response and clinical assessment. Ninety controls and 171 patients with various systemic infections were enrolled. We measured uTi enzymatically on a Cobas Fara (Roche Diagnostics). Patients were separated into bacterial, probable bacterial, viral, or probable viral groups based on the results of a complete blood count with differential (CBC), urinalysis (UA), and clinical assessment. In the bacterial (n=70) and control (n=90) groups, the uTi values (mean+/-SE) were 25.3+/-3.1 mg/L and 2.8+/-0.8 mg/L, respectively. uTi (at 2.7 mg/L) had a diagnostic sensitivity of 91% and specificity of 82% (AUC=0.889), whereas CRP (at a cutoff of 10 mg/L) had a sensitivity and specificity of 82% and 96%, respectively (AUC=0.921). As a marker of infection (positive in both bacterial and viral groups), uTi had a sensitivity of 91% (AUC=0.884) vs. 89% (AUC=0.828) for CRP. Our data indicate that uTi has sufficient clinical sensitivity for screening systemic infections, and may have diagnostic value as a noninvasive test.