Quantitative bioanalytical LC-MS/MS assay for S130 in rat plasma-application to a pharmacokinetic study.

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.

Nonclinical and clinical pharmacological characterization of the potent and selective cathepsin K inhibitor MIV-711.

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 (Ki: 0.98 nmol/L) with > 1300-fold selectivity towards other human cathepsins. MIV-711 inhibited human osteoclast-mediated bone resorption with an IC50 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.

Role of activation of lipid peroxidation in the mechanisms of acute methanol poisoning.

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.

The selective cathepsin K inhibitor MIV-711 attenuates joint pathology in experimental animal models of osteoarthritis.

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.

Analysis of the safety and efficacy of combined extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy for the treatment of complex renal calculus.

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.

Applicability of in vitro-in vivo translation of cathepsin K inhibition from animal species to human with the use of free-drug hypothesis.

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 IC50 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 EC50 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.

Effect of initial periodontal therapy on oxidative stress markers in gingival crevicular fluid, saliva, and serum in smokers and non-smokers with chronic periodontitis.

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.

On the role of survivin as a stem cell biomarker of canine hair follicle and related tumours.

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.

Oxidative stress in stable cystic fibrosis patients: do we need higher antioxidant plasma levels?

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.

[The levels of serum cystatin C in patients with tumors and healthy controls].

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.

[Cistatin C as a novel marker of renal function impairement and cardiovascular risk].

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.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from Angiostrongylus cantonensis.

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.

The imbalance of cathepsin B-like activity in acromegalic patients--preliminary report.

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.

Modulation of allergenicity of major house dust mite allergens Der f 1 and Der p 1 by interaction with an endogenous ligand.

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.

The importance of determining the aggressiveness of prostate cancer using serum and tissue molecular markers.

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.

Purification and characterization of a cysteine protease inhibitor from chum salmon (Oncorhynchus keta) plasma.

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: a promising marker and predictor of impaired renal function.

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.

Plasma cystatin-C and development of coronary heart disease: The PRIME Study.

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.

Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas.

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.

[Serum stefin A in patients with type 2 diabetes]. / Stefina A w osoczu krwi chorych na cukrzyce typu 2.

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.