Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Use of a type 1 genetic risk score for classification of diabetes type in young Australian adults: the Fremantle Diabetes Study Phase II.

The applicability of a UK-validated genetic risk score (GRS) was assessed in 158 participants in the Fremantle Diabetes Study Phase II diagnosed between 20 and <40 years of age with type 1 or type 2 diabetes or latent autoimmune diabetes of adults (LADA). For type 1 versus type 2/LADA, the area under the receiver operating characteristic curve (AUC) was highest for serum C-peptide (0.93) and lowest for the GRS (0.66). Adding age at diagnosis and body mass index to C-peptide increased the AUC minimally (0.96). The GRS appears of modest diabetes diagnostic value in young Australians.

Serum vitamin B12, distal symmetrical polyneuropathy and anaemia in type 2 diabetes: the Fremantle Diabetes Study Phase 2.

BACKGROUND: There are limited data relating to the effects of metformin-associated vitamin B12 deficiency on the risk of distal symmetrical polyneuropathy (DSPN) and megaloblastic anaemia in well-characterised community-based cohorts. AIMS: To assess inter-relationships between metformin therapy, vitamin B12 deficiency assessed using serum active B12 concentrations, and DSPN and anaemia in 1492 Fremantle Diabetes Study Phase 2 (FDS2) participants with type 2 diabetes. METHODS: Prevalence rates of vitamin B12 deficiency (total <80 pmol/L, active <23 pmol/L) and borderline deficiency (total ≥80 and ≤200 pmol/L, active ≥23 and ≤35 pmol/L) were determined using baseline sera. The relationship between vitamin B12 status and both DSPN and anaemia was assessed using multivariable analyses. RESULTS: Most FDS2 participants (94.4%) were vitamin B12 replete (total serum concentration >200 pmol/L, active >35 pmol/L), 2.0% were deficient (total <80 pmol/L, active <23 pmol/L) and the remainder (3.6%) borderline. Although metformin treatment increased the odds of deficiency (4.2%, 3.1% borderline) in a dose-dependent fashion (odds ratio (95% confidence interval) 39.4 (4.90-316) for >2000 mg daily compared with no treatment; P < 0.001), there was no significant association between vitamin B12 status and DSPN, anaemia (haemoglobin ≤130 g/L males, ≤120 g/L females), haemoglobin concentration or mean corpuscular volume (P ≥ 0.147). Metformin increased the likelihood of anaemia, especially at high doses, independent of vitamin B12 deficiency. CONCLUSIONS: Since nutritional sources likely attenuate metformin-associated vitamin B12 malabsorption and its clinical sequelae in developed countries such as Australia, there is no need for routine/opportunistic serum vitamin B12 screening in metformin-treated patients.

Assessment of venous pressure by compression sonography of the internal jugular vein during 3 days of bed rest.

Compression sonography has been proposed as a method for non-invasive measurement of venous pressures during spaceflight, but initial reports of venous pressure measured by compression ultrasound conflict with prior reports of invasively measured central venous pressure (CVP). The aim of this study is to determine the agreement of compression sonography of the internal jugular vein (IJVP) with invasive measures of CVP over a range of pressures relevant to microgravity exposure. Ten healthy volunteers (18-55 years, five female) completed two 3-day sessions of supine bed rest to simulate microgravity. IJVP and CVP were measured in the seated position, and in the supine position throughout 3 days of bed rest. The range of CVP recorded was in line with previous reports of CVP during changes in posture on Earth and in microgravity. The correlation between IJVP and CVP was poor when measured during spontaneous breathing (r = 0.29; R2  = 0.09; P = 0.0002; standard error of the estimate (SEE) = 3.0 mmHg) or end-expiration CVP (CVPEE ; r = 0.19; R2  = 0.04; P = 0.121; SEE = 3.0 mmHg). There was a modest correlation between the change in CVP and the change in IJVP for both spontaneous ΔCVP (r = 0.49; R2  = 0.24; P < 0.0001) and ΔCVPEE (r = 0.58; R2  = 0.34; P < 0.0001). Bland-Altman analysis of IJVP revealed a large positive bias compared to spontaneous breathing CVP (3.6 mmHg; SD = 4.0; CV = 85%; P < 0.0001) and CVPEE (3.6 mmHg; SD = 4.2; CV = 84%; P < 0.0001). Assessment of absolute IJVP via compression sonography correlated poorly with direct measurements of CVP by invasive catheterization over a range of venous pressures that are physiologically relevant to spaceflight. However, compression sonography showed modest utility for tracking changes in venous pressure over time. NEW FINDINGS: What is the central question of this study? Compression sonography has been proposed as a novel method for non-invasive measurement of venous pressures during spaceflight. However, the accuracy has not yet been confirmed in the range of CVP experienced by astronauts during spaceflight. What is the main finding and its importance? Our data show that compression sonography of the internal jugular vein correlates poorly with direct measurement of central venous pressures in a range that is physiologically relevant to spaceflight. However, compression sonography showed modest utility for tracking changes in venous pressure over time.

Canagliflozin Attenuates PromarkerD Diabetic Kidney Disease Risk Prediction Scores.

PromarkerD is a biomarker-based blood test that predicts kidney function decline in people with type 2 diabetes (T2D) who may otherwise be missed by current standard of care tests. This study examined the association between canagliflozin and change in PromarkerD score (Δ score) over a three-year period in T2D participants in the CANagliflozin cardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline and Year 3 in 2008 participants with preserved kidney function (baseline eGFR ≥60 mL/min/1.73 m2). Generalized estimating equations were used to assess the effect of canagliflozin versus placebo on PromarkerD scores. At baseline, the participants (mean age 62 years, 32% females) had a median PromarkerD score of 3.9%, with 67% of participants categorized as low risk, 14% as moderate risk, and 19% as high risk for kidney function decline. After accounting for the known acute drop in eGFR following canagliflozin initiation, there was a significant treatment-by-time interaction (p < 0.001), whereby participants on canagliflozin had decreased mean PromarkerD scores from baseline to Year 3 (Δ score: -1.0% [95% CI: -1.9%, -0.1%]; p = 0.039), while the scores of those on placebo increased over the three-year period (Δ score: 6.4% [4.9%, 7.8%]; p < 0.001). When stratified into PromarkerD risk categories, participants with high risk scores at baseline who were randomized to canagliflozin had significantly lower scores at Year 3 (Δ score: -5.6% [-8.6%, -2.5%]; p < 0.001), while those on placebo retained high scores (Δ score: 4.5% [0.3%, 8.8%]; p = 0.035). This post hoc analysis of data from CANVAS showed that canagliflozin significantly lowered PromarkerD risk scores, with the effect greatest in those T2D participants who were classified at study entry as at high risk of a subsequent decline in kidney function.

3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation.

Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell-cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures.

Volumetric mass density measurements of mesenchymal stem cells in suspension using a density meter.

To use regeneratively active cells for cell therapeutic applications, the cells must be isolated from their resident tissues. Different isolation procedures subject these cells to varying degrees of mechanical strain, which can affect the yield of cell number and viability. Knowledge of cell volumetric mass density is important for experimental and numerical optimization of these procedures. Although methods for measuring cell volumetric mass density already exist, they either consume much time and cell material or require a special setup. Therefore, we developed a user-friendly method that is based on the use of readily available instrumentation. The newly developed method is predicated on the linear relationship between the volumetric mass density of the cell suspension and the volumetric mass density, number, and diameter of the cells in the suspension. We used this method to determine the volumetric mass density of mesenchymal stem cells (MSCs) and compared it to results from the established density centrifugation.

Oxidative Stress Response in Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

Reactive oxygen species (ROS) can irreversibly damage biological molecules, a process known as oxidative stress. Elevated ROS levels are associated with immune cell activation. Sustained immune system activation can affect many different cells in the environment. One cell type that has been detected in almost all tissues of the body is mesenchymal stem/stromal cells (MSC). MSC possess proliferation and differentiation potential, thus facilitating regeneration processes. However, the regenerative capacity of MSC might be impaired by oxidative stress, and the effects of long-term oxidative stress on MSC functions are sparsely described. The examination of oxidative stress is often performed by exposure to H2O2. Since H2O2 is rapidly degraded, we additionally exposed the cell cultures to glucose oxidase (GOx), resulting in sustained exposure to H2O2. Using these model systems, we have focused on the effects of short- and long-term oxidative stress on viability, migration, differentiation, and signaling. All cellular functions examined were affected by the applied oxidative stress. The differences that occur between pulsed and sustained oxidative stress indicated higher oxidative stress in MSC upon direct H2O2 exposure, whereas the GOx-induced prolonged exposure to H2O2 seems to allow for better cellular adaptation. The mechanisms underlying these different responses are currently unknown.

The relationship between thyroid dysfunction, cardiovascular morbidity and mortality in type 2 diabetes: The Fremantle Diabetes Study Phase II.

AIMS: It is uncertain whether subclinical thyroid dysfunction is associated with cardiovascular disease (CVD) events and mortality in people with type 2 diabetes. The aim of this study was to determine whether undetected thyroid disease increases the risk of incident CVD and death in type 2 diabetes. METHODS: One thousand two hundred fifty participants with type 2 diabetes (mean age 65.3 years, 56.5% males, median diabetes duration 8.0 years) without known thyroid disease and not taking medications known to affect thyroid function were categorised, based on baseline serum free thyroxine (FT4) and thyrotropin (TSH) concentrations, as euthyroid, overt hypothyroid (increased TSH, low FT4), subclinical hypothyroid (increased TSH, normal FT4), overt thyrotoxic (decreased TSH, raised FT4) or subclinical thyrotoxic (decreased TSH, normal FT4). Incident myocardial infarction, incident stroke, all-cause and cardiovascular mortality were ascertained during a mean 6.2-6.7 years of follow-up. RESULTS: Most participants with newly-detected thyroid dysfunction had subclinical hypothyroidism (77.2%) while overt/subclinical thyrotoxicosis was infrequent. Compared to participants with TSH 0.34-2.9 mU/L, those with TSH > 5.1 mU/L were not at increased risk of incident myocardial infarction (adjusted hazard ratio (95% confidence limits) 1.77 (0.71, 2.87)), incident stroke (1.66 (0.58, 4.78)), all-cause mortality (0.78 (0.44, 1.37)) or cardiovascular mortality (1.16 (0.38, 3.58)). Independent baseline associates of subclinical hypothyroidism included estimated glomerular filtration rate and systolic blood pressure. CONCLUSIONS: Subclinical hypothyroidism was not independently associated with CVD events or mortality in community-dwelling people with type 2 diabetes despite its associations with CVD risk factors, questioning strategies to identify and/or treat mild thyroid dysfunction outside usual care.

Evaluation of the clinical utility of the PromarkerD in-vitro test in predicting diabetic kidney disease and rapid renal decline through a conjoint analysis.

BACKGROUND: Early identification of patients at risk of developing diabetic kidney disease or rapid renal decline is imperative for appropriate patient management, but traditional methods of predicting renal decline are limited. OBJECTIVE: This study evaluated the impact of PromarkerD, a biomarker-based blood test predicting the risk of diabetic kidney disease (DKD) and rapid renal decline. METHODS: Conjoint analysis clarified the importance of PromarkerD and other patient attributes to physician decisions for type 2 diabetes patients. Forty-two patient profiles were generated, with varying levels of albuminuria, estimated glomerular filtration rate (eGFR), blood pressure, hemoglobin A1c (HbA1c), age, and PromarkerD result. A web-based survey asked each physician to make monitoring/treatment decisions about eight randomly selected profiles. Data were analyzed using multivariable logit models. RESULTS: Two hundred three primary care physicians and 197 endocrinologists completed the survey. PromarkerD result was most important for increasing the frequency of risk factor monitoring. PromarkerD was second to HbA1c in importance for deciding to prescribe sodium/glucose cotransporter-2 inhibitors (SGLT2s) with a DKD indication, second to blood pressure for increasing the dose of lisinopril, and second to eGFR for replacing ibuprofen with a non-nephrotoxic medication. Compared with no PromarkerD results, a high-risk PromarkerD result was associated with significantly higher odds of increasing monitoring frequency (odds ratio [OR]: 2.56, 95% confidence interval: 1.90-3.45), prescribing SGLT2s (OR: 1.98 [1.56-2.52]), increasing lisinopril dose (OR: 1.48 [1.17-1.87]), and replacing ibuprofen (OR: 1.78 [1.32-2.40]). A low-risk PromarkerD result was associated with significantly lower odds of increasing monitoring frequency (OR: 0.48 [0.37-0.64]), prescribing SGLT2s (OR: 0.70 [0.56-0.88]), and replacing ibuprofen (OR: 0.75 [0.57-0.99]). CONCLUSION: PromarkerD could increase adoption of renoprotective interventions in patients at high risk for renal decline and lower the likelihood of aggressive treatment in those at low risk. Further studies are needed to assess patient outcomes with PromarkerD in real-world practice.

A Comparative Study on the Adipogenic Differentiation of Mesenchymal Stem/Stromal Cells in 2D and 3D Culture.

Mesenchymal stem/stromal cells (MSC) are capable of renewing the progenitor cell fraction or differentiating in a tissue-specific manner. Adipogenic differentiation of adipose-tissue-derived MSC (adMSC) is important in various pathological processes. Adipocytes and their progenitors are metabolically active and secrete molecules (adipokines) that have both pro- and anti-inflammatory properties. Cell culturing in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation for most cell types. Therefore, 3D culture systems have been developed to create an environment considered more physiological. Since knowledge about the effects of 3D cultivation on adipogenic differentiation is limited, we investigated its effects on adipogenic differentiation and adipokine release of adMSC (up to 28 days) and compared these with the effects in 2D. We demonstrated that cultivation conditions are crucial for cell behavior: in both 2D and 3D culture, adipogenic differentiation occurred only after specific stimulation. While the size and structure of adipogenically stimulated 3D spheroids remained stable during the experiment, the unstimulated spheroids showed signs of disintegration. Adipokine release was dependent on culture dimensionality; we found upregulated adiponectin and downregulated pro-inflammatory factors. Our findings are relevant for cell therapeutic applications of adMSC in complex, three-dimensionally arranged tissues.

Novel integrated workflow allows production and in-depth quality assessment of multifactorial reprogrammed skeletal muscle cells from human stem cells.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Effect of Nightly Lower Body Negative Pressure on Choroid Engorgement in a Model of Spaceflight-Associated Neuro-ocular Syndrome: A Randomized Crossover Trial.

IMPORTANCE: Astronauts returning from long-duration spaceflight experience ocular remodeling related to cephalad fluid shifts induced by microgravity. It is hypothesized that the absence of diurnal reductions in intracranial pressure in microgravity creates a low but persistent pressure gradient at the posterior aspect of the eye, which results in ocular remodeling and space-associated neuro-ocular syndrome (SANS) over many months. OBJECTIVE: To determine whether partial reintroduction of footward fluid shifts during simulated microgravity via lower body negative pressure (LBNP) during sleep attenuates choroid engorgement, an early marker of ocular remodeling related to SANS. DESIGN, SETTING, AND PARTICIPANTS: Between May 2019 and February 2020, participants with no major cardiovascular, kidney, or ophthalmic disease completed 3 days of supine (0°) bed rest with and 3 days without 8 hours of nightly LBNP in a randomized, crossover design. This single-center investigation took place at the UT Southwestern Medical Center. All analyses were conducted blinded to condition and time point. INTERVENTIONS: Eight hours of nightly LBNP (-20 mm Hg) vs no LBNP. MAIN OUTCOMES AND MEASURES: The primary outcome was the change in choroid area and volume after 3 days of bed rest measured by optical coherence tomography. RESULTS: Of 10 participants, 5 were female, the mean (SD) age was 29 (9) years, and the age range was 18 to 55 years. Central venous pressure increased from the seated to supine position (mean [SD], seated: -2.3 [2.0] vs supine: 6.9 [2.0] mm Hg; P < .001), leading to choroid engorgement over 3 days of bed rest (Δ area: +0.09 mm2 [95% CI, 0.04-0.13]; P = .001; Δ volume: +0.37 mm3 [95% CI, 0.19-0.55]; P = .001). Nightly LBNP caused a sustained reduction in supine central venous pressure (mean [SD], 5.7 [2.2] mm Hg to 1.2 [1.4 mm Hg]; P < .001) and attenuated the increase in choroid area (74%) (Δ: 0.02 mm2 [95% -0.02 to 0.06]; P = .01) and volume (53%) (Δ: 0.17 mm3 [95% CI, 0.01-0.34]; P = .05) compared with control. CONCLUSIONS AND RELEVANCE: Nightly LBNP reinstated a footward fluid shift and mitigated the increase in choroid area and volume. LBNP during sleep may be an effective countermeasure for ocular remodeling and SANS during long-duration space missions.

Assessment of biomarkers associated with rapid renal decline in the detection of retinopathy and its progression in type 2 diabetes: The Fremantle Diabetes Study Phase II.

AIMS: To determine whether biomarkers for diabetic kidney disease (DKD) can be used to determine the prevalence, progression and/or incidence of diabetic retinopathy (DR) complicating type 2 diabetes. METHODS: Proteomic biomarkers were measured in baseline fasting plasma from 958 Fremantle Diabetes Study Phase II participants whose baseline and, in those returning for follow-up (n = 764), Year 4 fundus photographs were graded for DR presence/severity. The performance of PromarkerD (three biomarkers and readily available clinical variables which identify prevalent DKD and predict incident DKD and estimated glomerular filtration rate decline ≥30% over four years) for detecting DR prevalence, progression and incidence was assessed using the area under the receiver operating curve (AUC). Logistic regression determined whether individual proteins were associated with DR outcomes after adjusting for the most parsimonious model. RESULTS: Plasma apolipoprotein A-IV (APOA4) was independently associated with moderate non-proliferative DR at baseline (OR (95% CI): 1.64 (1.01, 2.67), P = 0.047). Model discrimination was poor for all PromarkerD predicted probabilities against all DR outcomes (AUC ≤0.681). CONCLUSIONS: PromarkerD and its constituent biomarkers were not consistently associated with DR prevalence or temporal change. APOA4 was associated with prevalent DR, but not DR incidence or progression. Distinct pathophysiological mechanisms may underlie DKD and DR.

The New and the Old: Platform Cross-Validation of Immunoaffinity MASS Spectrometry versus ELISA for PromarkerD, a Predictive Test for Diabetic Kidney Disease.

PromarkerD is a proteomics derived test for predicting diabetic kidney disease that measures the concentrations of three plasma protein biomarkers, APOA4, CD5L and IBP3. Antibodies against these proteins were developed and applied to a multiplexed immunoaffinity capture mass spectrometry assay. In parallel, and facilitating current clinical laboratory workflows, a standard ELISA was also developed to measure each protein. The performance characteristics of the two technology platforms were compared using a cohort of 100 samples, with PromarkerD test scores demonstrating a high correlation (R = 0.97). These technologies illustrate the potential for large scale, high throughput clinical applications of proteomics now and into the future.

A robust multiplex immunoaffinity mass spectrometry assay (PromarkerD) for clinical prediction of diabetic kidney disease.

BACKGROUND: PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application. METHODS: A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories. RESULTS: Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer. CONCLUSIONS: An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients.

Changes in the Epidemiology of Hepatobiliary Disease Complicating Type 2 Diabetes over 25 Years: The Fremantle Diabetes Study.

OBJECTIVE: To determine whether the incidence/outcome of hepatobiliary disease (HBD) has increased over recent decades in community-based Australians with and without type 2 diabetes (T2D). METHODS: Longitudinal data from the Fremantle Diabetes Study Phase I (FDS1; recruitment 1993-1996; n = 1291 with T2D) and Phase II (FDS2; 2008-2011; n = 1509) were analyzed. Participants with T2D from both Phases were age-, sex-, and postcode-matched 1:4 to people without diabetes. Incident HBD and associated mortality were ascertained from hospitalization, cancer registration, and/or death certification codes. Incidence rates (IRs) and IR ratios (IRRs) for those with versus without diabetes in FDS1 and FDS2 were calculated. RESULTS: HBD IRs for people without diabetes did not change between Phases. The IRR (95% CI) for people with T2D in FDS2 versus FDS1 was 1.30 (1.01-1.68) with the highest IRRs in participants aged <65 years. Non-alcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) events were 54% greater in FDS2 than FDS1 in the presence of greater abdominal adiposity. NAFLD/NASH was coded in one in 11 HBD events in FDS2 and in 10% of HBD deaths (<4% of total mortality). CONCLUSIONS: HBD is more frequent in people with versus without T2D and this discrepancy is increasing. Hospitalizations/deaths due to NAFLD/NASH remain uncommon.

PromarkerD Predicts Renal Function Decline in Type 2 Diabetes in the Canagliflozin Cardiovascular Assessment Study (CANVAS).

The ability of current tests to predict chronic kidney disease (CKD) complicating diabetes is limited. This study investigated the prognostic utility of a novel blood test, PromarkerD, for predicting future renal function decline in individuals with type 2 diabetes from the CANagliflozin CardioVascular Assessment Study (CANVAS). PromarkerD scores were measured at baseline in 3568 CANVAS participants (n = 1195 placebo arm, n = 2373 canagliflozin arm) and used to predict incident CKD (estimated glomerular filtration rate (eGFR) <60 mL/min/1.73m2 during follow-up in those above this threshold at baseline) and eGFR decline ≥30% during the 4 years from randomization. Biomarker concentrations (apolipoprotein A-IV (apoA4), CD5 antigen-like (CD5L/AIM) and insulin-like growth factor-binding protein 3 (IGFBP3) measured by mass spectrometry were combined with clinical data (age, serum high-density lipoprotein (HDL)-cholesterol, eGFR) using a previously defined algorithm to provide PromarkerD scores categorized as low-, moderate- or high-risk. The participants (mean age 63 years, 33% females) had a median PromarkerD score of 2.9%, with 70.5% categorized as low-risk, 13.6% as moderate-risk and 15.9% as high-risk for developing incident CKD. After adjusting for treatment, baseline PromarkerD moderate-risk and high-risk scores were increasingly prognostic for incident CKD (odds ratio 5.29 and 13.52 versus low-risk, respectively; both p < 0.001). Analysis of the PromarkerD test system in CANVAS shows the test can predict clinically significant incident CKD in this multi-center clinical study but had limited utility for predicting eGFR decline ≥30%.