Mild traumatic brain injury (mTBI) and concussion from sporting/recreational activities are relatively common. However, assessment of mTBI is difficult and many incidents of mTBI and concussion are unrecognized and/or not reported. Levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) peptide, a product of the proteolytic degradation of AMPA receptors, have been found to be raised in mTBI and the development of point-of-care (POC) tests based on the recognition of the AMPAR peptide is underway. Such POC tests will be useful at the pitch sideline or in the combat field for aiding objective diagnosis and management of subtle brain injury.

by Prof. Svetlana A. Dambinova, Rozalyn Heath and Dr Galina A. Izykenova

Introduction
Mild traumatic brain injury (mTBI) including concussion is the most frequent form of injury in military and civilian settings with the highest prevalence among young adults aged 15 to 24 years. Each year sports and recreational activities contribute about 3.8 million cases of mTBI in the USA [1], whereas brain injuries caused by explosions are the most common combat wounds in the military arena.

Assessment of mTBI regardless of origin is complicated. Many primary mTBIs, and particularly concussions, go unrecognized or are not reported when there is no loss of consciousness. Additionally, without sufficient reports of previous incidents, soldiers and competitive athletes are often subjected to multiple concussions. There are several challenges to identify immediate (or primary acute and subacute within 24 hours and up to 2 weeks respectively) impact, secondary (beyond 14 days) and cumulative (brain-related seizures) consequences that might follow multiple concussions or mTBI.

Normally, acute subclinical concussions associated with micro-edema formation that is reversible and not visible on conventional computed tomography and magnetic resonance imaging (MRI) methods. Advanced neuroimaging techniques (diffusion tensor imaging, functional MRI, and positron emission tomography) that can register minor structural and microvascular changes are primarily used for research. These modalities are not available in emergency situations or for routine clinical evaluations and have a limited application in persons with metal implants [2] or claustrophobia [3].

Currently, there is an unmet diagnostic need to reveal brain micro-damage following concussion by use of a rapid and affordable assay detecting, for instance, neurotoxicity (immunoexcitotoxicity) biomarkers in the bloodstream [4]. Analogous to NR2 peptide as a biomarker for cortical lesions in transient ischemic attack (TIA)/strokes [5], we proposed that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) peptide marker is able to differentiate subtle brain injury in white matter associated with concussions.

Neurotoxicity biomarkers in mTBI
It is known that the family of ionotropic glutamate receptors (GluRs) implying N-methyl-D-aspartate (NMDAR), AMPAR and kainite receptors are involved in the regulation of synaptic connectivity in cortical/subcortical and brainstem areas [4,5]. Recently, it was shown that AMPAR represents a biomarker for the neurotoxicity cascade underlying subtle brain injury [6].

The family of location-specific GluRs is involved in more than 80% of cortical and subcortical neuronal communications underlying superior mental functions [7]. AMPAR is primarily distributed in the forebrain and subcortical pathways [8], and strategically located on surfaces of small cerebral arteries regulating blood circulations in white matter substructures [9]. Symptomatically the impact to brain may lead to executive brain dysfunctions associated with subcortical areas. Visual and cognitive deficits (including problems with memory, intellect, concentration and attention) might be an aftermath of subtle repetitive injuries to deep brain structures due to more severe cases of mTBI.

In acute and subacute phases of mTBI, a massive release of glutamate, which upregulates excitotoxic AMPARs has been detected [4]. The GluR1-subunit of N-terminal AMPAR fragments is rapidly cleaved by extracellular proteases and fragments carrying immune active epitopes released into the bloodstream through the compromised blood–brain barrier (BBB). This degradation product can be detected directly in the blood as AMPAR peptide fragments (molecular weight 5–7 kDa) [4]. The protective effects of a compromised BBB impacting neurotoxicity are exacerbated further when accompanied by a delayed immunological response generating peripheral anti-CNS antibodies [10].

Concussion assays development
To detect AMPAR peptide, magnetic-particle-based enzyme-linked immunosorbent assay (MP-ELISA) containing unique reagents has been developed. MP-ELISA involved a sandwich or ‘bridging’ assay where the suspended in solution microparticles coated by capture antibodies are binding to two epitopes of AMPAR peptide and reaction is revealed by probe-detection antibodies (Fig. 1). The probe is an enzyme that generates a colour reaction. The assay includes control samples (reference standards) produced synthetically or as a fusion human protein.

A feasibility study detecting the AMPAR peptide in a single blood draw taken from club sport athletes and professional football players in acute and subacute stage of concussions evaluated cut-offs of 0.4 and 1.0 ng/mL respectively for the assessment of single and recurrent concussions (Table 1) [11,12]. The predictive value of the test was assessed as 91% with a likelihood ratio of 11–12 for recognizing individuals with mTBI. If the test at a cut-off point of 0.4 ng/mL was negative, the post-test probability for a single concussion would be <4%.

AMPAR peptide concentrations in plasma for a military cohort suffering mTBI showed increased levels with an average concentration of 2.98 ng/mL [13]. In this study, the optimal cut-off value for recurrent mTBI was similar to professional players (Table 1), at which a positive predictive value of 93% was achieved. The trade-offs between true-positive and false-positive yielded in an area under the receiver operating characteristic (ROC) curve of 0.97.

Early experimental and clinical research of antibodies to AMPA receptors (AMPAR Ab) as an immunoexcitotoxicity biomarker has demonstrated their diagnostic value in detecting pathological brain-spiking activity and epileptic seizures [14,15] as a consequence of traumatic brain injury, thereby representing a prognostic risk factor [16]. Clinical studies of GluR1 antibodies in adult patients with different chronic neurological pathology (n=1866) performed in Russia, Germany, Ireland, Poland and the USA have demonstrated diagnostic potential (sensitivity of 86%-88% and specificity of 83–97%) in assessment of post-traumatic seizures.

In sport-related multiple concussions, AMPAR Ab values remained abnormally high in the blood of some athletes who had headaches and visual problems. It was suggested that this finding may reflect persistent changes in the subcortical areas of the brain. The diagnostic value of AMPAR Ab (sensitivity of 86–88%, specificity of 83–97% at 1.5 ng/ml cut-off) in assessment of seizures defined by electroencephalogram (EEG) with history of sustained single or multiple TBI have been demonstrated for children and adult patients indicating a development of ‘chronic’ conditions [13].

Sideline testing of AMPAR peptide and antibody
Recognizing the medical need for sideline testing for mTBI (largely for emergency care), a point-of-care (POC) testing platform has been recently undertaken for AMPAR peptide and antibody assays are being tested in clinical studies (www.drdbiotech.com).

A lateral flow sandwich assay to detect AMPAR Ab consists of a blood filtering sample pad, a pad containing gold nanoshells conjugated to protein A, a nitrocellulose strip with immobilized AMPAR peptide, a control line, and a cellulose absorbent. Applied to the blood filter, erythrocytes are removed from the sample, which passes to the conjugate pad where AMPAR IgGs are captured by protein A. When the complex reaches the peptide test line stripe on nitrocellulose, it binds to the test line yielding a visible signal with intensity proportional to the concentration of the AMPAR Ab presented in the sample (Fig. 2).

The control line then captures a portion of the remaining nanoshells regardless of the presence or absence of the peptide (Fig. 3).

The AMPAR peptide prototype test that works on a similar principle, employed gold nanoparticles as the signal-generating species covalently bound to specific antibodies against AMPAR peptide. This assay captures the AMPAR peptide from the blood sample between two different Abs, one immobilized on the nitrocellulose and the other on the gold nanoshells. This ‘bridging’ of the analyte leads to the immobilization of the particles at the test line producing a colour signal.

Conclusion
The laboratory assessment of concussion at the pitch sideline in competitive contact sports or on the field of combat is required to assist in the objective confirmation of when an injured athlete should return to play or a soldier may return to duty. Accurate diagnosis of concussion and appropriate management of subtle brain injury to prevent damage to the long-term health of athletes and others at risk of re-injury. Specific brain biomarkers detected by a rapid blood test would have important diagnostic/prognostic capabilities, particularly for concussion, to objectively evaluate early signs of further brain-function deterioration and assist in navigating personalized therapy when required. Clinical use of blood tests can reliably detect subtle brain impact, predict consequences and aid in triaging persons with persistent symptoms after a recurrent concussion for diffuse tensor or diffuse-weighted imaging modalities of MRI [17].

Early identification of concussion has the potential to become a key component of a successful treatment strategy and outcome monitoring. Advances in analytical assay technologies have made it possible to develop a rapid, cost-effective test that can be used to target populations and select a risk group for post-traumatic seizures to direct to the immediate attention of a specialist.

The opportunity of using ‘yes/no’ lateral flow tests without a need for an instrumental readout, being simply read by eye, would make a huge difference in supplying coaches with reliable, simple and rapid tests in return-to-play decisions. There is also a high demand for portable POC tests for mTBI diagnosis in military combat and civilian emergency settings.

References
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The authors
Svetlana A Dambinova*¹ DSc, PhD; Rozalyn Heath¹; Galina A Izykenova² PhD
¹Brain Biomarkers Research Laboratory, DeKalb Medical Center, Decatur, GA, USA
²GRACE Laboratories, LLC, Atlanta, GA, USA

*Corresponding author
E-mail: dambinova@aol.com

Biomarkers of diabetic nephropathy: A 2017 update
^{Papadopoulou-Marketou N, Kanaka-Gantenbein C, Marketos N, Chrousos GP, Papassotiriou I. Crit Rev Clin Lab Sci 2017; 54(5): 326–342}
Diabetic nephropathy (DN), also named diabetic kidney disease (DKD), is a devastating complication in patients with both type 1 and 2 diabetes mellitus (T1D and T2D) and its diagnosis has been traditionally based on the presence of micro-albuminuria (MA). The aim of this article is to update, through review of the relevant medical literature, the most promising biomarkers for early DKD detection. MA has historically been employed as an early marker of microvascular complications, indicating risk for advanced CKD. However, due to the inability of MA to adequately predict DKD, especially in young patients or in non-albuminuric DKD, additional biomarkers of glomerular and/or tubular injury have been proposed to uncover early renal dysfunction and structural lesions, even before MA occurs. Defining new predictive biomarkers to use alongside urinary albumin excretion (UAE) during the initial stages of DKD would provide a window of opportunity for preventive and/or therapeutic interventions to prevent or delay the onset of irreversible long-term complications and to improve outcomes by minimizing the rates of severe cardio-renal morbidity and mortality in DKD patients.

Urinary angiotensinogen and renin excretion are associated with chronic kidney disease
^{Juretzko A, Steinbach A, Hannemann A, Endlich K, Endlich N et al. Kidney Blood Press Res 2017; 42(1): 145–155}
BACKGROUND/AIMS: Several studies sought to identify new biomarkers for chronic kidney disease (CKD). As the renal renin-angiotensin system is activated in CKD, urinary angiotensinogen or renin excretion may be suitable candidates. We tested whether urinary angiotensinogen or renin excretion is elevated in CKD and whether these parameters are associated with estimated glomerular filtration rate (eGFR). We further tested whether urinary angiotensinogen or renin excretion may convey additional information beyond that provided by albuminuria.

METHODS: We measured urinary and plasma angiotensinogen, renin, albumin and creatinine in 177 CKD patients from the Greifswald Approach to Individualized Medicine project and in 283 healthy controls from the Study of Health in Pomerania. The urinary excretion of specific proteins is given as protein-to-creatinine ratio. Receiver operating characteristic (ROC) curves, spearman correlation coefficients and linear regression models were calculated.

RESULTS: Urinary angiotensinogen [2 511 (196–31 909) vs 18.6 (8.3–44.0) pmol/g, *P<0.01] and renin excretion [0.311 (0.135–1.155) vs 0.069 (0.045–0.148) pmol/g, *P<0.01] were significantly higher in CKD patients than in healthy controls. The area under the ROC curve was significantly larger when urinary angiotensinogen, renin and albumin excretion were combined than with urinary albumin excretion alone. Urinary angiotensinogen (ß-coefficient  −2.405, standard error 0.117, P<0.01) and renin excretion (ß-coefficient −0.793, standard error 0.061, P<0.01) were inversely associated with eGFR. Adjustment for albuminuria, age, sex, systolic blood pressure and body mass index did not significantly affect the results.

CONCLUSION: Urinary angiotensinogen and renin excretion are elevated in CKD patients. Both parameters are negatively associated with eGFR and these associations are independent of urinary albumin excretion. In CKD patients urinary angiotensinogen and renin excretion may convey additional information beyond that provided by albuminuria.

KIM-1 Is a potential urinary biomarker of obstruction: results from a prospective cohort study
^{Olvera-Posada D, Dayarathna T, Dion M, Alenezi H, Sener A et al. J Endourol 2017; 31(2): 111–118}
INTRODUCTION: Partial or complete obstruction of the urinary tract is a common and challenging urological condition that may occur in patients of any age. Serum creatinine is the most commonly used method to evaluate global renal function, although it has low sensitivity for early changes in the glomerular filtration rate or unilateral renal pathology. Hence, finding another measurable parameter that reflects the adaptation of the renal physiology to these circumstances is important. Several recent studies have assessed the use of new biomarkers of acute kidney injury (AKI), but the information among patients with stone disease and those with obstructive uropathy is limited.
MATERIAL AND METHODS: A prospective cohort study was conducted to determine the urinary levels of kidney injury molecule-1 (KIM-1), Total and Monomeric neutrophil gelatinase-associated lipocalin (NGAL) in patients with hydronephrosis secondary to renal stone disease, congenital ureteropelvic junction obstruction or ureteral stricture. Comparison between patients with hydronephrosis and no hydronephrosis was carried out along with correlation analysis to detect factors associated with biomarker expression.

RESULTS: Urinary levels of KIM-1 significantly decreased after hydronephrosis treatment in patients with unilateral obstruction (1.19 ng/mL vs 0.76 ng/mL creatinine, P=0.002), additionally KIM-1 was significantly higher in patients with hydronephrosis compared to stone disease patients without radiological evidence of obstruction (1.19 vs 0.64, P=0.006). Total and Monomeric NGAL showed a moderate correlation with the presence of leukocyturia. We found that a KIM-1 value of 0.735 ng/mg creatinine had a sensitivity of 75% and specificity of 67% to predict the presence of hydronephrosis in preoperative studies (95% CI 0.58-0.87, P = 0.006).

CONCLUSION: Our results show that KIM-1 is a promising biomarker of subclinical AKI associated with hydronephrosis in urological patients. NGAL values were influenced by the presence of leukocyturia, limiting its usefulness in this population.

Heparin-binding protein (HBP) improves prediction of sepsis-related acute kidney injury
^{Tverring J, Vaara ST, Fisher J, Poukkanen M et al. Ann Intensive Care 2017; 7(1): 105}
BACKGROUND: Sepsis-related acute kidney injury (AKI) accounts for major morbidity and mortality among the critically ill. Heparin-binding protein (HBP) is a promising biomarker in predicting development and prognosis of severe sepsis and septic shock that has recently been proposed to be involved in the pathophysiology of AKI. The objective of this study was to investigate the added predictive value of measuring plasma HBP on admission to the intensive care unit (ICU) regarding the development of septic AKI.

METHODS: We included 601 patients with severe sepsis or septic shock from the prospective, observational FINNAKI study conducted in seventeen Finnish ICUs during a 5-month period (1 September 2011-1 February 2012). The main outcome measure was the development of KDIGO AKI stages 2–3 from 12 h after admission up to 5 days. Statistical analysis for the primary endpoint included construction of a clinical risk model, area under the receiver operating curve (ROC area), category-free net reclassification index (cfNRI) and integrated discrimination improvement (IDI) with 95% confidence intervals (95% CI).

RESULTS: Out of 511 eligible patients, 101 (20%) reached the primary endpoint. The addition of plasma HBP to a clinical risk model significantly increased ROC area (0.82 vs 0.78, P=0.03) and risk classification scores: cfNRI 62.0% (95% CI 40.5–82.4%) and IDI 0.053 (95% CI 0.029–0.075).

CONCLUSIONS: Plasma HBP adds predictive value to known clinical risk factors in septic AKI. Further studies are warranted to compare the predictive performance of plasma HBP to other novel AKI biomarkers.

Prediction of contrast induced acute kidney injury using novel biomarkers following contrast coronary angiography
^{Connolly M, Kinnin M, McEneaney D, Menown I et al. QJM. 2017; doi: 10.1093/qjmed/hcx201}
BACKGROUND: Chronic kidney disease (CKD) is a risk factor for contrast-induced acute kidney injury (CI-AKI). Contrast angiography in CKD patients is a common procedure. Creatinine is a delayed marker of CI-AKI and delays diagnosis which results in significant morbidity and mortality.

AIM: Early diagnosis of CI-AKI requires validated novel biomarkers.

DESIGN: A prospective observation study of 301 consecutive CKD patients undergoing coronary angiography was performed. Samples for plasma neutrophil gelatinase-associated lipocalin (NGAL), serum liver fatty acid-binding protein (L-FABP), serum kidney injury marker 1 (KIM-1), serum interleukin 18 (IL-18) and serum creatinine were taken at 0, 1, 2, 4, 6 and 48 hours post contrast. Urinary NGAL and urinary cystatin C (CysC) were collected at 0, 6 and 48 hours. Incidence of major adverse clinical events (MACE) were recorded at 1 year. CI-AKI was defined as an absolute delta rise in creatinine of ≥26.5µmol/L or a 50% relative rise from baseline at 48 hours following contrast.
RESULTS: CI-AKI occurred in 28 (9.3%) patients. Plasma NGAL was most predictive of CI-AKI at 6 hours. L-FABP performed best at 4 hours.A combination of Mehran score >10, 4-hour L-FABP and 6-hour NGAL improved specificity to 96.7%. MACE was statistically higher at one year in CI-AKI patients (25.0% versus 6.2% in non CI-AKI patients).

CONCLUSIONS: Mehran risk score, 4 hour serum L-FAPB and 6 hour plasma NGAL performed best at early CI-AKI prediction. CI-AKI patients were four times more likely to develop MACE and had a trebling of mortality risk at 1 year.

Upregulation of long noncoding RNA PVT1 predicts unfavorable prognosis in patients with clear cell renal cell carcinoma
^{Bao X, Duan J, Yan Y, Ma X et al. Cancer Biomark 2017; doi: 10.3233/CBM-170251}
BACKGROUND: Renal cell carcinoma (RCC) is one of the most malignant genitourinary diseases worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs in the human genome that are involved in RCC initiation and progression.

OBJECTIVE: To investigate the expression of PVT1 in ccRCC and evaluate its correlation with clinicopathologic characteristics and patients’ survival.

METHODS: Quantitative real-time PCR was performed to examine PVT1 expression in 129 ccRCC tissue samples and matched adjacent normal tissue samples. The relationship of PVT1 expression with clinicopathologic characteristics and clinical outcome was evaluated.

RESULTS: We identified the lncRNA PVT1, which was upregulated in clear cell renal cell carcinoma (ccRCC) tissues when compared with corresponding controls. Furthermore, PVT1 expression was positively associated with gender, tumor size, pT stage, TNM stage, and Fuhrman grade. Kaplan-Meier survival analysis showed that patients with high PVT1 expression had shorter disease-free survival (DFS) and overall-survival (OS) than those with low PVT1 expression, and multivariate analysis identified PVT1 as an independent prognostic factor in ccRCC.

CONCLUSIONS: PVT1 may be an oncogene as well as may promote metastasis in ccRCC and could serve as a potential biomarker to predict the prognosis of ccRCC patients.

Urine S100 proteins as potential biomarkers of lupus nephritis activity
^{Turnier JL, Fall N, Thornton S, Witte D et al.}
BACKGROUND: Improved, non-invasive biomarkers are needed to accurately detect lupus nephritis (LN) activity. The purpose of this study was to evaluate five S100 proteins (S100A4, S100A6, S100A8/9, and S100A12) in both serum and urine as potential biomarkers of global and renal system-specific disease activity in childhood-onset systemic lupus erythematosus (cSLE).

METHODS: In this multicentre study, S100 proteins were measured in the serum and urine of four cSLE cohorts and healthy control subjects using commercial enzyme-linked immunosorbent assays. Patients were divided into cohorts on the basis of biospecimen availability: (1) longitudinal serum, (2) longitudinal urine, (3) cross-sectional serum, and (4) cross-sectional urine. Global and renal disease activity were defined using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and the SLEDAI-2K renal domain score. Nonparametric testing was used for statistical analysis, including the Wilcoxon signed-rank test, Kruskal-Wallis test, Mann-Whitney U test, and Spearman’s rank correlation coefficient.

RESULTS: All urine S100 proteins were elevated in patients with active LN compared with patients with active extrarenal disease and healthy control subjects. All urine S100 protein levels decreased with LN improvement, with S100A4 demonstrating the most significant decrease. Urine S100A4 levels were also higher with proliferative LN than with membranous LN. S100A4 staining in the kidney localized to mononuclear cells, podocytes, and distal tubular epithelial cells. Regardless of the S100 protein tested, serum levels did not change with cSLE improvement.

CONCLUSIONS: Higher urine S100 levels are associated with increased LN activity in cSLE, whereas serum S100 levels do not correlate with disease activity. Urine S100A4 shows the most promise as an LN activity biomarker, given its pronounced decrease with LN improvement, isolated elevation in urine, and positive staining in resident renal cells.