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Aim: An innovative Atg4B inhibitor, S130, exhibited a negative influence on colorectal cancer cells in vitro and in vivo . To assist reliable toxicodynamic and pharmacokinetic evaluation, an LC-MS/MS assay of S130 in rat plasma must be necessary. Results: An LC-MS/MS assay for determination of S130 in rat plasma has been first developed and fully verified whose values met the admissible limits as per the US FDA guidelines. Chromatographic separation was achieved by using an isocratic elution after 3 min. MS was conducted under the ESI + mode fitted with selected reaction monitoring. The calibration curve proved acceptable linearity over 0.50-800 ng/ml. Conclusion: The developed LC-MS/MS assay of S130 in rat plasma is easily applicable in pharmacokinetics study and the further toxicological evaluation.

Context: The role of activation of lipid peroxidation in the mechanisms of acute methanol poisoning has not been studied., Objective: We measured the concentrations of lipid peroxidation markers in acutely intoxicated patients with known serum concentrations of methanol and leukotrienes., Methods: Blood serum samples were collected from 28 patients hospitalized with acute intoxication and from 36 survivors 2 years after discharge. In these samples, concentrations of 4-hydroxy-trans-2-hexenal (HHE), 4-hydroxynonenal (HNE), and malondialdehyde (MDA) were measured using the method of liquid chromatography-electrospray ionization-tandem mass spectrometry., Results: The maximum acute serum concentrations of all three lipid oxidative damage markers were higher than the follow-up serum concentrations: HNE 71.7 ± 8.0 ng/mL versus 35.4 ± 2.3 ng/mL; p < .001; HHE 40.1 ± 6.7 ng/mL versus 17.7 ± 4.1 ng/mL; p < .001; MDA 80.0 ± 7.2 ng/mL versus 40.9 ± 1.9 ng/mL; p < .001. The survivors without methanol poisoning sequelae demonstrated higher acute serum concentrations of the markers than the patients with sequelae. A correlation between measured markers and serum leukotrienes was present: HNE correlated with LTC4 (r = 0.663), LTD4 (r = 0.608), LTE4 (r = 0.771), LTB4 (r = 0.717), HHE correlated with LTC4 (r = 0.713), LTD4 (r = 0.676), LTE4 (r = 0.819), LTB4 (r = 0.746), MDA correlated with LTC4 (r = 0.785), LTD4 (r = 0.735), LTE4 (r = 0.814), LTB4 (r = 0.674); all p < .001. Lipid peroxidation markers correlated with anion gap (r= -0.428, -0.388, -0.334; p = .026, .045, .080 for HNE, HHE, MDA, respectively). The follow-up serum concentrations of lipid oxidation markers measured in survivors with and without visual/neurological sequelae 2 years after discharge did not differ., Conclusion: Our results demonstrate that lipid peroxidation plays a significant role in the mechanisms of acute methanol poisoning. The acute concentrations of three measured biomarkers were elevated in comparison with the follow-up concentrations. Neuronal membrane lipid peroxidation seems to activate leukotriene-mediated inflammation as a part of the neuroprotective mechanisms. No cases of persistent elevation were registered among the survivors 2 years after discharge.

Background: Cathepsin K is an attractive therapeutic target for diseases in which bone resorption is excessive such as osteoporosis and osteoarthritis (OA). The current paper characterized the pharmacological profile of the potent and selective cathepsin K inhibitor, MIV-711, in vitro and in cynomolgus monkeys, and assessed translation to human based on a single dose clinical study in man., Methods: The potency and selectivity of MIV-711 were assessed in vitro using recombinant enzyme assays and differentiated human osteoclasts. MIV-711 was administered to healthy cynomolgus monkeys (3-30 µmol/kg, p.o.). Plasma levels of MIV-711 and the bone resorption biomarker CTX-I were measured after single dose experiments, and urine levels of CTX-I, NTX-I and CTX-II biomarkers were measured after repeat dose experiments. The safety, pharmacokinetics and pharmacodynamics (serum CTX-I) of MIV-711 were assessed in human healthy subjects after single ascending doses from 20 to 600 mg., Results: MIV-711 was a potent inhibitor of human cathepsin K (K i : 0.98 nmol/L) with > 1300-fold selectivity towards other human cathepsins. MIV-711 inhibited human osteoclast-mediated bone resorption with an IC 50 value of 43 nmol/L. Single oral doses of MIV-711 to monkeys reduced plasma levels of CTX-I in a dose-dependent fashion by up to 57% at trough. The effect on CTX-I was linearly correlated to the plasma exposure of MIV-711, while the efficacy duration outlasted plasma exposure. Repeat oral dosing with MIV-711 also reduced urinary levels of the bone resorption biomarkers CTX-I (by 93%) and NTX-I (by 71%) and the cartilage degradation biomarker CTX-II (by 71%). MIV-711 was safe and well-tolerated when given as single ascending doses to healthy subjects. MIV-711 reduced serum CTX-I levels in a dose-dependent manner by up to 79% at trough. The relationship between MIV-711 exposure and effects on these biomarkers in humans was virtually identical when compared to the corresponding monkey data., Conclusions: MIV-711 is a potent and selective cathepsin K inhibitor with dose-dependent effects on biomarkers of bone and cartilage degradation in monkey and human. Taken together, MIV-711 shows promise for the treatment of bone and cartilage related disorders in humans, such as OA. Trial Registration EudraCT number 2011-003024-12, registered on June 22nd 2011.

Background: MIV-711 is a highly potent and selective cathepsin K inhibitor. The current article summarizes the therapeutic effects of MIV-711 on joint pathology in rabbits subjected to anterior cruciate ligament transection (ACLT), and the prophylactic effects on joint pathology in dogs subjected to partial medial meniscectomy, two surgical models of osteoarthritis (OA)., Methods: Starting 1 week after surgery, rabbits were dosed daily via oral gavage with either MIV-711 or vehicle (n = 7/group) for 7 weeks. The four treatment groups were: (1) sham + vehicle; (2) ACLT + vehicle; (3) ACLT + MIV-711, 30 µmol/kg and (4) ACLT + MIV-711, 100 µmol/kg. Subchondral bone and articular cartilage structures were assessed by µCT, histomorphometry, and scoring. Dogs subjected to partial medial meniscectomy received either MIV-711 (30 µmol/kg) or vehicle (n = 15/group) via oral gavage once daily, starting 1 day before meniscectomy, for 28 days. Cartilage degradation was assessed at the macroscopic and microscopic levels. The exposures of MIV-711 were assessed in both studies and biomarkers reflecting bone resorption (HP-1 in rabbits, CTX-I in dogs) and cartilage degradation (CTX-II) were measured., Results: In ACLT rabbits, MIV-711 decreased HP-1 levels by up to 72% (p < 0.001) and CTX-II levels by up to 74% (p < 0.001) compared to ACLT vehicle controls. ACLT surgery significantly reduced the total thickness of the subchondral bone plate and reduced trabecular bone volume in the femur and tibia. These effects were reversed by MIV-711. ACLT resulted in cartilage thickening, which was attenuated by MIV-711. MIV-711 did not affect osteophyte formation or Mankin scores. In dogs, MIV-711 reduced CTX-I and CTX-II levels by 86% (p < 0.001) and 80% (p < 0.001), respectively. Synovial CTX-II levels were reduced by 55-57% (p < 0.001) compared to baseline. MIV-711-treated animals had 25-37% lower macroscopic scores in the femur condyles and 13-33% lower macroscopic scores in the tibial plateaus., Conclusions: MIV-711 prevents subchondral bone loss and partially attenuates cartilage pathology in two animal models of OA. These beneficial effects of MIV-711 on joint pathology are observed in conjunction with decreases in bone and cartilage biomarkers that have been shown to be clinically attainable in human. The data support the further development of MIV-711 for the treatment of OA.

Objective: To investigate the safety and efficacy of extracorporeal shock wave lithotripsy (ESWL) combined with percutaneous nephrolithotomy (PCNL) for treatment of complex renal calculus., Patients and Methods: Seventy-eight patients diagnosed with complex renal calculus and who accepted treatment in our hospital were consecutively selected. Patients were divided randomly into the observation group (n=40) treated by combined ESWL and PCNL and the control group (n=38) treated by PCNL. The effect of treatment between the two groups was compared., Results: The stone-free rate at 3 months after surgery was higher in the observation group than in the control group. There were no differences in the rates of complications (including infection, hemorrhage, collection system perforation and laceration, peripheral organ impairment, and urination extravasation). There were gradual decreases of serum creatinine in the observation group at 4 weeks after extubation of the double J catheter and at 3 months after surgery, while there were no apparent decreases in the control group. The levels of cysteine protease inhibitor and neutrophil gelatinase-associated lipocalin in both groups increased at 4 weeks after extubation of the double J catheter, and decreased at 3 months after surgery. The decreases were more apparent in the observation group compared with the control group, and the differences were statistically significant (p<0.05)., Conclusions: Combined use of ESWL and PCNL to treat complex renal calculus can improve the stone-free rate and renal function, and does not increase the complication rate. It is, therefore, safe and effective.

The correlation of in vitro inhibition of cathepsin K (CatK) activity and in vivo suppression of collagen I biomarkers was examined with three selective CatK inhibitors to explore the potential translatability from animal species to human. These inhibitors exhibited good in vitro potencies toward recombinant CatK enzymes across species, with IC 50 values ranging from 0.20 to 6.1 nM. In vivo studies were conducted in animal species following multiple-day dosing of the CatK inhibitors to achieve steady-state plasma drug concentration-time profiles. Measurement of urinary bone resorption biomarkers (cross-linked N-terminal telopeptide and helical peptide of type I collagen) revealed drug concentration-dependent suppression of biomarkers, with EC 50 values estimated to be 12 to 160 nM. Marked improvement in the correlation between in vitro and in vivo CatK activities was observed with the application of unbound (free) fraction in plasma, consistent with the conditions stipulated by the free-drug hypothesis. These results indicate that the in vitro-in vivo translation of CatK inhibition observed in animal species can translate to humans when the unbound fraction of the inhibitor is considered. Interestingly, residual levels of urinary bone resorption marker were detected as the suppression reached saturation (at an average of 82% inhibition), an apparent phenomenon observed regardless of the species, biomarker, or compound examined. Since cathepsin enzymes other than CatK were reported to catalyze cleavage of collagen I, it is hypothesized that CatK-mediated degradation of collagen I in bone represents ~82% of overall collagen I turnover in the body.

Background: The aim of this case-control study with an intervention arm is to determine the effect of initial periodontal treatment on oxidative stress biomarkers in smokers and non-smokers with chronic periodontitis (CP)., Methods: The study included 47 patients with CP (24 smokers [S+P+] and 23 non-smokers [S-P+]) and 46 periodontally healthy individuals (23 smokers [S+P-] and 23 non-smokers [S-P-]) for a total of 93 participants. Gingival crevicular fluid (GCF), serum, and saliva samples were obtained and clinical periodontal measurements were recorded at baseline and at the first and third months after periodontal therapy. 8-hydroxydeoxyguanosine (OHdG) and 4-hydroxynonenal (HNE) and enzyme activity of glutathione peroxidase (GSH-Px) were analyzed with enzyme-linked immunosorbent assay., Results: The level of 8-OHdD in GCF was found to be significantly higher in both periodontitis groups compared with both periodontally healthy groups. 8-OHdG and GSH-Px in saliva in both periodontitis groups were significantly increased compared with the S-P- group. In the S+P+ group, 4-HNE in GCF was found to be significantly higher than in periodontally healthy participants. After initial periodontal treatment, the levels of 8-OHdG in GCF and saliva were significantly decreased in both periodontitis groups., Conclusion: Initial periodontal therapy may be helpful for diminishing oxidative stress in periodontitis.

Background: Survivin has been identified as one of the most cancer-specific molecules, with a dual function of apoptosis inhibitor and orchestrator of cell division., Hypothesis/objectives: Based on our recent results obtained during the study of the role of survivin in epithelial-to-mesenchymal transition, we investigate its potential role in maintenance of stemness in both the normal canine hair follicle and related tumours., Methods: We performed a simultaneous evaluation, by immunofluorescence, of the expression of survivin and CK15. CK15 was selected as a marker for epidermal and hair follicle stem cells, based on its ability to identify hair follicle stem cells in the normal hair follicle and in canine follicular tumours. In this study, six cases were selected from the cases of hair follicle tumours evaluated in previous studies, based on the highest immunoreactivity for survivin and CK15. Three samples of healthy canine skin were also included as a normal control., Results: A partial co-localization of the molecules was observed in normal hair follicles, as well as in trichoepitheliomas and trichoblastomas. In particular, a different co-expression was observed in relationship to the hair follicle cycle stage., Conclusions and Clinical Importance: These findings suggest that survivin could play an important role in the maintenance of the hair follicle cycle as well as in tumour initiation and maintenance of cancer stem cells.

Unlabelled: Oxidative stress plays an important role in cystic fibrosis (CF). However, there is a lack of validated biomarkers of oxidative damage that correlate with the antioxidant needs of patients with CF., Objective: To investigate oxidative stress in stable pediatric CF patients and evaluate if vitamin supplementation may be tailored to individual needs and oxidative status., Results: Lipid-adducts 4-hydroxynonenal (HNE-L) and malonaldehyde (MDA-L) (chromolipids) were elevated in the majority of patients despite normal plasma vitamin E, A and C. HNE-L and MDA-L increased with age, while plasma vitamins decreased. The most relevant correlation was identified between vitamin C and chromolipids. Patients with pancreatic insufficiency (PI) showed significantly higher plasma chromolipids despite no differences in plasma vitamins., Conclusions: The majority of patients showed elevated plasma chromolipids that increased with age. Antioxidant vitamin reference ranges provide incomplete information on the redox status. CF patients with PI showed excessive oxidative stress damage., (Copyright © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Objective: To determine the association between serum cystatin C and tumors., Methods: Serum samples were obtained from 273 patients with tumors and 185 healthy volunteers. Serum cystatin C was determined by particle-enhanced turbidimetric immunoassay (PETIA). Student t-test, covariance analysis and multiple linear regressions were performed to examine the differences in the levels of cystatin C between the two groups., Results: The student t-test did not show significant statistical differences in the serum cystatin C levels between the two groups (P = 0.075). However, such differences became statistically significant (P < 0.01) after adjustment of age. The multiple liner regression demonstrated that healthy volunteers and men had higher levels of cystatin C than those with tumors and women. Cystatin C also increased with age and decreases with estimating glomerular filtration rate., Conclusion: Cystatin C may be associated with the genesis and development of tumors.

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, a novel cystatin molecule (AcCystatin) was cloned from a cDNA library of Angiostrongylus cantonensis fourth-stage larvae. The putative 14-kDa protein contained 120 residues with cystatin-conserved motifs known to interact with the active site of cysteine peptidases and showed high identities with cystatins from other nematodes. RT-PCR analysis revealed that the expression pattern of AcCystatin was equal at the time points of third-stage larvae, fourth-stage larvae, and adults of the parasite life cycle. The recombinant AcCystatin (rAcCystatin) expressed and purified from Escherichia coli has been demonstrated to possess an obvious inhibitory activity against cathepsin B and could significantly upregulate nitric oxide production from IFN-gamma activated RAW 264.7 macrophages. Sera from mice (non-permissive host) infected with A. cantonensis detected rAcCystatin by Western blot, while the sera from infected rats (permissive host) could not. The results implied that AcCystatin might be an immunoregulator in A. cantonensis infection.

Impairment of renal function beginning from subclinical stages is independent risk factor of development of cardiovascular diseases and cardiovascular death. Cystatin C is considered in recent years as an alternative marker of functional state of the kidney and risk of development of cardiovascular diseases. Cystatin C is a protease inhibiting protein with low molecular mass which is characterized by free glomerular filtration and is not subjected to tubular secretion. Formulas are elaborated for calculation glomerular filtration rate basing on cystatin C level. In this review we consider potential advantages of cystatin C for assessment of early impairment of renal function. We also present data on interrelation between cystatin C, metabolic parameters, and markers of target organs damage, on prognostic value of cystatin C in various populations of patients.

Objective: Acromegaly is a rare disease due to growth hormone (GH) excess. Patients must be carefully follow up because of mortality and co-morbidity increased risks. Since routinely used GH and insulin-like growth factor-1(IGF-1) estimations are not always sufficient, patients require assessment of organ- or tissue-specific tests. Cysteine proteases (CP), including cathepsin B, have been tested in a number of pathologies in respect of a role in pathogenesis and potential utility in the disease activity and prognosis assessment. There is lack of data on CP activity in acromegaly., Materials and Methods: In present study cathepsin B-like and cysteine peptidase inhibitor (CPI) activities have been tested in 29 acromegaly patients and in 15 healthy controls. Cathepsin B activity was assayed with N-bansoyl-DL-arginine-beta-naphthylamide (BANA) as substrate by the Barrett method. CPI activity was determined by measuring the inhibition of papain., Results: Serum cathepsin B activity (median: 1.38 U/ml) and CPI activity (median: 93.08 U/ml) were higher in acromegaly then in controls (0.93 U/ml and 82.55 U/ml, p=0.000017 and 0.00285, respectively). Neither cathepsin B nor CPI activity was correlated with GH or IGF-1 level. No correlation was recorded between cathepsin B and CPI activity., Conclusion: It was shown for the first time that cathepsin B and CPI activities are increased in acromegaly. These findings suggest to study cathepsin system as an adjuvant parameter in the assessment of the overall acromegaly complications. Moreover, CP may be involved in pathomechanism of organ complications in acromegaly and may interfere with IGF-1 action.

Although many allergens bind endogenous molecules other than Abs in the human body, whether the interaction can modulate allergenicity has been unknown. Here, we investigated the effect of the interaction of recombinant major mite group 1 allergens (Der f 1 and Der p 1), which belong to the papain-like cysteine protease family, with an endogenous protease inhibitor, cystatin A, on their allergenicity. Cystatin A bound reduced forms of the allergens, in which the cysteine residue at the catalytic center of the protease activity was reduced by treatment with L-cysteine, but did not bind oxidized forms. Cystatin A partially inhibited the binding of IgE in mite-allergic volunteers' sera to the reduced forms, but unexpectedly enhanced the basophil histamine-releasing activity. A catalytic site-mutant of Der f 1 behaved in terms of histamine release, similarly to the reduced form. Molecular modeling showed that cystatin A interacts with the allergens within a narrow area. The results indicate that interaction with cystatin A reduces the limited number of IgE epitopes of the allergens but enhances their biological activity to release histamine, suggesting a new concept, that interaction between allergens and their endogenous ligands modulates the allergenicity even toward enhancement in the effector phase. On the other hand, i.p. immunization without alum of mice with cystatin A-treated reduced Der f 1 induced less serum Der f 1-specific IgE than immunization with reduced Der f 1 alone, suggesting that endogenous protease inhibitors suppress the induction of allergen-specific IgE, which is dependent on the enzymatic activity of cysteine protease-allergens, in the sensitization process.

Incidental prostate cancer (PCa) has been demonstrated at autopsy in about 80% of men aged 80 years and above and also in 10%-15% of younger men aged 30-50 years in the United States. These data imply a wide variation in aggressiveness of prostate cancer, from indolent tumors to aggressive cancers that kill the patients. The use of prostate specific antigen (PSA) in screening for PCa may detect even indolent disease for which radical prostatectomy may not be necessary. Currently available criteria such as histological grade, PSA level, stage of the disease do not always predict outcome. Furthermore, only about 80% of men with metastatic PCa will respond to first line hormone manipulation and once the patient develops hormone resistant prostate cancer (HRPCa), survival remains poor. Recent genomic and proteomic studies have provided many novel molecular markers that may help to redefine prognostic parameters. This paper is a review of studies using these novel markers in order to determine whether prostate cancer patients with the following characteristics have more aggressive cancer than those without: a) high serum levels of cathepsin B, survivin, Her - 2 / neu, IGFBP-2; b) low serum stefin A, IGFBP-3, c) positive immuno-staining of primary tumors for Her-2/neu, survivin and cathepsin B / stefin A ratio > 1 and d) gene expression of AMACR, HER-2/neu, high Bcl-2: Bax ratio and EZH2 in cancer cells. These markers have been chosen for review because they are among the most promising markers emerging currently.

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.

Cystatin C is a relatively stable protein in serum and heparinized plasma that shows promise as a convenient measure of glomerular filtration rate (GFR). However, it is becoming clear that the relationship between cystatin C and GFR can depend on the clinical presentation. Factors influencing cystatin C levels are those that affect the rate of synthesis of the protein, such as thyroid status and the use of steroids. As with all laboratory tests, results should be interpreted in the light of the method's known limitations and in conjunction with other clinical and laboratory information. Nevertheless, accumulating evidence suggests that cystatin C is a useful biomarker for renal function, and may even be the method of choice in a range of clinical situations, from GFR surveillance in diabetics to the assessment of acute kidney injury in critically ill patients.

The pathogenesis of ischemic coronary events involves degradation of the extracellular matrix in atherosclerotic lesions. The cysteine protease inhibitor cystatin-C may be involved in this phenomenon. The association of plasma cystatin-C with the incidence of myocardial infarction-coronary death and angina, was examined in a nested case-control (two controls per case) design within the prospective cohort study (Prospective Epidemiological Study of Myocardial Infarction (PRIME Study)) which included 9,758 men aged 50-59 years who were free of coronary heart disease (CHD) on entry and followed for a 5-year period. Three hundred and thirteen participants suffered myocardial infarction or coronary death (n = 159) or angina pectoris (n = 154) during follow-up. Cystatin-C was positively correlated with body mass index (BMI), low-density lipoprotein (LDL)-cholesterol, triglycerides and several inflammatory markers such as fibrinogen (r = 0.18), C-reactive protein (CRP) (r = 0.24), interleukin-6 (= 0.20), tumor necrosis factor-alpha (TNFalpha) (r = 0.27) and two TNFalpha receptors: TNFR1A (r = 0.43) and TNFR1B (r = 0.41); and negatively with high-density lipoprotein (HDL)-cholesterol (r = -0.25). After adjustment for traditional risk factors (age, diabetes, smoking, hypertension, BMI, triglycerides, LDL- and HDL-cholesterol), cystatin-C was significantly associated with the occurrence of the first ischemic coronary event. However, this association was no longer significant when CRP was included in the analysis. A decrease in glomerular filtration rate did not explain higher cystatin-C in cases than in controls. Cystatin-C appears to participate in the inflammatory phenomenon observed in the atherosclerotic process. Cystatin-C is not a more predictive risk marker of CHD than CRP or interleukin-6, but could be useful in detecting moderate chronic renal disease.

Aim: Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors, and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals., Methods: The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC., Results: The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P<0.05). The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract., Conclusion: These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.

MDL 28170, Cbz-(L)-Val-(D,L)-Phe-H, which exists as a mixture of L,L- and L,D-diastereoisomers, is a calpain inhibitor currently investigated as a novel therapeutic agent for the treatment of ischemic stroke and head and spinal trauma. This report describes a capillary electrophoresis (CE) method that uses sodium dodecyl sulfate (SDS) micellar electrokinetic conditions for the separation of the L,L- and L,D-diastereoisomers of MDL 28170. The report also describes the applications of this CE method to the study of epimerization of the L,L- and L,D-diastereoisomers in pH 7.4 phosphate buffered saline solution (PBS), rat and human plasma at 37 degrees C. The relative percent-time courses obtained showed interconversion of the diastereoisomers in all three matrices studied. However, the epimerization process in rat and human plasma was found to be at least 50 times faster than that in PBS. The epimerization half-life of the L,L-diastereoisomer in rat plasma was approximately 30 min, which is about three-fold faster than the observed elimination half-life of the L,L-diastereoisomer reported in a pharmacokinetic study following intravenous bolus dosing.

Stefin A is the low-molecular, intracellular inhibitor of the human lysosomal cysteine proteinases, cathepsins B, H and L. The concentration of this inhibitor in plasma and in other biological fluids shows not only local expression and secretion but also immunity of the whole organism. The aim of our study was to examine if concentration of stefin A in plasma of patients with diabetes type 2 is different than in healthy subjects, depends on vascular complications, body mass index, glycaemic control and whether exists the relationship between activities of cathepsin B (CB) as well as N-acetyl-beta-D-glucosaminidase (NAG). In plasma of 62 diabetic patients and 14 control subjects, concentration of stefin A (using ELISA test) and activities of CB and NAG (using fluorescence methods) were investigated. Concentration of stefin A (4.95 micrograms/l) in comparison to control (2.80 micrograms/l) increased statistically significantly (p < 0.05). The highest increase of stefin A was discovered in patients with macrovascular complication (7.70 micrograms/l) and was significantly different (p < 0.01) in comparison with control group and patients with microangiopathy as well as both types of complications (micro- and macroangiopathy). Significantly higher concentration of stefin A was noted in patients with overweight and obesity (5.50 micrograms/l and 6.05 micrograms/l). No influence of glycaemic short and long term control on concentration of this inhibitor was observed. In patients divided into subgroups according to increasing plasma NAG activity, increase of stefin A was noted successively from subgroup K2 to K4 (NAG from 8.80 U/l to above 12.82 U/l). The results indicate contribution of stefin A in pathogenesis of vascular complications in patients with diabetes type 2 and its relationship with obesity.

We compared cystatin C, creatinine, and the Cockroft formula for assessment of early renal failure, defined as a (51)Cr-EDTA clearance < 80 mL/min, in 89 diabetic patients with various degrees of renal impairment (glomerular filtration rate [GFR], 11.4 to 196.5 mL/min). The relationships between cystatin C, creatinine, and (51)Cr-EDTA clearance were linearized by plotting the reciprocals of the values, and correlation coefficients were determined. Sensitivity and specificity for the diagnosis of early renal failure were calculated from receiver operating characteristic (ROC) curves. Over the whole population, cystatin C was as well correlated with GFR (r =.74) as was creatinine (r =.67) or the Cockroft formula (r =.88). Moreover, its diagnostic accuracy was comparable to that of the 2 other parameters. Its sensitivity (86.8%) was better than that of creatinine (77.4%) for screening GFR < 80 mL/min, although the Cockroft formula was more sensitive (96.2%). The study of albuminuric diabetics (n = 63) led to similar conclusions, except for a poor sensitivity of cystatin C. In the 36 patients whose plasma creatinine was < 1 mg/dL, 10 (27.7%) had GFR < 80 mL/min. The correlation of creatinine with GFR, its diagnostic accuracy, and sensitivity were significantly lower than those of cystatin C. In this population of patients with normal creatinine levels, the correlation coefficient of cystatin C, its sensitivity, and its diagnostic accuracy were comparable to those of the Cockroft formula. A moderate reduction in GFR may be present in diabetic patients with low creatinine levels. Although Cockroft formula remains the most reliable and the less expensive tool for the evaluation of renal function, cystatin C is a more reliable criterion for screening and assessment than creatinine and represents a useful alternative to the Cockcroft-Gault formula.

In a study performed in Tamale, in the Northern region of Ghana, cystatin C, a new and sensitive indicator of the glomerular filtration rate (GFR), was used to estimate the frequency of renal dysfunction in 78 children with uncomplicated, Plasmodium falciparum malaria. The excretion in urine of albumin, immunoglobulin G and alpha1-microglobulin was also investigated. Plasma concentrations of cystatin C were found to be elevated in 17% of the children, indicating subclinical impairment of renal function. As most (85%) of the children had glomerular as well as tubular patterns of proteinuria, it appears that both glomerulonephritis and damage to tubular cells often occur in P. falciparum malaria.

Purpose: We compared the diagnostic performance of cystatin C and beta-trace protein with serum creatinine and the Schwartz formula for the estimation of glomerular filtration rate in children with spina bifida., Materials and Methods: Cystatin C, beta-trace protein and serum creatinine in children who underwent routine measurement of glomerular filtration rate were obtained in a prospective study. A spina bifida group (27 measurements in 27 patients 1.4 to 20.0 years old, 15 female, 12 male) was compared with a group of controls (211 measurements in 201 patients with various other renal pathologies 1.0 to 20.2 years old, 89 female, 122 male). All patients underwent nuclear medicine clearance investigations with 99mtechnetium diethylenetetraminepentaacetic acid. Z scores were calculated for body mass index, height and weight. Receiver operating characteristic (ROC) analysis and ROC plots were done to assess their diagnostic accuracy., Results: Serum creatinine and the Schwartz formula did not correlate with glomerular filtration rate in the patients with spina bifida. Correlation coefficients for cystatin C were 0.45 and 0.84 in the spina bifida and control groups, respectively, while correlation coefficients for beta-trace protein were 0.31 and 0.75, respectively. For both groups ROC area was highest for cystatin C (up to 0.97 ROC area in control group)., Conclusions: Only cystatin C served as a suitable marker to estimate glomerular filtration rate in patients with spina bifida. Cystatin C proved to be a superior marker in patients with spina bifida. In the control group the Schwartz formula was comparable to cystatin C and beta-trace protein, although cystatin C remained superior.

Clinical biochemists have long known the analytical and clinical limitations of creatinine and creatinine clearance measurement in the assessment of glomerular filtration rate (GFR). This background is reviewed in the article before assessing the utility of cystatin C, the most promising replacement biochemical marker yet identified. Cystatin C has been used in clinical research studies for more than 20 years and yet has been introduced into clinical practice in very few centres, firstly in Lund in Sweden and now Carshalton in the UK. Why is this? The review compares our ability to measure creatinine and cystatin C and their relative sensitivity and specificity for changes in GFR. Comparison is made of cystatin C with creatinine as screening tests for early renal dysfunction and for monitoring its progression where issues of reference ranges and within-individual variation are important. The superiority of cystatin C as a screening test is probably accepted but there are still concerns about non-renal influences upon its circulating concentration, particularly steroid therapy, insufficient data on the influence of malignancy and a general lack of prospective clinical studies confirming the prognostic significance of cystatin C.

Cystatin C is a cysteine protease inhibitor produced by all nucleated cells. It is freely filtered by the glomerulus and is unaffected by nonrenal factors such as inflammation and gender. Because of greater sensitivity and specificity, cystatin C has been proposed to replace creatinine as a marker of glomerular filtration rate (GFR) in humans. The aims of this study were to validate an automated assay in canine plasma and to evaluate the usefulness of cystatin C as a marker of GFR in dogs. Western blotting was used to demonstrate cross-reactivity of an anti-human cystatin C antibody. An immunoturbidimetric assay was used to detect cystatin C in 25 clinically healthy dogs and 25 dogs with renal failure. Mean cystatin C concentration in the healthy dogs and the dogs with renal failure was 1.08 +/- 0.16 mg/L and 4.37 +/- 1.79 mg/L respectively. Intra- and interassay variability was <5%. The assay was linear (r = .974) between 0.14 and 7.53 mg/L. Both cystatin C and creatinine concentrations were measured in banked, frozen serum from 20 remnant kidney model dogs and 10 volume-depleted dogs for which GFR measurements by exogenous creatinine clearance had been determined previously. In the remnant kidney model, cystatin C was better correlated with GFR than creatinine (r = .79 versus .54) but was less well correlated with GFR in volume-depleted dogs (r = .54 versus .95). GFR measurements were repeated in the remnant kidney model dogs 60 days after initial GFR measurements. At this time, cystatin C and creatinine concentrations correlated equally well with GFR (r = .891 versus .894, respectively). Cystatin C concentration is a reasonable alternative to creatinine for screening dogs with decreased GFR due to chronic renal failure.

Serum levels of T-kininogen increase dramatically as rats approach the end of their lifespan. Stable expression of the protein in Balb/c 3T3 fibroblasts leads to a dramatic inhibition of cell proliferation, as well as inhibition of the ERK signaling pathway. T-kininogen is a potent inhibitor of cysteine proteinases, and we have described that the inhibition of ERK activity occurs, at least in part, via stabilization of the MAP kinase phosphatase, MKP-1. Since fibroblasts are not a physiological target of T-kininogen, we have now purified the protein from rat serum, and used it to assess the effect of T-kininogen on endothelial cells. Adding purified T-kininogen to EAhy 926 hybridoma cells resulted in inhibition of basal ERK activity levels, as estimated using appropriate anti-phospho ERK antibodies. Furthermore, exogenously added T-kininogen inhibited the activation of the ERK pathway induced by either bradykinin or T-kinin. We conclude that the age-related increase in hepatic T-kininogen gene expression and serum levels of the protein could have dramatic consequences on endothelial cell physiology, both under steady state conditions, and after activation by cell-specific stimuli. Our results are consistent with T-kininogen being an important modulator of the senescent phenotype in vivo.

Human cystatin C is a low molecular weight protein which has been proposed as a better marker of glomerular filtration rate than creatinine. To be able to interpret results obtained in different patient populations it is necessary to define cystatin C reference values. We measured serum concentration of cystatin C in 1223 subjects using a particle-enhanced nephelometric assay. Subjects were aged 4 to 79 years and were selected among apparently healthy individuals who came to the Centre for Preventive Medicine in Vandoeuvre-Lès-Nancy, France. We observed a Gaussian distribution of cystatin C concentration in serum. We did not find any effect of age or gender in children, hormonal status in women (puberty, menopause, oral contraceptives or hormone replacement therapy) or alcohol intake. Cystatin C concentration was slightly lower in female than in male adults below the age of 60 years. Cystatin C levels significantly increased above the age of 60 in both males and females, probably due to physiological aging of renal function. No other significant differences were observed between males and females. Using multiple regression analysis, moderate correlations were observed between body mass index and cystatin C, and between smoking and cystatin C, but these were not biologically significant. According to the literature, only methylprednisolone and cyclosporin A increased and decreased cystatin C levels, respectively. The reference values for cystatin C obtained in a carefully selected population were 0.75+/-0.089 mg/l for children aged 4-19 years, 0.74+/-0.100 mg/l for males and 0.65+/-0.085 mg/l for females (aged 20-59 years), and 0.83+/-0.103 mg/l for older individuals (> or =60 years).

To date, little evidence is available to define the role of cystatin C in patients with renal transplants. Thus, to assess, whether cystatin C (CysC) provides better information on renal function than other markers, CysC, creatinine clearance (CrCl), serum creatinine (SCr), beta2-microglobulin (beta2-M), and 125I-Iothalamate clearance were determined in 30 patients. Correlation and ROC curves were obtained and characteristics like sensitivity and specificity were calculated. Further, to evaluate the usefulness of these markers for monitoring, intraindividual coefficients of variation for CysC and SCr measurements were compared in 85 renal transplant patients. CysC correlated best with GFR, whereas SCr, CrCl and beta2-M all had lower correlation coefficients. CysC was superior to SCr, even when renal function equations of were used. The diagnostic accuracy of CysC was significantly better than SCr. but did not differ significantly from CrCl and beta2-M. Together, our data show that in patients with renal transplants, CysC has a similar diagnostic value as CrCl. However, it is superior to determinations of SCr. The intraindividual variation of CysC is significantly greater than that of SCr. This might be due to better ability of CysC to reflect temporary changes especially in mildly impaired GFR, most critical for early detection of rejection and other function impairment. In conclusion, CysC allows for easy and accurate assessment of renal function (GFR) in steady state renal transplant patients and is clearly superior to the commonly used serum creatinine.

A sandwich-type ELISA has been developed for quantification of the complex between the cysteine proteinase cathepsin B (CB) and its reversible tight-binding inhibitor cystatin C (CC) in normal and pathological sera. The assay is based on a combination of catching Ab (3E1), raised against CB, and a horseradish peroxidase-labelled detection Ab (1A2), raised against CC. Only the CB/CC complex is able to evoke a signal in this assay. The detection limit of the assay was 15.5 nM and the working range between 31.3-200 nM. The within and between-run coefficients of variance (CV) varied from 4.7% to 9.4% and 11% to 12.8%, respectively, demonstrating satisfactory reproducibility of the method. The concentration of the CB/CC complex was determined in sera from 90 healthy controls, 32 patients with non-cancerous lung diseases, 148 patients with lung and 32 patients with colorectal cancer. The CB/CC complex was significantly less abundant in sera of patients bearing malignant lung tumours than in those with non-cancerous lung diseases or healthy controls (p<0.001). In colorectal cancer sera its level was significantly lower in advanced stages C and D than in early Dukes' stages A and B (p=0.02). Our results show that the increased levels of CB in malignant sera are not impaired effectively by CC and support the hypothesis of hindered inhibitory capability during cancer progression.

Background: Cystatin C (MW 13kDa) serum concentration reflects glomerular filtration rate better than creatinine. Like other low-molecular weight proteins it is not eliminated by dialysis. Still, cystatin C serum concentrations do not rise progressively in end-stage renal failure and rarely exceed 10 mg/L (i.e. 8 times the upper limit of normal)., Objective: To study cystatin C kinetics in a rat model of end-stage renal failure., Methods: Sequential bilateral nephrectomy was performed seven days apart in 13 male Sprague-Dawley rats as described by Levine and Saltzman. Serum cystatin C (Cystatin C PET-kit, DAKO), creatinine and total protein were measured in daily intervals after the second nephrectomy. Linearity of the anti-human cystatin C assay for rat cystatin C was tested using dilutions of uremic rat serum. Rats were sacrificed for signs of severe uremia on days 10 (n=5), 11 (n=4) and 12 (n = 5)., Results: At baseline, mean (+/- SE) cystatin C was 1.59+/-0.041 mg/L, creatinine 19.6+/-1.2 micromol/L. Following bilateral nephrectomy, cystatin C immediately rose to 3.82+/-0.15 mg/L, creatinine to 312+/-20 micromol/L. During the following days, cystatin C concentration stabilized to 4 mg/L approximately whereas creatinine continued to rise to 822+/-185 kmol/L on day 12. Correction for the decrease in serum total protein concentration from 48.9+/-2.3 g/L to 37.4+/-3.6 g/L did not alter these results., Conclusion: The kinetics of cystatin C and creatinine in this rat model of end-stage renal failure are in accordance with human data suggesting a change in cystatin C production or extra-renal elimination in severe chronic uremia.

The assessment of the glomerular filtration rate (GFR) is the most commonly used test of renal function. Cystatin C, a cysteine protease inhibitor, which can be measured by light scattering immunoassay, possesses many of the attributes required of the ideal GFR marker. This paper reviews so far obtained results dealing with cystatin C measurement, reference intervals and its diagnostic significance in nephropathy. Although serum cystatin C can generally be recommended as a marker of GFR, especially to detect mild GFR reductions, further clinical studies should be performed to confirm the validate in renal transplants and cancer patients.

The aim of this study was to assess parameters of renal function and other determinants of plasma homocysteine in type 2 diabetic patients without coronary heart disease (CHD). Fasting plasma homocysteine, serum cystatin C and serum creatinine were determined in 183 (75 men, 108 women) Type 2 diabetic patients without clinical evidence of CHD. Creatinine clearance was calculated and parameters such as blood pressure, body mass index (BMI), and glycated haemoglobin (HbA(1c)) were assessed. The urine albumin:creatinine ratio was used to classify patients as normo-, micro- or macroalbuminuric. One hundred and ten patients were normoalbuminuric, 67 patients were microalbuminuric and six patients were macroalbuminuric. There was no statistically significant difference in plasma homocysteine concentration between patients with normoalbuminuria and microalbuminuria. There was a trend towards increasing plasma homocysteine with decreasing glomerular filtration rate (GFR) (r=-0.46; P<0.0001). There was statistically significant correlation between plasma homocysteine and age (r=0.37), serum cystatin C (r=0.47), and serum creatinine (r=0.56). Plasma homocysteine concentration was significantly higher in patients with BMI<30 kg/m(2) and showed significant inverse correlation with weight (r=-0.16; P=0.03) and body mass index (r=-0.24; P=0.001). Homocysteine and serum creatinine were significantly higher in males than females and higher in smokers than non smokers but was not associated with glycemic control and duration of diabetes. In conclusion, elevated homocysteine concentration in patients with type 2 DM without CHD is related to age, gender, smoking, BMI and GFR. Follow up studies will provide further information on the association between hyperhomocysteinemia and the development of cardiovascular disease.

Aim: Serum cystatin C (SCyst) has been proposed as a novel indicator of GFR., Patients and Methods: We compared SCyst, serum creatinine (SCreat) and Cockcroft and Gault's estimated clearance (CCG) using inulin clearance (Cin) as gold standard. 140 subjects (161 samples; aged 39 +/- 14; male/female: 79/82) underwent simultaneous measurements., Results: A highly significant correlation r = 0.70, 0.74, 0.77 (p < 0.0001) was found between 1/SCyst, 1/SCreat, C(CG), respectively, and Cin. Receiver-operating characteristic (ROC) analysis was performed on SCyst, SCreat and C(CG) using a Cin cut-off of 90 ml/min/1.73 m2. Best fit for SCyst was 0.90 mg/l with a sensitivity of 75% and a specificity of 92%. The area under the ROC curve was not significantly greater for SCreat or C(CG) than for SCyst (p = 0.91,0.13, respectively). When relationship between Cin and SCyst was plotted, experimental data deviated from the theoretical model, suggesting that cystatin C may not be solely filtered. Additional patients were selected in our database on the basis of discordant SCreat/GFR values: false negative (n = 46 samples, 31 patients) and false positive (n = 16 samples, 9 patients). In this highly selected subgroup, 38% of the SCreat false positive had normal SCyst values and 48% of the false negative SCreat had abnormally elevated SCyst., Conclusion: This study suggests that SCyst is not more sensitive than SCreat or C(CG) for detecting renal failure, however, SCyst could be proposed as a confirmatory test for patients with elevated SCreat.

The effect of asthma pathogenesis on serum cystatin C, a potent inhibitor of cysteine proteinases and a newly proposed marker of the renal function, has not been yet determined. The objectives were to determine the 24-h pattern of cystatin C and creatinine concentrations in sera of asthmatic patients in order to test whether their concentrations might reflect circadian rhythms, the disease severity and the effect of therapy. Serum concentrations of cystatin C and creatinine were determined in steroid-independent and steroid-dependent asthmatics before and after 1 week of treatment with methylprednisolone and cyclosporin A, respectively. Samples were collected every 4 h during a 24-h period. Little or no significant effects of time on cystatin C and creatinine concentrations over a 24-h period were observed in healthy and asthmatic sera. However, significantly higher cystatin C concentrations were found in asthmatic patients compared to controls which suggests its role in the pathogenesis of asthma. Methylprednisolone increased and cyclosporin A decreased serum cystatin C concentrations after 1 week of therapy. Additionally these results support the need for the evaluation of cystatin C as a marker of glomerular filtration rate determination in asthma.

Cysteine proteinase inhibitors (IPC) concentration was measured by the modified Barrett method using papaine in urine, amniotic fluid and serum obtained from the healthy labored women and from labored women in pregnancy complicated by EPH-gestosis. It was noticed the statistically significant increase in the IPC concentration in the material from the pregnant women with EPH-gestosis comparing to the women, which pregnancy had the physiologically normal course.

Background: Cystatin C is a proteinase inhibitor with a low molecular weight. The serum levels of cystatin C are mainly dependent on glomerular filtration rate (GFR) making cystatin C an endogenous parameter of GFR. The aim of the study was to elucidate the applicability of serum cystatin C as a parameter of GFR in patients with normal to moderately impaired kidney function and to estimate a reference interval for serum cystatin C., Patients and Methods: Forty-six patients (25 males and 21 females) aged 22 to 83 years with various kidney diseases and 250 blood donors (164 males and 86 females) aged 19 to 64 years were included. Cystatin C was measured by an automated particle-enhanced nephelometric immunoassay, serum creatinine by an enzymatic and by Jaffé method, urine creatinine by an enzymatic method, and GFR by 99mTc-DTPA clearance., Results: Serum levels ofcystatin C and creatinine showed increments with decreasing values of 99mTc-DTPA clearance and a linear relationship was found between 99mTc-DTPA clearance and l/serum cystatin C, l/serum creatinine (enzymatic method), and creatinine clearance. Comparison of the non-parametric receiver-operating characteristic (ROC) plots for serum cystatin C (area under the curve (AUC) = 0.996; SE = 0.005), serum creatinine (enzymatic method) (AUC = 0.899; SE = 0.044), serum creatinine (Jaffé method) (AUC = 0.870; SE = 0.051), measured creatinine clearance (AUC = 0.959; SE = 0.025), and estimated creatinine clearance (0.950; SE = 0.029) revealed significant differences for serum cystatin C and serum creatinine (enzymatic and Jaffé method) (p values: 0.03 and 0.01). No significant differences were demonstrated between serum cystatin C and measured and estimated creatinine clearance (p value: 0.14 and 0.12). The non-parametric reference interval for serum cystatin C was calculated to be 0.51-1.02 mg/l (median: 0.79 mg/l; range: 0.33 - 1.07 mg/l)., Conclusion: Serum cystatin C seems to be a better parameter of GFR than serum creatinine in adults with various types of kidney disease with normal to moderately impaired kidney function.

Introduction: Cystatin C, a cysteine protease inhibitor, has been implicated in the neurodegenerative and repair processes of the nervous system, and the deposition of the same protein together with beta amyloid peptide was found as cerebral amyloid angiopathy (CAA) in different types of dementias., Objective and Methods: Because of the differential diagnostic importance, serum and cerebrospinal fluid (CSF) cystatin C levels of 24 late onset Alzheimer's demented (AD) and 16 ischemic type of vascular demented (VD) probands were compared with 17 aged control (AC) persons., Results: The serum and CSF cystatin levels were found in the normal range in all groups. The ischemic VD probands had the tendency to have higher cystatin C levels than the AD. No correlation has been found with the severity and duration of dementia and with the other measured parameters., Conclusion: These results indicate that lower than normal CSF cystatin C level is not a diagnostic marker in ischemic VD and CAA related to AD.

Serum cystatin C, a cysteine proteinase inhibitor, has been proposed as a marker of glomerular filtration rate (GFR). Serum cystatin C, serum creatinine and creatinine clearance were measured in 226 patients with various nephropathies, covering the entire range of renal function, to evaluate the efficacy of cystatin C as a screening test to detect reduced creatinine clearance in comparison to creatinine. Subgroups of 53 patients with glomerular and 26 patients with tubular impairment were compared to assess whether cystatin C performed differently in either glomerular or tubular impairment. Cystatin C detected reduced creatinine clearance with higher sensitivity (97 vs. 83%), and higher negative predictive value (96 vs. 87%) compared to creatinine. In parallel, 95% sensitivity of cystatin C as derived from receiver-operating characteristic plot was significantly higher (p < 0.05). In the subgroups with glomerular or tubular impairment, cystatin C and creatinine did not significantly differ with regard to efficacy. Serum cystatin C is as efficacious as serum creatinine to detect reduced GFR as measured by creatinine clearance. The efficacy of cystatin C as a screening test may even be superior compared to creatinine. In addition, the efficacy of cystatin C is independent of either glomerular or tubular impairment., (Copyright 2000 S. Karger AG, Basel)

The levels of cysteine proteinase inhibitors stefin A, stefin B, and cystatin C were determined using ELISAs in sera obtained preoperatively from 345 patients with colorectal cancer and in control sera from 125 healthy blood donors. The levels of stefin A and cystatin C were found to be moderately increased in patient sera (1.4-fold and 1.6-fold, respectively; P < 0.0001), whereas the level of stefin B remained statistically unchanged when compared with controls. The medians were 4.3 ng/ml versus 3.2 ng/ml for stefin A, 1.2 ng/ml versus 1.7 ng/ml for stefin B, and 679 ng/ml versus 425 ng/ml for cystatin C. In patient sera, a weak correlation of cystatin C with age (r = 0.34; P < 0.001) and gender (P = 0.01) was found. Stefin A and cystatin C levels were independent of Dukes' stage, whereas stefin B correlated significantly with Dukes' stage, its level being the highest in stage D (P < 0.007). Stefin B and cystatin C correlated with survival, whereas stefin A was not a significant prognostic factor in this study. Using medians as cutoff values, patients with high levels of stefin B and patients with high levels of cystatin C exhibited a significantly higher risk of death than those with low levels of inhibitors (hazard ratio = 1.6; 95% confidence interval, 1.2-2.2; P = 0.002 for stefin B; hazard ratio = 1.3; 95% confidence interval, 1.0-1.8; P = 0.04 for cystatin C). Our results reveal a correlation between high levels of extracellular cysteine proteinase inhibitors and short survival in patients with colorectal cancer, and the data thus support previous studies suggesting a contributing role of protease inhibitors in the progression of cancer.

The inhibition of cysteine proteinase (papain) by human, bovine, horse, rabbit, guinea pig, rat, hamster, and mouse plasma, and pig, sheep, goat, and dog sera was investigated. The rat and mouse plasma and the pig serum showed such high inhibitory capacity as 813, 380, and 508%, respectively, of the human plasma. On the other hand, the horse, guinea pig, and rabbit plasma indicated 36, 40, and 54%, respectively, of the human plasma. A cysteine proteinase inhibitor (s) was separated from alpha-macroglobulin by Sephacryl S-300, and the inhibitory activity of all of the mammalian plasma/sera except for the guinea pig plasma appeared at around the 50-150-kDa region. The guinea pig plasma exhibited well-resolved two peaks of 100 and 200 kDa.

The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves breakdown of the elastic laminae. Elastolytic cysteine proteases, including cathepsins S and K, are overexpressed at sites of arterial elastin damage, but whether endogenous local inhibitors counterbalance these proteases is unknown. We show here that, whereas cystatin C is normally expressed in vascular wall smooth muscle cells (SMCs), this cysteine protease inhibitor is severely reduced in both atherosclerotic and aneurysmal aortic lesions. Furthermore, increased abdominal aortic diameter among 122 patients screened by ultrasonography correlated inversely with serum cystatin C levels. In vitro, cytokine-stimulated vascular SMCs secrete cathepsins, whose elastolytic activity could be blocked when cystatin C secretion was induced by treatment with TGF-beta(1). The findings highlight a potentially important role for imbalance between cysteine proteases and cystatin C in arterial wall remodeling and establish that cystatin C deficiency occurs in vascular disease.

Protein inhibitors capable of inhibiting BCP (Bombyx cysteine proteinase) were found in the larval-pupal hemolymph of Bombyx mori. Two forms of the inhibitors, named BCPI (BCP inhibitor) alpha and BCPI beta, were purified from the pupal hemolymph by heat treatment and column chromatographies on CM-cellulose, Toyopearl HW-50, Phenyl-Sepharose, and Mono Q. Purified BCPI beta gave a single protein band with a molecular mass of 10,500 daltons on SDS-PAGE. BCPI alpha is mostly composed of the same molecular mass protein as BCPI beta. Both forms were inhibitory towards other cysteine proteinases such as cathepsins L,B and papain but had no effects on trypsin and pepsin. Both forms inhibited the processing of the enzymatically inactive proform of BCP (pro-BCP) to the activated mature BCP. BCPI alpha and BCPI beta shared many other features such as molecular mass determined by gel filtration, antigenicity, and HPLC profiles. NH(2)-terminal amino acid sequencing of the purified inhibitors revealed that three amino acid residues were different in the BCPI alpha and BCPI beta sequences, all others being identical. The hemolymph BCP inhibitor increased activity approximately four- to fivefold at the time of spinning and maintained this level of activity during pupation., (Copyright 1999 Wiley-Liss, Inc.)

Cystatin C is a small basic protein with a MW of 13,359 Daltons, consisting of a non-glycosylated polypeptide chain containing 120 amino-acid residues. Cystatin C is produced in all the nucleated cells of the human body and its output rate is constant. The kidney is the main catabolic site of cystatin C, since the protein, by virtue of its low MW and its positive charge at normal pH, is freely filtered by the glomerulus and almost completely reabsorbed, catabolised and broken down in the cells of the proximal convoluted tubule. It is practically entirely filtered via the glomerular membrane, without any significant tubular secretion. The constant production rate of cystatin C in all the tissues, its elimination via the glomerular filter and its non-dependence on many extrinsic factors, including sex, age, diet, inflammation, are potentially ideal conditions for an endogenous biochemical marker of glomerular filtration. A recent method for determining cystatin C, is based on an immune reaction, could increase its clinical application. Not many studies have been conducted to date on cystatin C in children. The cystatin C concentration was higher during the first few days of life (range: 1.64-2.59 mg/L) with a rapid reduction during the first 4 months. Beyond the first year of life, cystatin C concentration became constant, with a reference range of 0.7-1.38 mg/L. On the basis of the data currently available, neonatal serum cystatin C would appear to derive from the newborn itself. In fact no correlations were found between maternal and neonatal serum cystatin C values. Cystatin C determination appears to be at least equivalent to serum creatinine measurement for the assessment of glomerular filtration rate in children. Further extended studies are needed to investigate these aspects more thoroughly in neonates.