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Background: The early and accurate diagnosis of preeclampsia is crucial to avoid serious complications for both the mother and baby. However, the current diagnostic methods are limited, and there is a need for new diagnostic biomarkers. Previous studies have shown that cathepsin D (CTD) participates in the pathophysiology of preeclampsia and is present in urine samples, making it a potential biomarker for the disease. This study aimed to compare urinary and serum levels of CTD in preeclamptic and normotensive women and analyze its potential role as a diagnostic biomarker in preeclampsia., Methods: The study included thirty-nine patients with preeclampsia and twelve normotensive pregnant women as controls. Biomarkers were determined using Multiplex Assay kit, and serum prolactin (Prl) and urinary TNF-α levels were also evaluated. Statistical analysis was conducted using the Mann-Whitney U test., Results: We found that urinary and serum CTD levels were significantly higher in the preeclampsia group than in the normotensive group, suggesting that CTD could be a diagnostic biomarker for preeclampsia. No significant differences were found in the levels of serum prolactin or urinary TNF-α between the two groups., Conclusions: The study provides evidence that non-invasive biological samples such as urine can be used to improve new therapeutic strategies for the early management of preeclampsia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Objective: To evaluate the clinical and laboratory correlation of biomarkers with anti- and pro-apoptotic activity with the severity of motor and non-motor symptoms depending on the progression rate of Parkinson's disease (PD)., Material and Methods: A wide range of non-motor symptoms (emotional-affective, cognitive, psychotic and behavioral disorders, fatigue, sleep disorders and autonomic disorders) was evaluated using validated scales and a number of serum neuromarkers responsible for neuroplasticity and neuronal survival processes (BDNF, PDGF, cathepsin D) in 71 patients with PD (mean age 65 (55; 70) years, disease duration 7 (4; 9) years, age of onset 57 (49; 62) years)., Results: The concentration of biomarkers (BDNF, PDGF and cathepsin D) was the lowest in the group of patients with a rapid PD progression rate ( p <0.001, p =0.001 and p =0.031, respectively), the severity of motor and most non-motor symptoms was higher ( p =0.023 and p =0.001, respectively) compared to middle and slow progression rate. There were correlations between BDNF concentration and the severity of depression ( r =-0.63, p <0.001), apathy ( r =-0.48, p <0.001), impulsive behavioral disorders ( r =0.500, p <0.001), level of cognitive functions ( r =0.54, p <0.001), motor symptoms ( r =-0.43, p <0.001); between PDGF level and the severity of motor manifestations of PD ( r =-0.30, p =0.011), depression ( r =-0.70, p <0.001), apathy ( r =-0.460, p <0.001), the degree of severity of behavioral disorders ( r =0.742, p <0.001). No significant correlations were observed between the level of cathepsin D and the severity of clinical manifestations of PD, which indicates the connection of cathepsin D with the general pathogenesis of PD., Conclusion: The possibility of using serum proteins of the neurotrophin subfamily and the protein associated with autophagy, cathepsin D, as biomarkers that determine the prognosis of PD, is considered.

Objective: Hepatocellular carcinoma (HCC) represents a global health concern, particularly in Southeast Asia where hepatitis B virus (HBV) infection is common. In this study, we applied tissue-based proteomics to identify novel serological proteins for HCC and validated their performance in serum specimens., Methods: In a discovery set, liver tissue specimens of HBV-related HCC, intrahepatic cholangiocarcinoma (iCCA) and colorectal cancer with liver metastasis (CRLM) were analyzed using mass spectrometry (LTQ-Orbitrap-XL). A subset of proteins that showed highly expressed in HCC were then confirmed by Western blotting. Additionally, clinical significance of selected candidate proteins was tested in serum samples of 80 patients with HBV-related HCC, 50 patients with HBV-related liver cirrhosis and 30 healthy controls., Results: Based on LTQ-Orbitrap-XL mass spectrometer, various differentially expressed proteins (DEPs) between tumor and adjacent non-tumor tissues were identified. These included 77 DEPs for HCC, 77 DEPs for iCCA and 55 DEPs for CRLM. Among selected candidate proteins, annexin A2 and cathepsin D were confirmed to be overexpressed in HCC tissue by Western blot analysis. In a validate cohort, serum cathepsin D level, but not annexin A2, was significantly higher in HCC compared with the non-HCC groups. Serum cathepsin D level was also positively correlated with tumor size and tumor stage. Additionally, the combined assay of serum cathepsin D and alpha-fetoprotein had a high sensitivity in detecting early HCC (83%) and intermediate/advanced HCC (96%). Moreover, patients with low serum cathepsin D (<305 ng/mL) displayed significantly better overall survival than those whose serum levels were high (≥305 ng/mL)., Conclusions: Proteomics and subsequent validation revealed cathepsin D as a novel biomarker for HCC. Apart from its diagnostic role, serum cathepsin D might also serve as a prognostic biomarker of HCC. Additional large-scale studies are needed to verify our findings.

Hidradenitis suppurativa is a chronic inflammatory dermatosis with presentations ranging from painful nodules and abscesses to draining tunnels. Using an unbiased proteomics approach, we assessed cardiovascular-, cardiometabolic-, and inflammation-related biomarkers in the serum of patients with moderate-to-severe hidradenitis suppurativa. The serum of patients with hidradenitis suppurativa clustered separately from that of healthy controls and had an upregulation of neutrophil-related markers (Cathepsin D, IL-17A, CXCL1). Patients with histologically diagnosed dermal tunnels had higher serum lipocalin-2 levels compared with those without tunnels. Consistent with this, patients with tunnels had a more neutrophilic-rich serum signature, marked by Cathepsin D, IL-17A, and IL-17D alterations. There was a significant serum‒skin correlation between proteins in the serum and the corresponding mRNA expression in skin biopsies, with healthy-appearing perilesional skin demonstrating a significant correlation with neutrophil-related proteins in the serum. CSF3 mRNA levels in lesional skin significantly correlated with neutrophil-related proteins in the serum, suggesting that CFS3 in the skin may be a driver of neutrophilic inflammation. Clinical significantly correlated with the levels of lipocalin-2 and IL-17A in the serum. Using an unbiased, large-scale proteomic approach, we demonstrate that hidradenitis suppurativa is a systemic neutrophilic dermatosis, with a specific molecular signature associated with the presence of dermal tunnels., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cathepsin D (CTSD) is an aspartyl proteinase that plays an important role in protein degradation, antigen processing and apoptosis. It has been associated with several pathologies such as cancer, Alzheimer's disease and inflammatory disorders. Its function in lung diseases remains, however, controversial. In the current study, we determined CTSD activity in serum of patients with chronic obstructive pulmonary disease (COPD) and evaluated the correlations between this proteinase and inflammatory and oxidative parameters. We also investigated the impact of a CTSD C224T polymorphism on enzyme activity and clinicopathological parameters., Methods: Our population included 211 healthy controls and 138 patients with COPD. CTSD activity, MMPs (-1/-7/-12), cytokines (IL-6, TNF-α), malondialdehyde (MDA), nitric oxide and peroxynitrite levels were measured in patients and controls using standard methods. Genotyping of CTSD C224T polymorphism was determined using PCR-RFLP., Results: Our results showed an increased CTSD activity in COPD patients compared to healthy controls (4.87 [3.99-6.07] vs. 3.94 [2.91-5.84], respectively, p < 0.001). COPD smokers presented also a higher CTSD activity when compared to nonsmokers (4.91[3.98-6.18] vs. 4.65[4.16-5.82], respectively, p = 0.01), while no differences were found when subjects were compared according to their GOLD stages. The activity of this proteinase was not dependent on the C224T polymorphism because we did not found any influence of this SNP on proteinase activity among patients and controls. Furthermore, our data provide the first evidence of the interrelationships between CTSD activity and both MMPs and TNF-α levels (MMP-1[r = - 0.4; p = 0.02], MMP-7[r = 0.37; p = 0.04], MMP-12[r = 0.43; p = 0.02], TNF-α [r = 0.89, p = 0.001]) in COPD smokers. There were no correlations, however, between CTSD activity and oxidative stress parameters in controls and patients., Conclusion: Our findings suggest that CTSD could be a relevant marker for COPD disease. Alteration of CTSD activity may be related to increased MMPs and TNF-α levels, particularly in COPD smokers.

The aim of the study is to evaluate oxidant-antioxidant balance as well as lysosomal and anti-protease activities in ovarian cancer since it has been emphasized that the crucial inducing factor of carcinogenesis may be reactive oxygen/nitrogen species or, more precisely, oxidative stress-induced inflammation. The study involved 15 women with ovarian cancer, aged 59.9 ± 7.8 years, and 9 healthy women aged 56.3 ± 4.3 years (controls). The study material was venous blood collected from fasting subjects. In erythrocytes, the activities of superoxide dismutase, glutathione peroxidase, and catalase, as well as concentrations of conjugated dienes (CDs) and thiobarbituric acid reactive substances (TBARS), were investigated. CD, TBARS, and vitamins A and E plasma concentrations were also determined. Moreover, total antioxidant capacity and concentrations of 4-hydroxynonenal adducts and 8-iso-prostaglandin F2α, as well as activities of acid phosphatase, arylsulfatase, cathepsin D, and α 1 -antitrypsin, were studied in serum. The vitamin E and 8-iso-prostaglandin F2α concentrations as well as arylsulfatase activity were lower in the women with cancer compared to the controls ( p = 0.006, p = 0.03, p = 0.001, respectively). In contrast, cathepsin D activity was lower in the controls ( p = 0.04). In the peripheral blood of the women with cancer, oxidant-antioxidant and lysosomal disturbances were observed.

Inherited retinal dystrophies (IRDs) are rare but highly heterogeneous genetic disorders that affect individuals and families worldwide. However, given its wide variability, its analysis of the driver genes for over 50% of the cases remains unexplored. The present study aims to identify novel driver genes, disease-causing variants, and retinitis pigmentosa (RP)-associated pathways. Using family-based whole-exome sequencing (WES) to identify putative RP-causing rare variants, we identified a total of five potentially pathogenic variants located in genes OR56A5, OR52L1, CTSD, PRF1, KBTBD13, and ATP2B4. Of the variants present in all affected individuals, genes OR56A5, OR52L1, CTSD, KBTBD13, and ATP2B4 present as missense mutations, while PRF1 and CTSD present as frameshift variants. Sanger sequencing confirmed the presence of the novel pathogenic variant PRF1 (c.124&#95;128del) that has not been reported previously. More causal-effect or evidence-based studies will be required to elucidate the precise roles of these SNPs in the RP pathogenesis. Taken together, our findings may allow us to explore the risk variants based on the sequencing data and upgrade the existing variant annotation database in Taiwan. It may help detect specific eye diseases such as retinitis pigmentosa in East Asia.

Previously, we have shown that hepatic lipid accumulation induces the secretion of cathepsin D (CTSD), and that plasma CTSD levels are associated with increased inflammation and disease severity in nonalcoholic fatty liver disease (NAFLD). Although it is clear that the liver is a major source of plasma CTSD, it is unknown whether other metabolically active organs such as the muscle, also associate with plasma CTSD levels in NAFLD patients. Therefore, the aim of this study was to explore the relation between lipid accumulation in the muscle (myosteatosis) and plasma CTSD levels in forty-five NAFLD patients. We observed that hepatic steatosis positively associated with plasma CTSD levels, confirming the previously established link between plasma CTSD and the liver. Furthermore, a positive association between myosteatosis and plasma CTSD levels was observed, which was independent of sex, age, BMI, waist circumference and hepatic steatosis. By establishing a positive association between myosteatosis and plasma CTSD levels, our findings suggest that, in addition to the liver, the muscle is also linked to plasma CTSD levels in NAFLD patients. The observed link between myosteatosis and plasma CTSD levels supports the concept of a significant role of the skeletal muscle in metabolic disturbances in metabolic syndrome-related disorders., (© 2021 Lingling Ding et al., published by De Gruyter.)

The current diagnosis of Parkinson's disease (PD) mostly relies on clinical rating scales related to motor dysfunction. Given that clinical symptoms of PD appear after significant neuronal cell death in the brain, it is required to identify accessible, objective, and quantifiable biomarkers for early diagnosis of PD. In this study, a total of 20 patients with idiopathic PD and 20 age-matched patients with essential tremor according to the UK Brain Bank Criteria were consecutively enrolled to identify peripheral blood biomarkers for PD. Clinical data were obtained by clinical survey and assessment. Using albumin-depleted and immunoglobulin G-depleted plasma samples, we performed immunoblot analysis of seven autophagy-related proteins and compared the levels of proteins to those of the control group. We also analyzed the correlation between the levels of candidate proteins and clinical characteristics. Finally, we validated our biomarker models using receiver operating characteristic curve analysis. We found that the levels of BCL2-associated athanogene 2 (BAG2) and cathepsin D were significantly decreased in plasma of patients with PD (P = 0.009 and P = 0.0077, respectively). The level of BAG2 in patients with PD was significantly correlated with Cross-Culture Smell Identification Test score, which indicates olfactory dysfunction. We found that our biomarker model distinguishes PD with 87.5% diagnostic accuracy (area under the curve (AUC) = 0.875, P < 0.0001). Our result suggests BAG2 and cathepsin D as candidates for early-diagnosis plasma biomarkers for PD. We provide the possibility of plasma biomarkers related to the autophagy pathway, by which decreased levels of BAG2 and cathepsin D might lead to dysfunction of autophagy., (© 2020 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Background and Aims: An increased amount of visceral adipose tissues has been related to atherosclerosis and future cardiovascular events. The present study aims to investigate how the abdominal fat distribution links to plasma levels of cardiovascular-related proteins., Method and Results: In the Prospective investigation of Obesity, Energy and Metabolism (POEM) study (n = 326, all aged 50 years), abdominal visceral (VAT) and subcutaneous (SAT) adipose tissue volumes were quantified by MRI. Eighty-six cardiovascular-related proteins were measured by the proximity extension assay (PEA). Similar investigations were carried out in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (n = 400, all aged 75 years). In the discovery dataset (POEM), 10 proteins were related to the VAT/SAT-ratio using false discovery rate <.05. Of those, Cathepsin D (CTSD), Interleukin-1 receptor antagonist protein (IL-1RA) and Growth hormone (GH) (inversely) were related to the VAT/SAT-ratio in the validation in PIVUS following adjustment for sex, BMI, smoking, education level and exercise habits (p < 0.05). In a secondary analysis, a meta-analysis of the two samples suggested that 15 proteins could be linked to the VAT/SAT-ratio following adjustment as above and Bonferroni-correction of the p-value., Conclusion: Three cardiovascular-related proteins, cathepsin D, IL-1RA and growth hormone, were being associated with the distribution of abdominal adipose tissue using a discovery/validation approach. A meta-analysis of the two samples suggested that also a number of other cardiovascular-related proteins could be associated with an unfavorable abdominal fat distribution., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Although Alzheimer's disease (AD) is the most common neurodegenerative disease, there are still no drugs available to treat or prevent AD effectively. Here, we examined changes in levels of selected proteins implicated in the pathogenesis of AD using plasma samples of control subjects and patients with cognition impairment. To precisely categorize the disease, fifty-six participants were examined with clinical cognitive tests, amyloid positron emission tomography (PET) scan, and white matter hyperintensities scored by magnetic resonance imaging. Plasma cathepsin D levels of the subjects were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Correlation of plasma cathepsin D levels with AD-related factors and clinical characteristics were examined by statistical analysis. By analyzing quantitative immunoblot and ELISA, we found that the plasma level of cathepsin D, a major lysosomal protease, was decreased in the group with amyloid plaque deposition at the brain compared to the control group. The level of plasma cathepsin D was negatively correlated with clinical dementia rating scale sum of boxes (CDR-SB) scores. In addition, our integrated multivariable logistic regression model suggests the high performance of plasma cathepsin D level for discriminating AD from non-AD. These results suggest that the plasma cathepsin D level could be developed as a diagnostic biomarker candidate for AD.

Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM-MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM-MS determination of multiple enzymes were synthesized by chemical coupling of alkyl-umbelliferone building blocks with the corresponding peptidyl-substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM-MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl-(4-alkyl-umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins-D, -F, and -B), to provide a differentiation of proteolytic specificities., (© 2020 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

Background and Aims: Monocyte chemoattractant protein-1 (MCP-1) and cathepsin-D are progressively raised in type 2 diabetes mellitus (T2DM) with both non proliferative and proliferative retinal disease. This study aimed to evaluate the effect of antidiabetic medications on MCP-1 and cathepsin-D., Methods: 60 patients of T2DM without retinopathy and 60 of diabetic retinopathy were enrolled to receive metformin (500 mg-1000 mg) combined with either glimepiride (1 mg-2 mg) or insulin. The effect of antidiabetic medications on serum MCP-1 and cathepsin-D was assessed., Results: Mean MCP-1 (pg/ml) and cathepsin-D (ng/ml) levels were significantly lower in patients of T2DM with and without retinopathy treated with metformin + insulin (468.52 ± 272.84 vs 234.30 ± 180.58; p < 0.01 and 460.15 ± 128.52 vs 517.33 ± 213.49; p = 0.214) as compared to patients treated with metformin + glimepiride (1434.02 ± 105.27 vs 1256.27 ± 76.76; p < 0.01 and 1689.36 ± 752.57 vs 919.69 ± 675.05; p = < 0.01). No significant correlation of MCP-1 and cathepsin-D with HbA1c, fasting and post prandial blood glucose were found., Conclusion: Patients treated with metformin and insulin combination had lower serum MCP-1 and cathepsin-D levels which suggests that this combination may be more effective in reducing the progression of diabetic retinopathy. (CTRI/2018/05/013601)., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Aims: Cathepsin D is a ubiquitous lysosomal protease that is primarily secreted due to oxidative stress. The role of circulating cathepsin D in heart failure (HF) is unknown. The aim of this study is to determine the association between circulating cathepsin D levels and clinical outcomes in patients with HF and to investigate the biological settings that induce the release of cathepsin D in HF., Methods and Results: Cathepsin D levels were studied in 2174 patients with HF from the BIOSTAT-CHF index study. Results were validated in 1700 HF patients from the BIOSTAT-CHF validation cohort. The primary combined outcome was all-cause mortality and/or HF hospitalizations. Human pluripotent stem cell-derived cardiomyocytes were subjected to hypoxic, pro-inflammatory signalling and stretch conditions. Additionally, cathepsin D expression was inhibited by targeted short hairpin RNAs (shRNA). Higher levels of cathepsin D were independently associated with diabetes mellitus, renal failure and higher levels of interleukin-6 and N-terminal pro-B-type natriuretic peptide (P < 0.001 for all). Cathepsin D levels were independently associated with the primary combined outcome [hazard ratio (HR) per standard deviation (SD): 1.12; 95% confidence interval (CI) 1.02-1.23], which was validated in an independent cohort (HR per SD: 1.23, 95% CI 1.09-1.40). In vitro experiments demonstrated that human stem cell-derived cardiomyocytes released cathepsin D and troponin T in response to mechanical stretch. ShRNA-mediated silencing of cathepsin D resulted in increased necrosis, abrogated autophagy, increased stress-induced metabolism, and increased release of troponin T from human stem cell-derived cardiomyocytes under stress., Conclusions: Circulating cathepsin D levels are associated with HF severity and poorer outcome, and reduced levels of cathepsin D may have detrimental effects with therapeutic potential in HF., (© 2019 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Objective: Type 2 diabetes mellitus is a metabolic disorder characterized by insulin resistance. Previous studies in patients demonstrated that plasma levels of cathepsin D (CTSD), which is optimally active in the acidic environment of lysosomes, correlate with insulin resistance. As plasma pH is slightly reduced in type 2 diabetic patients and we have previously shown that plasma CTSD activity is causally linked to insulin levels in vivo , it is likely that the activity of CTSD in plasma will be increased in type 2 diabetes compared to healthy individuals. However, so far the interaction between CTSD activity and levels to postprandial metabolic derangements in type 2 diabetes is not known., Methods: Eighteen type 2 diabetes and 16 age-matched healthy males were given 2 consecutive standardized mixed meals, after which blood samples were collected. Plasma metabolic parameters as well as CTSD levels and activity were measured, and changes in plasma pH was assessed., Results: In line with the elevation of plasma free fatty acids (FFA) levels in male type 2 diabetics patients, plasma pH in type 2 diabetic individuals was decreased compared to male healthy individuals. While plasma CTSD levels were similar, plasma CTSD activity was increased in male type 2 diabetic compared to male healthy individuals. Besides, plasma CTSD activity rather than levels significantly correlated with indicators of type 2 diabetes (HbA1c, HOMA-IR and glucose). Furthermore, FFA was also independently associated with plasma CTSD activity (standardized β = 0.493, p = 0.007)., Conclusions: Despite similar plasma CTSD levels, type 2 diabetic male individuals showed increased plasma CTSD activity compared to healthy males, which was independently linked to plasma FFA levels. Our data therefore point toward plasma CTSD as a metabolic regulator in male type 2 diabetes., (Copyright © 2020 Ding, Houben, Oligschlaeger, Bitorina, Verwer, Tushuizen and Shiri-Sverdlov.)

Background: Malignant pleural effusion (MPE) causes substantial symptomatic burden in advanced malignancy. Although pleural fluid cytology is a commonly accepted gold standard of diagnosis, its low diagnostic yield is a challenge for clinicians. The aim of this study was to determine whether pro-cathepsin D can serve as a novel biomarker to discriminate between MPE and benign pleural effusion (BPE)., Methods: This study included 81 consecutive patients with exudative pleural effusions who had underwent thoracentesis or pleural biopsy. Pleural fluid and serum were collected as a standard procedure for all individuals at the same time. The level of pro-cathepsin D was measured by the sandwich enzyme-linked immunosorbent assay method., Results: Though there were no significant differences in plasma pro-cathepsin D between the two groups, the level of pleural fluid pro-cathepsin D was significantly higher in the MPE group than the BPE group (0.651 versus 0.590 pg/mL, P = 0.034). The discriminative power of pleural fluid pro-cathepsin D for diagnosing MPE was moderate, with 81% sensitivity and 53% specificity at a pro-cathepsin D cut-off ≥0.596 pg/mL (area under the curve: 0.656). Positive and negative predictive values for MPE were 38 and 89%, respectively, with pro-cathepsin D cut-off value (> 0.596 pg/mL)., Conclusions: The level of pleural fluid pro-cathepsin D was found to be significantly higher in MPE than in BPE. Although results of this study could not support the sole use of pleural fluid pro-cathepsin D to diagnose MPE, pleural fluid pro-cathepsin D can be added to pre-existing diagnostic methods for ruling-in or ruling-out MPE.

Deep electromagnetic stimulation (DEMS) and low-frequency ultrasound (US) are new physical therapy methods used in the rehabilitation of the musculoskeletal system and wound healing. They are applied locally to treat the injured tissues. The beneficial effects of these methods in supportive care have been documented, but accurate biochemical effects are not known. The goal was to assess the effect of single DEMS and US sessions on the oxidant-antioxidant equilibrium, as well as the activities of lysosomal hydrolases and α 1 -antitrypsin (AAT) in peripheral blood of juvenile injured amateur athletes. In the athletes with low back pain (DEMS treated, N = 16) and pain in the shoulder or ankle joint (US treated, N = 14), as well as in healthy control amateur athletes (DEMS treated, N = 14; US treated, N = 17), before the sessions and 30 minutes and 24 hours after them, the levels of the following parameters were determined: thiobarbituric acid reactive substances (TBARS) in erythrocytes and plasma, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in erythrocytes, as well as acid phosphatase (AcP), arylsulfatase (ASA), cathepsin D (CTS D), and α 1 -antitrypsin (AAT) in serum. After both procedures, the levels of parameters changed in a negligible manner, excluding the cathepsin D activity, which was statistically significantly lower 30 min and 24 h after US in the control athletes compared to the baseline activity determined directly before the procedure (47.5% and 55.7% differences, respectively). Similar tendency was observed after DEMS ( p > 0.05). The procedures, especially low-frequency US, decrease lysosomal proteolytic activity and do not significantly disrupt the oxidant-antioxidant and lysosomal equilibriums in the peripheral blood both of healthy and injured athletes. No systemic acute-phase response of AAT was also detected in the athletes after both procedures. This trial is registered with CTRI/2018/01/011344., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Łukasz Sielski et al.)

The Prostate Specific Antigen (PSA) test suffers from low specificity for the diagnosis of Prostate Cancer (PCa). We originally discovered two cancer-related proteins thrombospondin-1 (THBS1) and cathepsin D (CTSD) using a mass-spectrometry-based proteomics approach. The two serum proteins were shown to improve the diagnosis of high-grade PCa. Thus, we developed quantitative ELISAs for the determination of their concentration in human serum. Here we report their analytical performance in terms of limit of detection, specificity, precision, linearity and interferences, which were determined based on CLSI guidelines. Further, we investigated the influence of pre-analytical factors on concentration measurements. For this, blood from 4-6 donors was collected in different tubes and stored at room temperature for different times prior to centrifugation at different centrifugal forces and temperatures. Stability of THBS1 and CTSD under different storage temperatures was also evaluated. Our results show that the assays are specific, linear and sensitive enough to allow measurement of clinical samples. Precision in terms of repeatability and total within-laboratory coefficient of variation (CV) are 5.5% and 8.1% for THBS1 and 4.3% and 7.2% for CTSD, respectively. Relative laboratory-to-laboratory differences were -6.3% for THBS1 and -3% for CTSD. Both THBS1 and CTSD were stable in serum samples, with 80-120% recoveries of concentrations across donors, sample preparation and storage. In conclusion, the ELISAs as part of the novel commercial in vitro diagnostic test Proclarix are suitable for the use in clinical practice. THBS1 and CTSD can be accurately measured for their intended use independent of the lot and laboratory when conditions consistent with routine practice for PSA sampling and storage are used., Competing Interests: AM, AA and RS are inventors of the following patent application (WO2018011212, Method of detecting proteins in human samples and uses of such methods) as well as RS on patent application (WO2009138392, Method for biomarker and drug-target discovery for prostate cancer diagnosis and treatment as well as biomarker assays determined therewith). RS is co-founder of Proteomedix. Authors AM, AA, AW, RH, MR, BG, RS are employed by Proteomedix and contributed to the development of the marketed product Proclarix (Proteomedix, Switzerland), which includes the THBS1 and CTSD assays described in this manuscript. Some of the authors hold stock options and received salaries (AM, AA, AW, RH, MR, BG) and founder shares (RS) of Proteomedix. TK and SW received payments from Proteomedix for statistical consultancy and are employed by ACOMED Statistik. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Vasoinhibin is generated in the pituitary gland and in multiple target tissues by proteolytic cleavage of prolactin by matrix metalloproteinases and cathepsin D. A dysregulation of vasoinhibin generation appears to contribute to diabetic retinopathy and diabetic macular edema, retinopathy of prematurity, peripartum cardiomyopathy, and preeclampsia. Here, we investigate whether vasoinhibin is generated by matrix metalloproteinases and cathepsin D in human serum., Methods: The abundance of matrix metalloproteinases 1, 2, 3, 8, 9, 10, 13, tissue inhibitors of metalloproteinases 1, 2, 4, and the activity of cathepsin D in serum samples were determined. Samples from healthy male (n = 3) and female (n = 2) subjects, pregnant subjects (n = 2), and patients with type 2 diabetes mellitus (n = 2) were investigated. The samples were incubated with recombinant prolactin at 37°C, under different pH, time, and buffer conditions. Prolactin and cleaved prolactin products were investigated by SDS-PAGE and western blotting., Results: Matrix metalloproteases-1, -2, -3, -8, -9, -10, -13, TIMP-1, -2, and -4, and the activity of cathepsin D were detected in all sera. Full-length prolactin incubated with human sera, containing endogenous matrix metalloproteinases and cathepsin D, remained intact at neutral pH during a time frame from 1 to 24 hours. Partial enzymatic cleavage of prolactin resulting in the generation of a vasoinhibin-like 17 kDa peptide was observed in samples incubated at pH 3.4. Heat inactivation of the serum and the addition of an MMP inhibitor suppressed the generation of the 17 kDa peptide, indicating that its generation was MMP-mediated., Conclusions: Vasoinhibin generation by enzymatic cleavage of prolactin by matrix metalloproteases or cathepsin D does not occur in human serum at physiological pH. A limited proteolysis of prolactin, resulting in the generation of a vasoinhibin-like peptide with an apparent molecular weight of 17 kDa occurs in serum at acidic pH. The generation of vasoinhibin may require the cellular and tissue microenvironments.

Cathepsins are the major lysosomal proteases that maintain intracellular homeostasis. Herein, we investigated the alterations in myocardial cathepsin expression during aging, cardiac hypertrophy, and sudden cardiac death (SCD). Cardiac tissue and blood were sampled from autopsy cases. Subjects were classified into three groups: SCD with cardiac hypertrophy (SCH), compensated cardiac hypertrophy (CCH), and control. Immunoblotting was performed for the major cardiac cathepsins and their targets: cathepsin B, D, and L (CTSB/D/L), p62, ATP synthase subunit c (ATPSC), and α-synuclein (ASNC). Immunohistochemical analysis and ELISA using serum samples were performed for CTSD. Cardiac CTSB and CTSD were upregulated with age (r = 0.63 and 0.60, respectively), whereas the levels of CTSL, p62, ATPSC, and ASNC remained unchanged. In age-matched groups, cardiac CTSD was significantly downregulated in SCH (p = 0.006) and CTSL was moderately downregulated in CCH (p = 0.021); however, p62, ATPSC, and ASNC were not upregulated in cardiac hypertrophy. Immunohistochemistry also revealed decreased myocardial CTSD levels in SCH, and serum CTSD levels were relatively lower in SCH cases. Overall, these results suggest that upregulation of cardiac CTSB and CTSD with age may compensate for the elevated proteolytic demand, and that downregulation of CTSD is potentially linked to SCH., Competing Interests: The authors have declared that no competing interests exist.

Glutaric acidemia type I (GA1) is caused by severe deficiency of glutaryl-CoA dehydrogenase activity, resulting in an accumulation of glutaric acid and glutarylcarnitine (C5DC) in the organism. Patients affected by GA1 are asymptomatic in the neonate period but usually manifest chronically progressive neurodegeneration apart from severe encephalopathic crises associated with acute striatum necrosis. Neurological manifestations like dyskinesia, dystonia, hypotonia, muscle stiffness, and spasticity are present. Treatment is based on protein/lysine restriction and l-carnitine supplementation. In this work, we evaluated markers of neurodegeneration and inflammation, namely BDNF (brain-derived neurotrophic factor), NCAM (neuronal adhesion molecule), PDGF-AA (platelet-derived growth factor), and cathepsin-d in plasma of six treated GA1 patients. We first found marked increases of plasma C5DC concentrations in GA1 patients, as well as increased levels of the markers BDNF and cathepsin-d as compared to those of age-matched healthy children. Furthermore, C5DC concentrations were highly correlated with the levels of cathepsin-d. These results may demonstrate that brain tissue degeneration is present in GA1 patients and that there is a relationship between increased metabolites concentrations with this process. To the best of our knowledge, this is so far the first study showing altered peripheral parameters of neurodegeneration and inflammation in GA1 patients., (© 2020 International Society for Developmental Neuroscience.)

Aims/hypothesis: Insulin resistance in skeletal muscle and liver plays a major role in the pathophysiology of type 2 diabetes. The hyperinsulinaemic-euglycaemic clamp is considered the gold standard for assessing peripheral and hepatic insulin sensitivity, yet it is a costly and labour-intensive procedure. Therefore, easy-to-measure, cost-effective approaches to determine insulin sensitivity are needed to enable organ-specific interventions. Recently, evidence emerged that plasma cathepsin D (CTSD) is associated with insulin sensitivity and hepatic inflammation. Here, we aimed to investigate whether plasma CTSD is associated with hepatic and/or peripheral insulin sensitivity in humans., Methods: As part of two large clinical trials (one designed to investigate the effects of antibiotics, and the other to investigate polyphenol supplementation, on insulin sensitivity), 94 overweight and obese adults (BMI 25-35 kg/m 2 ) previously underwent a two-step hyperinsulinaemic-euglycaemic clamp (using [6,6- 2 H 2 ]glucose) to assess hepatic and peripheral insulin sensitivity (per cent suppression of endogenous glucose output during the low-insulin-infusion step, and the rate of glucose disappearance during high-insulin infusion [40 mU/(m 2 × min)], respectively). In this secondary analysis, plasma CTSD levels, CTSD activity and plasma inflammatory cytokines were measured., Results: Plasma CTSD levels were positively associated with the proinflammatory cytokines IL-8 and TNF-α (IL-8: standardised β = 0.495, p < 0.001; TNF-α: standardised β = 0.264, p = 0.012). Plasma CTSD activity was negatively associated with hepatic insulin sensitivity (standardised β = -0.206, p = 0.043), independent of age, sex, BMI and waist circumference, but it was not associated with peripheral insulin sensitivity. However, plasma IL-8 and TNF-α were not significantly correlated with hepatic insulin sensitivity., Conclusions/interpretation: We demonstrate that plasma CTSD activity, but not systemic inflammation, is inversely related to hepatic insulin sensitivity, suggesting that plasma CTSD activity may be used as a non-invasive marker for hepatic insulin sensitivity in humans.

Background: Inflammation has been shown to accompany Metabolic Syndrome (MetS) and its features. Cathepsin D is one of a proinflammatory mediator. In the current study, we aimed to investigate the effect of curcumin supplementation on serum cathepsin D levels in patients with MetS., Methods: The current study was conducted on 18-65 years old individuals with MetS diagnosed according to the International Diabetes Federation guidelines. A total of 80 participants were randomly divided into treatment and control groups. The first group (n=40) was given 2 capsules containing 500 mg of phosphatidylcholine complex of curcumin, and the other group (n=40) was given two 500 mg placebo capsules for 6 weeks. Before (week 0) and after (week 6) the intervention, anthropometric indices and blood pressure were measured and blood samples were taken. Serum cathepsin D was measured using an ELISA kit., Results: There was no significant difference between treatment and control groups in terms of weight, body mass index, waist circumference and serum cathepsin D levels before and after the intervention. In addition, there was no significant difference between pre- and post-trial values of serum cathepsin D., Conclusion: The present results do not suggest any effect of curcumin on cathepsin D levels in patients with MetS., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims/hypothesis: The pathogenesis of type 2 diabetes is not fully understood. We investigated whether circulating levels of preselected proteins were associated with the outcome 'diabetes' and whether these associations were causal., Methods: In 2467 individuals of the population-based, cross-sectional EpiHealth study (45-75 years, 50% women), 249 plasma proteins were analysed by the proximity extension assay technique. DNA was genotyped using the Illumina HumanCoreExome-12 v1.0 BeadChip. Diabetes was defined as taking glucose-lowering treatment or having a fasting plasma glucose of ≥7.0 mmol/l. The associations between proteins and diabetes were assessed using logistic regression. To investigate causal relationships between proteins and diabetes, a bidirectional two-sample Mendelian randomisation was performed based on large, genome-wide association studies belonging to the DIAGRAM and MAGIC consortia, and a genome-wide association study in the EpiHealth study., Results: Twenty-six proteins were positively associated with diabetes, including cathepsin D, retinal dehydrogenase 1, α-L-iduronidase, hydroxyacid oxidase 1 and galectin-4 (top five findings). Three proteins, lipoprotein lipase, IGF-binding protein 2 and paraoxonase 3 (PON-3), were inversely associated with diabetes. Fourteen of the proteins are novel discoveries. The Mendelian randomisation study did not disclose any significant causal effects between the proteins and diabetes in either direction that were consistent with the relationships found between the protein levels and diabetes., Conclusions/interpretation: The 29 proteins associated with diabetes are involved in several physiological pathways, but given the power of the study no causal link was identified for those proteins tested in Mendelian randomisation. Therefore, the identified proteins are likely to be biomarkers for type 2 diabetes, rather than representing causal pathways.

In Gaucher disease, several macrophage-specific biomarkers have been validated for use in the clinic. However, Gaucher disease is more complex involving system-wide pathophysiology beyond the macrophage, and based on gene array analysis in our Gaucher disease mouse model and other emerging pathophysiologic insights, we evaluated serum levels of cathepsins D and S, YKL-40 and progranulin in Gaucher disease patients. We assessed their biomarker potential in Gaucher disease and compared them to established Gaucher disease biomarkers, chitotriosidase, chemokine ligand 18 (CCL18), and other indicators of disease severity and response to therapy. Mean YKL-40 and cathepsin D and S levels were significantly higher in Gaucher disease patients compared to healthy controls; in contrast, mean progranulin levels were lower in Gaucher disease patients compared to healthy controls. Enzyme replacement therapy resulted in a significant reversal of elevated cathepsin D and S but there was no change in progranulin and YKL-40 levels. Patients with persistent splenomegaly after long-term enzyme replacement therapy had significantly higher serum YKL-40 than patients with smaller spleens (63.0 ± 6.4 ng/ml vs. 46.4 ± 4.3 ng/ml, p = .03). Serum YKL-40 levels were higher in subjects with severe bone involvement (Hermann Score 3 to 5) compared to those with milder bone involvement (Hermann Score 1 to 2) (70.1 ± 4.3 ng/ml vs. 48.1 ± 3.7 ng/ml, p = .0002). YKL-40 was only weakly associated with chitotriosidase (r = 0.2, p = .008) and CCL18 (r = 0.3, p = .0004), and cathepsin S was moderately associated with chitotriosidase (r = 0.4, p = .01) and CCL18 (r = 0.6, p < .0001). Receiver operating curves for progranulin and YKL-40 demonstrated areas under the curves of 0.80 and 0.70, respectively. In conclusion, while these biomarkers do not meet robust properties of established macrophage-specific biomarkers, they may inform severity of skeletal disease, contribution of fibrosis to residual splenomegaly, and other disease manifestations. These findings, including markedly low progranulin levels that do not change upon enzyme replacement therapy, are intriguing to prompt further investigations to decipher their role in pathophysiology and relevance to diverse phenotypes of Gaucher disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Newer therapeutic agents for type 2 diabetes mellitus can improve cardiovascular outcomes, but diabetes remains underdiagnosed in patients with myocardial infarction (MI). We sought to identify proteomic markers of undetected dysglycaemia (impaired fasting glucose, impaired glucose tolerance, or diabetes mellitus) to improve the identification of patients at highest risk for diabetes. Materials and methods: In this prospective cohort, 626 patients without known diabetes underwent oral glucose tolerance testing (OGTT) during admission for MI. Proximity extension assay was used to measure 81 biomarkers. Multivariable logistic regression, adjusting for risk factors, was used to evaluate the association of biomarkers with dysglycaemia. Subsequently, lasso regression was performed in a 2/3 training set to identify proteomic biomarkers with prognostic value for dysglycaemia, when added to risk factors, fasting plasma glucose, and glycated haemoglobin A1c. Determination of discriminatory ability was performed in a 1/3 test set. Results: In total, 401/626 patients (64.1%) met the criteria for dysglycaemia. Using multivariable logistic regression, cathepsin D had the strongest association with dysglycaemia. Lasso regression selected seven markers, including cathepsin D, that improved prediction of dysglycaemia (area under the receiver operator curve [AUC] 0.848 increased to 0.863). In patients with normal fasting plasma glucose, only cathepsin D was selected (AUC 0.699 increased to 0.704). Conclusions: Newly detected dysglycaemia, including manifest diabetes, is common in patients with acute MI. Cathepsin D improved the prediction of dysglycaemia, which may be helpful in the a priori risk determination of diabetes as a motivation for confirmatory OGTT.

Objectives: To investigate and further validate if two novel cancer-related glycoproteins, discovered by a genetic-guided proteomics approach, can distinguish benign disease from prostate cancer (PCa) in men with enlarged prostates., Patients and Methods: A retrospective study was performed that included men with a total prostate-specific antigen (PSA) concentration of 2.0-10 ng/mL, negative digital rectal examination and enlarged prostate (volume ≥35 mL). Serum samples were collected between 2011 and 2016 at a single centre from 474 men before they underwent prostate biopsy. Serum concentrations of thrombospondin 1 (THBS1) and cathepsin D (CTSD) glycoproteins were combined with the percentage of free PSA to total PSA ratio (%fPSA) to predict any or significant cancer at biopsy., Results: The multivariable logistic regression model including THBS1, CTSD and %fPSA discriminated among biopsy-positive and biopsy-negative patients in the validation set with an area under the curve (AUC) of 0.86 (P < 0.001, 95% confidence interval (CI) 0.82-0.91), while %fPSA alone showed an AUC of 0.64 (P < 0.001, 95% CI 0.57-0.71). At 90% sensitivity for PCa, the specificity of the model was 62%, while %fPSA had a specificity of 23%. For high grade (Gleason score ≥ 7 in prostatectomy specimen) PCa, the specificity was 48% at 90% sensitivity, with an AUC of 0.83, (P < 0.001, 95% CI 0.77 to 0.88). Limitations of the study include the retrospective set-up and single-centre cohort., Conclusions: A model combining two cancer-related glycoproteins (THBS1 and CTSD) and %fPSA can improve PCa diagnosis and may reduce the number of unnecessary prostate biopsies because of its improved specificity for PCa when compared to %fPSA alone., (© 2018 The Authors BJU International © 2018 BJU International Published by John Wiley & Sons Ltd on behalf of BJU International.)

Background: The study aims to investigate the significance of predictors including HbA1c, Lipid, S. Creatinine, Adiponectin, Cat D, HSP70, HSP47, and 25-hydroxy vitamin D and to evaluate the rate of healing of ulcers among the diabetic foot patients., Methods: A retrospective method is used to quantitatively assess the role of HbA1c, Lipid, S. Creatinine, Adiponectin, Cat D, HSP70, HSP47, and 25-hydroxy vitamin D in the healing process of ulcer among diabetic patients. A total of 192 diabetic patients were included, who underwent ulcer healing diagnosis., Results: The results have shown a positive and significant correlation between Adiponectin and Cat D with group 2. The findings have shown higher prevalence of Cad D among patients with group 1, HSP70 among patients with group 2, HSP47 among patients with group 2, and 25-hydroxy vitamin D among patients with group 1., Conclusion: The study concluded that insignificant predictors should be assessed more comprehensively to reveal its efficacy on healing of ulcers with different grades., (Copyright © 2018 Diabetes India. Published by Elsevier Ltd. All rights reserved.)

Objective: The aim of this work was to assess baseline serum levels of established biomarkers related to inflammation and oxidative stress in samples from alkaptonuric subjects enrolled in SONIA1 (n = 40) and SONIA2 (n = 138) clinical trials (DevelopAKUre project)., Methods: Baseline serum levels of Serum Amyloid A (SAA), IL-6, IL-1β, TNFα, CRP, cathepsin D (CATD), IL-1ra, and MMP-3 were determined through commercial ELISA assays. Chitotriosidase activity was assessed through a fluorimetric method. Advanced Oxidation Protein Products (AOPP) were determined by spectrophotometry. Thiols, S-thiolated proteins and Protein Thiolation Index (PTI) were determined by spectrophotometry and HPLC. Patients' quality of life was assessed through validated questionnaires., Results: We found that SAA serum levels were significantly increased compared to reference threshold in 57.5% and 86% of SONIA1 and SONIA2 samples, respectively. Similarly, chitotriosidase activity was above the reference threshold in half of SONIA2 samples, whereas CRP levels were increased only in a minority of samples. CATD, IL-1β, IL-6, TNFα, MMP-3, AOPP, thiols, S-thiolated protein and PTI showed no statistically significant differences from control population. We provided evidence that alkaptonuric patients presenting with significantly higher SAA, chitotriosidase activity and PTI reported more often a decreased quality of life. This suggests that worsening of symptoms in alkaptonuria (AKU) is paralleled by increased inflammation and oxidative stress, which might play a role in disease progression., Conclusions: Monitoring of SAA may be suggested in AKU to evaluate inflammation. Though further evidence is needed, SAA, chitotriosidase activity and PTI might be proposed as disease activity markers in AKU., (Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: Coronary artery disease (CAD) cannot be sufficiently explained by the presence of traditional risk factors. Cathepsin D has been proposed to serve as a surrogate marker of atherosclerosis but its alterations in CAD patients have not been studied., Objective: To evaluate serum cathepsin D concentrations in relation to the presence and severity of CAD., Materials and Methods: A total of 104 subjects were recruited; 71 patients with suspected CAD and 33 healthy subjects. Thirty-four patients had >50% coronary stenosis of at least one artery (CAD+); the remaining 37 patients had <50% stenosis (CAD-) based on angiography. CAD+ patients were sub-divided into three sub-groups with single (SVD; n=15), double (2VD; n=9), and triple vessel (3VD; n=10) disease. Serum soluble cathepsin D concentrations were determined using an enzyme-linked immunosorbent assay (ELISA)., Results: Serum cathepsin D concentrations were significantly higher in the CAD+ compared with healthy control (p=0.016) but not CAD- group (p=0.098). Within the CAD+ group, patients with 3VD had significantly higher serum cathepsin D concentrations compared with the SVD group (p=0.025), and also compared with the CAD- (p=0.011) and SVD (p=0.001) groups. No significant associations were found between serum cathepsin D concentrations and potential confounders including age, sex, blood pressure, smoking history and dyslipidemia., Conclusion: Serum cathepsin D concentrations may be associated with the presence of CAD., (Copyright © 2018 Cardiological Society of India. Published by Elsevier B.V. All rights reserved.)

Prostate Cancer (PCa) diagnosis is currently hampered by the high false-positive rate of PSA evaluations, which consequently may lead to overtreatment. Non-invasive methods with increased specificity and sensitivity are needed to improve diagnosis of significant PCa. We developed and technically validated four individual immunoassays for cathepsin D (CTSD), intercellular adhesion molecule 1 (ICAM1), olfactomedin 4 (OLFM4), and thrombospondin 1 (THBS1). These glycoproteins, previously identified by mass spectrometry using a Pten mouse model, were measured in clinical serum samples for testing the capability of discriminating PCa positive and negative samples. The development yielded 4 individual immunoassays with inter and intra-variability (CV) <15% and linearity on dilution of the analytes. In serum, ex vivo protein stability (<15% loss of analyte) was achieved for a duration of at least 24 hours at room temperature and 2 days at 4°C. The measurement of 359 serum samples from PCa positive (n = 167) and negative (n = 192) patients with elevated PSA (2-10 ng/ml) revealed a significantly improved accuracy (P <0.001) when two of the glycoproteins (CTSD and THBS1) were combined with %fPSA and age (AUC = 0.8109; P <0.0001; 95% CI = 0.7673-0.8545). Conclusively, the use of CTSD and THBS1 together with commonly used parameters for PCa diagnosis such as %fPSA and age has the potential to improve the diagnosis of PCa.

Aim: Young onset type 2 diabetes patients (T2DM-Y) have been shown to possess an increased risk of developing microvascular complications particularly diabetic retinopathy. However, the molecular mechanisms are not clearly understood. In this study, we investigated the serum levels of monocyte chemotactic protein 1 (MCP-1) and cathepsin-D in patients with T2DM-Y without and with diabetic retinopathy., Methods: In this case-control study, participants comprised individuals with normal glucose tolerance (NGT=40), patients with type 2 diabetes mellitus (T2DM=35), non-proliferative diabetic retinopathy (NPDR=35) and proliferative diabetic retinopathy (PDR=35). Clinical characterization of the study subjects was done by standard procedures and MCP-1 and cathepsin-D were measured by ELISA., Results: Compared to control individuals, patients with T2DM-Y, NPDR and PDR exhibited significantly (p<0.001) higher levels of MCP-1. Cathepsin-D levels were also significantly (p<0.001) higher in patients with T2DM-Y without and with diabetic retinopathy. Correlation analysis revealed a positive association (p<0.001) between MCP-1 and cathepsin-D levels. There was also a significant negative correlation of MCP1/cathepsin-D with C-peptide levels. The association of increased levels of MCP-1/cathepsin-D in patients with DR persisted even after adjusting for all the confounding factors., Conclusion: As both MCP-1 and cathepsin-D are molecular signatures of cellular senescence, we suggest that these biomarkers might be useful to predict the development of retinopathy in T2DM-Y patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Alkaptonuria (AKU; OMIM:203500) is a classic Mendelian genetic disorder described by Garrod already in 1902. It causes urine to turn black upon exposure to air and also leads to ochronosis as well as early osteoarthritis. Our objective is the implementation of a Precision Medicine (PM) approach to AKU. We present here a novel ApreciseKUre database facilitating the collection, integration and analysis of patient data in order to create an AKU-dedicated "PM Ecosystem" in which genetic, biochemical and clinical resources can be shared among registered researchers. In order to exploit the ApreciseKUre database, we developed an analytic method based on Pearson's correlation coefficient and P value that generates as refreshable correlation matrix. A complete statistical analysis is obtained by associating every pair of parameters to examine the dependence between multiple variables at the same time., Short Conclusions: Employing this analytic approach, we showed that some clinically used biomarkers are not suitable as prognostic biomarkers in AKU for a more reliable patients' clinical monitoring. We believe this database could be a good starting point for the creation of a new clinical management tool in AKU, which will lead to the development of a deeper knowledge network on the disease and will advance its treatment. Moreover, our approach can serve as a personalization model paradigm for other inborn errors of metabolism or rare diseases in general.

Background: Cathepsin D has been recently implicated in insulin resistance and cardiovascular disease. This study was designed to investigate the relationship between cathepsin D and newly diagnosed type 2 diabetes., Methods: Circulating cathepsin D levels and metabolic variables were measured in 98 cases and 98 controls. Myocardial performance index "Tei index" that reflects both left ventricular systolic and diastolic function was measured with Doppler echocardiography in cases., Results: Newly diagnosed type 2 diabetes demonstrated significantly higher circulating cathepsin D concentrations than controls (median level: 227 ng/ml versus 174 ng/ml, P < 0.01). In newly diagnosed type 2 diabetes, a significant correlation was found between cathepsin D levels and HOMA-IR (homeostatic model assessment of insulin resistance) ( r = 0.25, P = 0.01). In contrast, no significant correlation was found between cathepsin D levels and clinical parameters in the control group (all P > 0.05). Interestingly, correlation analysis revealed a positive association between cathepsin D levels and Tei index in type 2 diabetes ( r = 0.22, P = 0.03)., Conclusions: Increased levels of circulating cathepsin D are closely linked with the presence of type 2 diabetes, and cathepsin D might serve as a novel biomarker for cardiac dysfunction in newly diagnosed type 2 diabetes.

Non-alcoholic steatohepatitis (NASH) is characterized by liver lipid accumulation and inflammation. The mechanisms that trigger hepatic inflammation are poorly understood and subsequently, no specific non-invasive markers exist. We previously demonstrated a reduction in the plasma lysosomal enzyme, cathepsin D (CatD), in children with NASH compared to children without NASH. Recent studies have raised the concept that non-alcoholic fatty liver disease (NAFLD) in adults is distinct from children due to a different histological pattern in the liver. Yet, the link between plasma CatD to adult NASH was not examined. In the current manuscript, we investigated whether plasma CatD in adults correlates with NASH development and regression. Biopsies were histologically evaluated for inflammation and NAFLD in three complementary cohorts of adults (total n = 248). CatD and alanine aminotransferase (ALT) were measured in plasma. Opposite to our previous observations with childhood NASH, we observed increased levels of plasma CatD in patients with NASH compared to adults without hepatic inflammation. Furthermore, after surgical intervention, we found a reduction of plasma CatD compared to baseline. Our observations highlight a distinct pathophysiology between NASH in children and adults. The observation that plasma CatD correlated with NASH development and regression is promising for NASH diagnosis.

The local endocrine environment of the breast may have stronger relations to breast cancer risk than systemic hormones. Nipple aspiration fluid (NAF) provides a window into this milieu. We hypothesized that the correlations between proteins and steroid hormones in NAF are stronger, and specific relationships may reveal links to breast cancer risk. NAF and blood samples were obtained simultaneously from 54 healthy women and from the contralateral unaffected breast of 60 breast cancer patients. The abundance of five proteins, superoxide dismutase (SOD1), C-reactive protein (CRP), chitinase-3-like protein 1 (YKL40), cathepsin D (CatD), and basic fibroblast growth factor (bFGF) in NAF was measured using ELISA. The NAF and serum concentrations of estradiol, estrone, progesterone, androstenedione, testosterone, and dehydroepiandrostrerone (DHEA) were measured using ELISA or RIA. The correlations between proteins and hormones revealed that NAF proteins correlated with each other: SOD1 with CRP (R = 0.276, P = 0.033) and CatD (R = 0.340, P = 0.0036), and bFGF with CRP (R = 0.343, P = 0.0021). NAF proteins displayed significant correlations with NAF steroids, but not with serum steroids: SOD1 with DHEA (R = 0.333, P = 0.019), YKL40 with testosterone (R = 0.389, P = 0.0012), and bFGF negatively correlated with testosterone (R = -0.339, P = 0.015). The regulation of YKL40 and bFGF by testosterone was confirmed in breast cancer cell lines. In summary, NAF proteins were more strongly related to local hormone levels than to systematic hormone levels. Some proteins were specifically correlated with different NAF steroids, suggesting that these steroids may contribute to breast cancer risk through different mechanisms.

Aim: Nowadays, cathepsins have been reported to be related to aging. The aim of this study is to evaluate the association between serum levels of cathepsin D (CTSD) and human aging., Methods: In the present study, we analyzed the serum levels of CTSD and its relation with levels of sirtuin1 (SIRT1) and endothelial nitric oxide synthase (eNOS) activity, which were known having an important role in aging. This study recruited 90 healthy subjects (62 men and 28 women), which were subdivided into three groups with respect to age: young (about 19 years old, n = 30), middle-age (about 40 years old, n = 30), and aged (above 65 years old, n = 30). Altered serum levels of CTSD and SIRT1 were measured by enzyme-linked immunosorbent assay, and eNOS activity was assessed by the conversion of 14(C)-L-arginine to 14(C)-L-citrulline., Results: Elderly subjects had significantly lower CTSD, SIRT1, and eNOS than younger ones. Serum levels of CTSD were negatively correlated with age. There was a statistically significant positive correlation between serum levels of CTSD, eNOS, and SIRT1., Conclusions: This study shows lower serum CTSD values in elderly subjects than in younger subjects. This is the first to demonstrate age-related changes in cathepsin D levels in humans and the association between SIRT1 and eNOS.

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). We have previously reported increased monocyte (Mono) trafficking into the retinas of diabetic animals. In this study, we have examined the effect of activated Monos on retinal endothelial cells (ECs). The U937 Mϕ-conditioned medium (CM) significantly decreased the transendothelial resistance of EC monolayers as measured by electric cell-substrate impedance sensing (P= 0.007). The CM was fractioned, and the effective fraction (30-100 kDa) was analyzed by liquid chromatography-mass spectrometry, and cathepsin D (CD) was identified as a major secreted product. Immunoprecipitated CD resulted in decreased resistance in ECs (P= 0.006). The specificity of CD in mediating alterations of the EC barrier was confirmed using small interfering RNA. The decreased resistance correlated with a significantly increased gap between ECs. CD altered the Ras homolog gene family, member A/Rho-associated kinase pathway with increased stress actin filament formation in the EC layer. Increased CD levels were found in the retinas of diabetic mice (3-fold) and serum samples of patients with diabetic macular edema (1.6-fold) measured by Western blot and ELISA. These findings suggest an important role for Mϕ-derived CD in altering the blood-retinal barrier and reveal a potential therapeutic target in the treatment of DR.-Monickaraj, F., McGuire, P. G., Nitta, C. F., Ghosh, K., Das, A. Cathepsin D: an Mϕ-derived factor mediating increased endothelial cell permeability with implications for alteration of the blood-retinal barrier in diabetic retinopathy., (© FASEB.)

Background: The diagnostic and prognostic significance of increased cathepsin B (CTSB) and cathepsin D (CTSD) concentration in the serum of cancer patients were evaluated for some tumor types. High expression of CTSD and CTSB was detected in biopsy tissues from nasopharyngeal carcinoma (NPC). However, whether CTSD and CTSB serve as diagnostic and prognostic markers of NPC remains unclear., Methods: Serum samples were collected from 40 healthy volunteers and 80 NPC patients enrolled in the study. CTSB and CTSD in the serum samples were detected using enzyme-linked immunosorbent assay (ELISA). Concomitantly, the relationship between CTSB and CTSD concentrations and clinicopathological prognosis was assessed. The sensitivity and specificity of the two components in the diagnosis of NPC were evaluated in 80 NPC patients., Results: ELISA analysis showed that in the sera obtained from NPC patients, the CTSB concentration was 12.5 ± 3.5 mg/L (median, 12.4 mg/L), and the CTSD concentration was 15.7 ± 8.7 mg/L (median, 14.7 mg/L). CTSB and CTSD levels were significantly higher in the NPC patient population compared to the healthy control population (p = 0.001; p = 0.001, respectively). The presence of CTSB and CTSD in the serum of the patients with NPC correlated with the tumor node metastasis (TNM) scores (p = 0.001). Other parameters were not identified to be of significance. Receiver operating characteristic (ROC) analysis showed that a cut off CTSB concentration of 12.4 mg/L had 61.9% sensitivity and 63.2% specificity in the prediction of progression-free survival (Area under the curve (AUC) = 0.525; 95% CI, 39.7-65.2; p = 0.704); whereas a cut off CTSD concentration of 14.7 mg/L had 66.7% sensitivity, and 58.5% specificity (AUC = 0.552; 95% CI, 42.3-68.1; p = 0.42)., Conclusions: Serum CTSB and CTSD concentrations were found to have a diagnostic value in NPC. However, the CTSB and CTSD serum levels had no prognostic role for the outcome in NPC patients.

Insulin resistance (IR) is a precursor of type 2 diabetes (T2D), and improved risk prediction and understanding of the pathogenesis are needed. We used a novel high-throughput 92-protein assay to identify circulating biomarkers for HOMA of IR in two cohorts of community residents without diabetes (n = 1,367) (mean age 73 ± 3.6 years). Adjusted linear regression identified cathepsin D and confirmed six proteins (leptin, renin, interleukin-1 receptor antagonist [IL-1ra], hepatocyte growth factor, fatty acid-binding protein 4, and tissue plasminogen activator [t-PA]) as IR biomarkers. Mendelian randomization analysis indicated a positive causal effect of IR on t-PA concentrations. Two biomarkers, IL-1ra (hazard ratio [HR] 1.28, 95% CI 1.03-1.59) and t-PA (HR 1.30, 1.02-1.65) were associated with incident T2D, and t-PA predicted 5-year transition to hyperglycemia (odds ratio 1.30, 95% CI 1.02-1.65). Additional adjustment for fasting glucose rendered both coefficients insignificant and revealed an association between renin and T2D (HR 0.79, 0.62-0.99). LASSO regression suggested a risk model including IL-1ra, t-PA, and the Framingham Offspring Study T2D score, but prediction improvement was nonsignificant (difference in C-index 0.02, 95% CI -0.08 to 0.12) over the T2D score only. In conclusion, proteomic blood profiling indicated cathepsin D as a new IR biomarker and suggested a causal effect of IR on t-PA., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Aim: Increased serum levels of the activated aspartic lysosomal endopeptidase cathepsin D (CatD) have been found in patients with acute myocardial infarction (AMI). However, to date there have been no analyses of clinical follow-up data measuring the enzyme course and its role in the development of post-MI heart failure. This study aimed to evaluate the role of serum CatD activity in the development of heart failure in patients with ST-segment elevation acute myocardial infarction (STEMI)., Patients and Methods: Eighty-eight consecutive patients (79.5 % men, mean age 57.4 ± 10.2 years) with STEMI were included in this study. Serum CatD activity was measured directly after primary percutaneous coronary intervention (PCI), before discharge, and at the 6-month follow-up. Patients were monitored for major adverse cardiovascular events (MACE), defined as hospitalization due to cardiovascular causes, recurrent nonfatal myocardial infarction, unplanned PCI, new-onset heart failure, and cardiovascular mortality., Results: Serum CatD activity was significantly higher in patients with AMI after PCI and during follow-up (FU) than that in age-matched controls (16.2 ± 7.5 and 29.8 ± 8.9 vs. 8.5 ± 4.2 RFU; p < 0.001 for each time point). At the 6-month follow-up, serum CatD activity in these patients was inversely related to new-onset cardiac dysfunction compared with patients with preserved and improved LVEF after treatment (23.1 ± 3.2 vs. 28.8 ± 7.0 and 29.7 ± 5.0 RFU respectively, p < 0.01). Patients suffering from MACE during a follow-up period of 6 months had lower serum levels of activated CatD than those without any MACE (23.8 ± 4.6 vs 29.6 ± 6.9 RFU; p < 0.001)., Conclusions: Serum CatD activity as a marker of healthy endogenous phagocytosis and remodeling was impaired in patients with new-onset cardiac dysfunction, and lower levels of serum CatD were associated with MACE at the 6-month post-MI follow-up.

Objective: Diverse autolysosomal proteins were quantified in neurally derived blood exosomes from patients with Alzheimer disease (AD) and controls to investigate disordered neuronal autophagy., Methods: Blood exosomes obtained once from patients with AD (n = 26) or frontotemporal dementia (n = 16), other patients with AD (n = 20) both when cognitively normal and 1 to 10 years later when diagnosed, and case controls were enriched for neural sources by anti-human L1CAM antibody immunoabsorption. Extracted exosomal proteins were quantified by ELISAs and normalized with the CD81 exosomal marker., Results: Mean exosomal levels of cathepsin D, lysosome-associated membrane protein 1 (LAMP-1), and ubiquitinylated proteins were significantly higher and of heat-shock protein 70 significantly lower for AD than controls in cross-sectional studies (p ≤ 0.0005). Levels of cathepsin D, LAMP-1, and ubiquitinylated protein also were significantly higher for patients with AD than for patients with frontotemporal dementia (p ≤ 0.006). Step-wise discriminant modeling of the protein levels correctly classified 100% of patients with AD. Exosomal levels of all proteins were similarly significantly different from those of matched controls in 20 patients 1 to 10 years before and at diagnosis of AD (p ≤ 0.0003)., Conclusions: Levels of autolysosomal proteins in neurally derived blood exosomes distinguish patients with AD from case controls and appear to reflect the pathology of AD up to 10 years before clinical onset. These preliminary results confirm in living patients with AD the early appearance of neuronal lysosomal dysfunction and suggest that these proteins may be useful biomarkers in large prospective studies., (© 2015 American Academy of Neurology.)

Objective: To assess the activity of arylsulfatase (AS), acid phosphatase (ACP), cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood serum and synovial fluid (SF) of patients with hip or knee osteoarthritis (OA)., Methods: The study included 43 subjects with OA (35 hip OA, 8 knee OA), submitted total joint replacement. The control group consisted of 58 subjects with no past history of musculoskeletal disorders., Results: The OA blood serum samples showed a significantly higher level of lysosomal enzymes activity than in the control group (AS by 17.8%, AAT by 42.4%); only the CAT-D activity decreased by 50%). AS, ACP and CAT-D activities were about two-fold higher in SF when compared with blood of OA patients. The differences between the genders were visible in the SF: Total protein concentration, activity of ACP (both higher in OA men) and activity of CAT-D (higher in OA women). Between the involved hip and knee, there were no significant differences in all estimated parameters in the blood serum of the OA group. In regard to the SF, only ACP activity was significantly higher in patients with a hip involved., Conclusions: The osteoarthritic SF enzymatic profile differs from that in normal joints. The OA in joints is not reflected in the systemic response. Our preliminary results suggest further studies on role of lysosomal enzymes (ACP and AS) as biomarkers for the detection of osteoarthritis.

Objectives: Nonalcoholic steatohepatitis (NASH) is the most severe form of a hepatic condition known as nonalcoholic fatty liver disease (NAFLD). NASH is histologically characterized by hepatic fat accumulation, inflammation, and ballooning, and eventually coupled with fibrosis that, in turn, may progress to end-stage liver disease even in young individuals. Hence, there is a critical need for specific noninvasive markers to predict hepatic inflammation at an early age. We investigated whether plasma levels of cathepsin D (CatD), a lysosomal protease, correlated with the severity of liver inflammation in pediatric NAFLD., Methods: Liver biopsies from children (n=96) with NAFLD were histologically evaluated according to the criteria of Kleiner (NAFLD activity score) and the Brunt's criteria. At the time of liver biopsy, blood was taken and levels of CatD, alanine aminotransferase (ALT), and cytokeratin-18 (CK-18) were measured in plasma., Results: Plasma CatD levels were significantly lower in subjects with liver inflammation compared with steatotic subjects. Furthermore, we found that CatD levels were gradually reduced and corresponded with increasing severity of liver inflammation, steatosis, hepatocellular ballooning, and NAFLD activity score. CatD levels correlated with pediatric NAFLD disease progression better than ALT and CK-18. In particular, CatD showed a high diagnostic accuracy (area under receiver operating characteristic curve (ROC-AUC): 0.94) for the differentiation between steatosis and hepatic inflammation, and reached almost the maximum accuracy (ROC-AUC: 0.998) upon the addition of CK-18., Conclusions: Plasma CatD holds a high diagnostic value to distinguish pediatric patients with hepatic inflammation from children with steatosis.

We aimed to investigate the role of cathepsin D, an inflammatory and atherosclerotic mediator, in endothelial dysfunction in chronic kidney disease. The study included 65 patients with stage 2&ndash;4 chronic kidney disease (35 females, 30 males; mean age, 55.8&plusmn;15.6 years). Serum creatinine and cathepsin D levels and glomerular filtration rates (GFRs) were determined, and brachial flow-mediated dilation (FMD) percentage was measured by two-dimensional gray scale and color flow Doppler and vascular imaging. FMD &le;6% was considered to indicate endothelial dysfunction. Mean GFR, median creatinine levels, and median cathepsin D levels were 40.2&plusmn;11.2mL/min/1.73m2, 1.7mg/dL, and 819.75ng/mL, respectively. Endothelial dysfunction was present in 30 of the 65 patients (46.2%). There was a significant difference between groups with and without endothelial dysfunction in terms of cathepsin D (p=0.001) and creatinine (p=0.03) levels, and negative and significant correlations were found between brachial artery FMD% and cathepsin D (r=&minus;0.359, p=0.003) and creatinine (r=&minus;0.304, p=0.014) levels. Cathepsin D, which is known to be associated with atherosclerosis, may play a role in the process of endothelial dysfunction. Further studies are essential to determine the exact function of cathepsin D in endothelial dysfunction in chronic kidney disease and to determine its value as a tool for early diagnosis and target for treatment of cardiovascular diseases in patients with chronic kidney disease.

The activity of α1-antitrypsin (AAT) and the lysosomal enzymes, cathepsin D (CTS D), arylsulfatase (ASA), and acid phosphatase, (AcP) was determined in patients with COPD (GOLD category A). Moreover, the diagnostic usefulness of these parameters in blood serum was assessed along with establishing whether smoking cessation affects these parameters. The study included 70 patients with COPD who ceased smoking (study group) and two control groups of 33 subjects each: nonsmokers without COPD (control I) and patients with COPD who continued smoking (control II). In control I, blood was taken once and in control II, at the start of the experiment and after the 1st, 2nd, and 3rd months. AAT in the patients exhibited higher activity than in the healthy subjects at all time points. AAT activity in the patients before the start of the experiment was ~80% higher (P < 0.001) than in control I. No statistically significant differences in CTS D, ASA, and AcP activity were found. COPD involves increased AAT activity and unchanged activities of the assessed lysosomal enzymes. Three-month tobacco abstinence does not affect these parameters in peripheral blood. Determining the AAT levels in blood serum can be used in the diagnostics of COPD.

Objective: To assess the activity of cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood in patients with hip or knee osteoarthritis, and to explore whether these two enzymes could be served as serum biomarkers for cartilage degeneration., Methods: Th e activity of CAT-D and AAT in blood serum of 44 women and 26 men with hip or knee osteoarthritis was determined by the method of ELISA before total joint replacement and on the 10th day aft er the surgery. One hundred healthy volunteers were chosen as the control. All data were analyzed by using SPSS19.0 software., Results: Compared with the controls, the activity of CAT-D in patients with osteoarthritis was decreased by 25% (P<0.05) and 50% (P<0.05) before and after the surgery, respectively. The activity of AAT in the osteoarthritis patients before the surgery was not significantly changed compared with the control group (P>0.05), but it was increased by 80% after the surgery than that in the control group (P<0.05). There was no significant difference in the activities of 2 enzymes between hip and knee osteoarthritis (P>0.05). The gender, hypertension, diabetes and age did not affect the activities of the 2 enzymes (P>0.05)., Conclusion: AAT might be a possible inflammatory indicator in the osteoarthritis. CAT-D and AAT enzymes are not affected by gender, age, hypertension and diabetes, etc, and they might be served as potential biomarkers for cartilage degradation.

Unlabelled: The key role of cathepsin D and B is intralysosomal digestion of used cellular proteins and other proteins that enter cells through endocytosis. Under pathological conditions like cancer formation and growth, cathepsins from lysosomes are released. The aim of the study was to determin of cathepsin D and B activities in serum of patients with urothelial bladder cancer depending on disease severity and determination of its' changes after transurethral resection of tumor., Material and Methods: Experiment involved 50 patients. Blood samples were obtained from 18 healthy volunteers and 32 urothelial bladder cancer patients. Samples from people with suspected urothelial bladder cancer were collected three times: before the surgery, 2 weeks and 6 weeks after the surgical treatment., Results: Our research showed that cathepsin D activity, measured as the level increment of acid soluble tyrosine, is the highest before the surgery in muscle invasive bladder tumor (pT2) (57,9 nmol/ml). 2 weeks and 6 weeks after the surgical treatment, cathepsin D activity is decreased. In case of cathepsin B activity, measured as the level of released p-nitroaniline, decreased, 2 weeks and 6 weeks after the surgical treatment in both cases of disease severity., Conclusion: Cathepsin D and B activities in the serum of patients with urothelial bladder cancer are directly proportional to disease severity and significantly higher compared with control group. Transuretral resection of the tumor leads to diminution of their activities in second and 6th week after the procedure.