20S constitutive proteasome, 20S immunoproteasome, and cathepsin S are high-sensitivity and independent markers of immunological activity in relapsing-remitting type of multiple sclerosis.

Research on the markers of autoimmune response in multiple sclerosis (MS) is still of great importance. The aim of our study was the evaluation of plasma 20S constitutive proteasome, 20S immunoproteasome, and cathepsin S concentrations as potential biomarkers of a relapsing-remitting type of MS (RRMS). Surface plasmon resonance imaging (SPRI) biosensors were used for the evaluation of protein concentrations. Plasma 20S constitutive proteasome, 20S immunoproteasome, and cathepsin S concentrations were significantly higher in RRMS patients compared to the control group. All three parameters were characterized by excellent usefulness in differentiating MS patients from healthy individuals (AUC equal to or close to 1.000). The plasma concentration of analyzed parameters was not correlated with severity of disability in the course of RRMS (EDSS value), the number of years from the first MS symptoms, the number of years from MS diagnosis, or the number of relapses within the 24-month observational period. Our study has shown that plasma concentrations of 20S constitutive proteasome, 20S immunoproteasome, and cathepsin S have promising potential in differentiating RRMS patients from healthy individuals. All of the analyzed parameters were found to be independent of the time of MS relapse and the severity of neurological symptoms. Hence, their potential as highly sensitive and independent circulating markers of RRMS suggests a stronger association with immunological activity (inflammatory processes) than with the severity of the disease.

Application of Surface Plasmon Resonance Imaging Biosensors for Determination of Fibronectin, Laminin-5, and Type IV Collagen in Plasma, Urine, and Tissue of Renal Cell Carcinoma.

Laminin, fibronectin, and collagen IV are pivotal extracellular matrix (ECM) components. The ECM environment governs the fundamental properties of tumors, including proliferation, vascularization, and invasion. Given the critical role of cell-matrix adhesion in malignant tumor progression, we hypothesize that the concentrations of these proteins may be altered in the plasma of patients with clear cell renal cell carcinoma (ccRCC). This study aimed to evaluate the serum, urine, and tissue levels of laminin-5, collagen IV, and fibronectin among a control group and ccRCC patients, with the latter divided into stages T1-T2 and T3-T4 according to the TNM classification. We included 60 patients with histopathologically confirmed ccRCC and 26 patients diagnosed with chronic cystitis or benign prostatic hyperplasia (BPH). Collagen IV, laminin-5, and fibronectin were detected using Surface Plasmon Resonance Imaging biosensors. Significant differences were observed between the control group and ccRCC patients, as well as between the T1-T2 and T3-T4 subgroups. Levels were generally higher in plasma and tissue for fibronectin and collagen IV in ccRCC patients and lower for laminin. The ROC (Receiver operating characteristic) analysis yielded satisfactory results for differentiating between ccRCC patients and controls (AUC 0.84-0.93), with statistical significance for both fibronectin and laminin in plasma and urine. Analysis between the T1-T2 and T3-T4 groups revealed interesting findings for all examined substances in plasma (AUC 0.8-0.95). The results suggest a positive correlation between fibronectin and collagen levels and ccRCC staging, while laminin shows a negative correlation, implying a potential protective role. The relationship between plasma and urine concentrations of these biomarkers may be instrumental for tumor detection and staging, thereby streamlining therapeutic decision-making.

SPRi Biosensor for Simultaneous Determination of HIF-1α, Angiopoietin-2, and Interleukin-1ß in Blood Plasma.

A new analytical method, based on SPRi biosensors, has been developed for the simultaneous determination of the pro-angiogenic factors HIF-1α, angiopoietin-2 (ANG-2), and interleukin-1ß (IL-1ß) in biological fluids. These proteins take part in the process of angiogenesis, i.e., the creation of new blood vessels, which is a key stage of cancer development and metastasis. A separate validation process was carried out for each individual compound, indicating that the method can also be used to study one selected protein. Low values of the limit of detection (LOD) and quantification (LOQ) indicate that the developed method enables the determination of very low concentrations, in the order of pg/mL. The LOD values obtained for HIF-1α, ANG-2, and IL-1ß were 0.09, 0.01, and 0.01 pg/mL, respectively. The LOQ values were 0.27, 0.039, and 0.02 pg/mL, and the response ranges of the biosensor were 5.00-100.00, 1.00-20.00, and 1.00-15.00 pg/mL. Moreover, determining the appropriate validation parameters confirmed that the design offers high precision, accuracy, and sensitivity. To prove the usefulness of the biosensor in practice, determinations were made in plasma samples from a control group and from a study group consisting of patients with diagnosed bladder cancer. The preliminary results obtained indicate that this biosensor can be used for broader analyses of bladder cancer. Each of the potential biomarkers, HIF-1α, ANG-2, and IL-1ß, produced higher concentrations in the study group than in the control group. These are preliminary studies that serve to develop hypotheses, and their confirmation requires the analysis of a larger number of samples. However, the constructed biosensor is characterized by its ease and speed of measurement, and the method does not require special preparation of samples. SPRi biosensors can be used as a sensitive and highly selective method for determining potential blood biomarkers, which in the future may become part of the routine diagnosis of cancers.

Changes in the Concentrations of Proangiogenic Cytokines in Human Brain Glioma and Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) and glioma are some of the most common malignancies, with ALL most often affecting children and glioma affecting adult men. Proangiogenic cytokines and growth factors play an important role in the development of both of these tumors. Glioma is characterized by an extremely extensive network of blood vessels, which continues to expand mainly in the process of neoangiogenesis, the direct inducers of which are cytokines from the family of vascular endothelial growth factors, i.e., vascular endothelial growth factor (VEGF-A) and its receptor vascular endothelial growth factor receptor 2 (VEGF-R2), as well as a cytokine from the fibroblast growth factor family, fibroblast growth factor 2 (FGF-2 or bFGF). Growth factors are known primarily for their involvement in the progression and development of solid tumors, but there is evidence that local bone marrow angiogenesis and increased blood vessel density are also present in hematological malignancies, including leukemias. The aim of this study was to examine changes in the concentrations of VEGF-A, VEGF-R2, and FGF-2 (with a molecular weight of 17 kDa) in a group of patients divided into specific grades of malignancy (glioma) and a control group; changes of VEGF-A and FGF-2 concentrations in childhood acute lymphoblastic leukemia and a control group; and to determine correlations between the individual proteins as well as the influence of the patient's age, diet, and other conditions that may place the patient in the risk group. During the statistical analysis, significant differences in concentrations were found between the patient and control groups in samples from people with diagnosed glioma and from children with acute lymphoblastic leukemia, but in general, there are no significant differences in the concentrations of VEGF-A, VEGF-R2, and FGF-2 between different grades of glioma malignancy. Among individuals treated for glioma, there was no significant impact from the patient's gender and age, consumption of food from plastic packaging, frequency of eating vegetables and fruit, smoking of tobacco products, the intensity of physical exercise, or the general condition of the body (Karnofsky score) on the concentrations of the determined cytokines and receptor. The listed factors do not bring about an actual increase in the risk of developing brain glioma.

Comparison of Fluidic and Non-Fluidic Surface Plasmon Resonance Biosensor Variants for Angular and Intensity Modulation Measurements.

Fluidic and non-fluidic surface plasmon resonance measurements were realized for the same type of sensory layer and using the same mouse IgG antibody and anti-mouse IgG antibody biomolecular system. A comparison of the thicknesses of the anti-mouse IgG antibody layers bound to the ligand at increasing analyte concentrations ranging from 0.0 µg mL-1 to 5.0 µg mL-1 in the non-fluidic and the fluidic variant showed that the thickness of the bound anti-mouse antibody layers in the fluidic variant was approximately 1.5-3 times larger than in the non-fluidic variant. The greater thicknesses of the deposited layers were also reflected in the larger increment of the resonant angle in the fluidic variant compared to the non-fluidic variant in the considered range of analyte concentrations. The choice between fluidic and non-fluidic surface plasmon resonance biosensors may be justified by the availability of analyte volume and the intended modulation technique. When working with limited analyte, non-fluidic biosensors with intensity modulation are more advantageous. For larger analyte quantities, fluidic biosensors with angular modulation are recommended, primarily due to their slightly higher sensitivity in this measurement mode.

New Biosensor for Determination of Neuropilin-1 with Detection by Surface Plasmon Resonance Imaging.

Neuropilin-1 is transmembrane protein with soluble isoforms. It plays a pivotal role in both physiological and pathological processes. NRP-1 is involved in the immune response, formation of neuronal circuits, angiogenesis, survival and migration of cells. The specific SPRI biosensor for the determination of neuropilin-1 was constructed using mouse monoclonal antibody that captures unbound NRP-1 form body fluids. The biosensor exhibits linearity of the analytical signal between 0.01 and 2.5 ng/mL, average precision value 4.7% and recovery between 97% and 104%. The detection limit is 0.011 ng/mL, and the limit of quantification is 0.038 ng/mL. The biosensor was validated by parallel determination of NRP-1 in serum and saliva samples using the ELISA test, with good agreement of the results.

An Array SPRi Biosensor for the Determination of Follicle-Stimulating Hormone in Blood Plasma.

Follicle-stimulating hormone (FSH) regulates the development, growth, pubertal maturation and reproductive processes of the human body. The determination of serous FSH concentration is significant as an alternative to testicular biopsy in the case of boys suffering from cryptorchidism after orchidopexy, and as a means of determining the menopausal stage in women. The aim of this investigation is to develop a specific array surface plasmon resonance imaging (SPRi) biosensor for the determination of FSH in body liquids such as blood plasma, obtaining sufficient sensitivity to determine FSH at levels characteristic for that hormone in blood plasma, without any signal enhancement. The biosensor consists of a mouse monoclonal anti-FSH antibody attached to the gold surface of a chip via a cysteamine linker. Its linear response range is from 0.08 mIU mL-1 (LOQ) to 20 mIU mL-1, and well covers most of the range of FSH activities found in blood without dilution. The precision of measurement is between 3.2% and 13.1% for model samples, and between 3.7% and 5.6% for spiked plasma samples. Recoveries are in the range from 94% to 108%. The biosensor has good selectivity, and is validated by comparison with ECLE, with good agreement of the results.

A New Approach to the Quantification of Fibroblast Growth Factor 23-An Array Surface Plasmon Resonance Imaging Biosensor.

A new biosensor based on the "surface plasmon resonance imaging (SPRi)" detection technique for the quantification of "fibroblast growth factor 23 (FGF23)" has been developed. FGF23 is mainly produced in bone tissues as a phosphaturic hormone that forms a trimeric complex with "fibroblast growth factor receptor 1 (FGFR1)" and αKlotho upon secretion. FGF23 stimulates phosphate excretion and inhibits the formation of active vitamin D in the kidneys. FGF23 has been shown to play a role in bone carcinogenesis and metastasis. The newly developed method, based on the array SPRi biosensor, was validated-the precision, accuracy, and selectivity were acceptable, and yielded less than ±10% recovery. The rectilinear response of the biosensor ranges from 1 to 75 pg/mL. The limit of detection was 0.033 pg/mL, and the limit of quantification was 0.107 pg/mL. The biosensor was used to determine FGF23 concentrations in the blood plasma of healthy subjects and patients with "clear cell" renal cell carcinoma (ccRCC). The obtained results were compared with those measured through an "enzyme-linked immunosorbent assay (ELISA)". The determined Pearson correlation coefficients were 0.994 and 0.989, demonstrating that the newly developed biosensor can be used as a competitive method for the ELISA.

Development of an SPRi Test for the Quantitative Detection of Cadherin 12 in Human Plasma and Peritoneal Fluid.

A new method for the determination of cadherin 12 (CDH12)-an adhesive protein that has a significant impact on the development, growth, and movement of cancer cells-was developed and validated. The method is based on a biosensor using surface plasmon resonance imaging (SPRi) detection. A quartz crystal microbalance was used to analyze the characteristics of the formation of successive layers of the biosensor, from the linker monolayer to the final capture of CDH12 from solution. The association equilibrium constant (KA = 1.66 × 1011 dm3 mol-1) and the dissociation equilibrium constant (KD = 7.52 × 10-12 mol dm-3) of the anti-CDH12 antibody-CDH12 protein complex were determined. The determined analytical parameters, namely the values determining the accuracy, precision, and repeatability of the method, do not exceed the permissible 20% deviations specified by the aforementioned institutions. The proposed method is also selective with respect to possible potential interferents, occurring in up to 100-fold excess concentration relative to the CDH12 concentration. The determined Limit of Quantification (LOQ = 4.92 pg mL-1) indicates the possibility of performing quantitative analysis in human plasma or peritoneal fluid without the need to concentrate the samples; however, particular attention should be paid to their storage conditions, as the analyte does not exhibit high stability. The Passing-Bablok regression model revealed good agreement between the reference method and the SPRi biosensor, with ρSpearman values of 0.961 and 0.925.

Evaluation of Proteasome and Immunoproteasome Levels in Plasma and Peritoneal Fluid in Patients with Endometriosis.

Endometriosis is a chronic disease in which the endometrium cells are located outside the uterine cavity. The aim of this study was to evaluate circulating 20S proteasome and 20S immunoproteasome levels in plasma and peritoneal fluid in women with and without endometriosis in order to assess their usefulness as biomarkers of disease. Concentrations were measured using surface plasmon resonance imaging biosensors. Patients with suspected endometriosis were included in the study-plasma was collected in 112 cases and peritoneal fluid in 75. Based on the presence of endometriosis lesions detected during laparoscopy, patients were divided into a study group (confirmed endometriosis) and a control group (patients without endometriosis). Proteasome and immunoproteasome levels in both the plasma (p = 0.174; p = 0.696, respectively) and the peritoneal fluid (p = 0.909; p = 0.284, respectively) did not differ between those groups. There was a statistically significant difference in the plasma proteasome levels between patients in the control group and those with mild (Stage I and II) endometriosis (p = 0.047) and in the plasma immunoproteasome levels in patients with ovarian cysts compared to those without (p = 0.017). The results of our study do not support the relevance of proteasome and immunoproteasome determination as biomarkers of the disease but suggest a potentially active role in the pathogenesis of endometriosis.

An Immunosensor for the Determination of Cortisol in Serum and Saliva by Array SPRi.

Cortisol is a hormone which plays an essential role in the immune, endocrine, cardiovascular, renal and skeletal systems. Its level increases in response to stress, illness, injury or exhaustion, and it is therefore a significant diagnostic biomarker of stress. An immunosensor for the determination of cortisol by SPRi array was developed. The receptive part of the immunosensor is mouse monoclonal antibody against cortisol, immobilized via cysteamine linker. The optimum pH of the immunosensor is 7.4, and the optimum concentration of the antibody is 50 ng mL-1. The immunosensor is specific for cortisol, and its linear response ranges from 0.20 ng mL-1 (LOQ) to 8 ng mL-1. The precision of the determination was between 3.1% and 3.3%, and the recovery between 99% and 102%. The immunosensor was validated by simultaneous determination of cortisol in serum and saliva samples by a standard method, with good agreement between the results.

Plasma and Peritoneal Fluid Fibronectin and Collagen IV Levels as Potential Biomarkers of Endometriosis.

Laparoscopy as a diagnostic tool for patients with suspected endometriosis is associated with several potentially life-threatening complications. Therefore, it is imperative to identify reliable, non-invasive biomarkers of the disease. The aim of this study was to analyse the concentrations of fibronectin and type IV collagen in peritoneal fluid and plasma to assess their role as potential biomarkers in the diagnosis of endometriosis. Fibronectin and collagen IV protein levels were assessed by surface plasmon resonance imaging (SPRi) biosensors with the usage of monoclonal antibodies. All patients enrolled in the study were referred for laparoscopy for the diagnosis of infertility or chronic pelvic pain (n = 84). The study group included patients with endometriosis confirmed during surgery (n = 49). The concentration of fibronectin in the plasma (329.3 ± 98.5 mg/L) and peritoneal fluid (26.8 ± 11.1 µg/L) in women with endometriosis was significantly higher than in the control group (251.2 ± 84.0 mg/L, 7.0 ± 5.9 µg/L). Fibronectin levels were independent of endometriosis stage (p = 0.874, p = 0.469). No significant differences were observed in collagen IV levels (p = 0.385, p = 0.465). The presence of elevated levels of fibronectin may indicate abnormalities in cell-ECM signalling during the course of endometriosis, and may be a potential biomarker for early detection.

Two Biosensors for the Determination of VEGF-R2 in Plasma by Array SPRi.

Vascular endothelial growth factor receptor 2 (VEGF-R2) is a marker of angiogenesis and metastasis of cancer. Two biosensors for the determination of VEGF-R2 in plasma have been developed. One of them is based on a pure gold chip, and the other on a silver/gold bimetallic chip; both have the receptor, monoclonal rabbit antibody specific for human VEGF-R2, attached to the chip via a cysteamine linker. The biosensor with the gold chip exhibits linearity of the analytical signal between 0.03 and 2 ng/mL, a precision of 1.4% and recovery between 99% and 102%. The biosensor with the bimetallic chip exhibits linearity between 0.03 and 1 ng/mL, a precision of 2.2% and recovery between 99% and 103%. Both biosensors tolerate a 1:100 excess of VEGF, VEGF-R1 and VEGF-R3. Both biosensors were validated by parallel determination of VEGF-R2 in 27 different plasma samples using the ELISA immunosensor assay, with very good agreement of the results. Thermodynamic parameters of the interaction of VEGF-R2 with the antibody were determined by QCM (Quartz Crystal Microbalance) and SPRi (Surface Plasmon Resonance imaging) measurements.

Sensitivity Analysis of Single- and Bimetallic Surface Plasmon Resonance Biosensors.

Comparative analysis of the sensitivity of two surface plasmon resonance (SPR) biosensors was conducted on a single-metallic Au sensor and bimetallic Ag-Au sensor, using a cathepsin S sensor as an example. Numerically modeled resonance curves of Au and Ag-Au layers, with parameters verified by the results of experimental reflectance measurement of real-life systems, were used for the analysis of these sensors. Mutual relationships were determined between ∂Y/∂n components of sensitivity of the Y signal in the SPR measurement to change the refractive index n of the near-surface sensing layer and ∂n/∂c sensitivity of refractive index n to change the analyte's concentration, c, for both types of sensors. Obtained results were related to experimentally determined calibration curves of both sensors. A characteristic feature arising from the comparison of calibration curves is the similar level of Au and Ag-Au biosensors' sensitivity in the linear range, where the signal of the AgAu sensor is at a level several times greater. It was shown that the influence of sensing surface morphology on the ∂n/∂c sensitivity component had to be incorporated to explain the features of calibration curves of sensors. The shape of the sensory surface relief was proposed to increase the sensor sensitivity at low analyte concentrations.

An SPRi Biosensor for Determination of the Ovarian Cancer Marker HE4 in Human Plasma.

Human epididymis protein 4 (HE4) is an ovarian cancer marker. Various cut-off values of the marker in blood are recommended, depending on the method used for its determination. An alternative biosensor for HE4 determination in blood plasma has been developed. It consists of rabbit polyclonal antibody against HE4, covalently attached to a gold chip via cysteamine linker. The biosensor is used with the non-fluidic array SPRi technique. The linear range of the analytical signal response was found to be 2-120 pM, and the biosensor can be used for the determination of the HE4 marker in the plasma of both healthy subjects and ovarian cancer patients after suitable dilution with a PBS buffer. Precision (6-10%) and recovery (101.8-103.5%) were found to be acceptable, and the LOD was equal to 2 pM. The biosensor was validated by the parallel determination of a series of plasma samples from ovarian cancer patients using the Elecsys HE4 test and the developed biosensor, with a good agreement of the results (a Pearson coefficient of 0.989). An example of the diagnostic application of the developed biosensor is given-the influence of ovarian tumor resection on the level of HE4 in blood serum.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging.

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the "EDC/NHS protocol". The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL-1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 µL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2-16%, a recovery of 101-104% depending on CEA concentration, a detection limit of 0.12 ng mL-1 and a quantification limit of 0.40 ng mL-1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

Plasma concentration of MMP-1 and MMP-2 in boys with cryptorchidism and its lack of correlation with INSL3 and inhibin B.

The matrix metalloproteinases are enzymes capable of remodeling of extracellular matrix, and modulate the behavior of cells. Maturation of gubernaculum and spermatogenesis demand proper equilibrium of metalloproteinases and their inhibitors. The aim of this survey was to investigate the levels of matrix metalloproteinase type 1 (MMP-1) and matrix metalloproteinase type 2 (MMP-2) in the plasma of children with unilateral cryptorchidism along with levels of Insulin-like Peptide 3 (INSL3) and inhibin B. INSL3 have a role in gubernaculum development. Inhibin B is produced by Sertoli cells, and its levels reflect the status of the testis germinative epithelium. Fifty boys with an undescended testicle, aged 1-4 years (median = 2.4 years) were enrolled into the study. Fifty boys with inguinal hernia aged 1-4 years, served as controls (median age = 2.1 years). Investigators assessed the MMP-1 and MMP-2 concentrations using Surface Plasmon Resonance Imaging. The levels of INSL-3 and inhibin B were assessed using commercial enzyme-linked immunosorbent assay ELISA. The median concentration of MMP-1 and MMP-2 in the blood plasma of patients with unilateral cryptorchidism, was nearly 2-folds higher than in controls. The great area under the ROC curve with the cut off value of 0.865 for MMP-1, and 0.819 for MMP-2, indicates the high clinical sensitivity and specificity of the test of plasma levels of MMP-1 and MMP-2 for boys with cryptorchidism. The increased plasma levels of MMP-1 and MMP-2, probably reflect the level of apoptosis of the germ cells in undescended testicles, in response to the heat stress during the period of prepubertal testis development. In the group of cryptorchid boys, we found slightly lower concentrations of INSL3, without statistical significance and without correlation with MMP-1 and MMP-2 levels. There were no significant differences in the levels of inhibin B in the group of boys with cryptorchidism and boys with inguinal hernia and it also did not correlate with MMP-1 and MMP-2 concentrations.

A New Analytical Method for Determination of Cathepsin L Based on the Surface Plasmon Resonance Imaging Biosensor.

The purpose of this study was to develop a new method for a determination of the cathepsin L-biosensor based on the Surface Plasmon Resonance Imaging technique. The cathepsin L is an endopeptidase, which degrades proteins and plays an important role in various processes occurring in the human body. The detection technique, Surface Plasmon Resonance Imaging, is an optical, label-free technique, which can be used for quantitative determination of the different proteins. In order to bind the enzyme, the cathepsin L inhibitor-RKLLW-NH2 was used. The validation process showed that parameters: precision, accuracy, and selectivity of the method were acceptable. The analytically useful range of the standard curve was 0.50 ng/mL-15.00 ng/mL. The detection and quantification limit of method was 1.67 pg/mL and 5.07 pg/mL, respectively. The usefulness of the developed method was confirmed by the determination of the cathepsin L concentration in the blood plasma of some healthy persons and in the blood plasma of patients. The obtained results were compared with the results obtained by the ELISA. It was found that the correlation between these two methods was very strong, what suggest that the developed method can be used as the competitive method to the ELISA.

Concentration of Proteasome in the Blood Plasma of Children with Acute Appendicitis, Before and After Surgery, and Its Correlation with CRP.

OBJECTIVE: The determination of 20S proteasome concentration in the blood plasma of children with appendicitis and its correlation with CRP. DESIGN AND SETTING: Thirty-one children with acute appendicitis, were randomly included into the study (age 5 years up to 17 years, mean age 11.5 + 1 years). PARTICIPANTS: There were 17 girls and 14 boys. Eighteen healthy, age-matched subjects, admitted for planned surgeries served as controls. Exclusion criteria were: severe preexisting infections, immunological or cardiovascular diseases that required long-term medication, and complicated cases of appendicitis with perforation of appendix and/or peritonitis. MAIN OUTCOME MEASURES: The 20S proteasome concentrations in the blood plasma of patients with acute appendicitis were highest before the surgery and were above the range of concentrations measured in controls, and the difference was statistically significant. RESULTS: The 20S proteasome concentration measured 24 and 72 h after the operation, slowly decreased over time, and still did not reach the normal range, when compared with the concentration measured in controls (p < 0.05). CONCLUSIONS: 20S proteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation. The method of operation-classic open appendectomy, or laparoscopic appendectomy, does not influence the general trend in 20S proteasome concentration.

Utility of cystatin C as a potential bladder tumour biomarker confirmed by surface plasmon resonance technique.

BACKGROUND & OBJECTIVES: The determination of cystatin C (cysC) may be helpful in diagnosis and monitoring of cancer because the pathogenesis of cancer is linked with an increased activity of cysteine peptidases (cathepsins) and a decrease of cysC concentration. This study was aimed to examine the utility of cysC as a marker of bladder cancer (BCa) to be used in the diagnosis. METHODS: This study was conducted with 90 patients with BCa and 27 healthy people. Patients with other cancers, inflammation process and impaired renal function were excluded from the study. The concentrations of cysC in the plasma and urine were measured by surface plasmon resonance imaging technique. RESULTS: The concentration of cysC in the serum taken from the patients with BCa [0.35±0.02 µg/ml (range: 0.20-0.78 µg/ml)] was significantly (P<0.001) lower than the serum cysC concentration of the healthy people [0.68±0.05 µg/ml (range: 0.52-0.89 µg/ml)]. The urinary cysC concentration of the BCa patients [0.19±0.01 µg/ml (range: 0.09-0.34 µg/ml)] was not significantly different from the urinary cysC concentration of the healthy people [0.24±0.02 µg/ml (range: 0.16-0.33 µg/ml)]. Receiver operating characteristic (ROC) curve showed that BCa patients with cysC concentration <0.54 µg/ml [sensitivity: 87%; specificity: 92%; area under the curve (AUC) of ROC: 0.927; P=0.02] could be optimally separated from healthy people. The ROC curve further showed that superficial low-grade patients with cysC concentration lower than 0.36 µg/ml (sensitivity: 0.63%; specificity: 0.58%; AUC of ROC: 0.635; P=0.08) could not be optimally separated from high-risk tumour patients. INTERPRETATION & CONCLUSIONS: BCa patients have lower serum cysC concentration than the control group. Serum cysC may be considered as a potential marker of BCa but not its aggressiveness.