The identification of discontinuous epitope in the human cystatin C - Monoclonal antibody HCC3 complex.

Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments - α-helical part, hCC (17-28) and ß4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. SIGNIFICANCE OF THE STUDY: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.

Nanostructure of Edge Dislocations in a Smectic-C* Liquid Crystal.

We report on the first direct nanoscale imaging of elementary edge dislocations in a thermotropic smectic-C* liquid crystal with the Burgers vector equal to one smectic layer spacing d. We find two different types of dislocation profiles. In the dislocation of type A, the layers deformations lack mirror symmetry with respect to the plane perpendicular to the Burgers vector; the dislocation core size is on the order of d. In the dislocation of type S, the core is strongly anisotropic, extending along the Burgers vector over distances much larger (by a factor of 4) than d. The difference is attributed to a different orientation of the molecular tilt plane with respect to the dislocation's axis; the asymmetric layers distortions are observed when the molecular tilt plane is perpendicular to the axis and the split S core is observed when the molecules are tilted along the line.

Serum amyloid a protein and C-reactive protein in normal pregnancy and preeclampsia.

AIMS: To study plasma levels of serum amyloid A protein and C-reactive protein in pregnant women with and without preeclampsia and non-pregnant women. Plasma levels of haptoglobin, orosomucoid and ceruloplasmin were also analyzed. METHODS: The study included 295 women with uncomplicated pregnancies, 57 women diagnosed with preeclampsia, and 58 healthy non-pregnant women. Plasma concentrations of acute phase proteins were analyzed by particle-enhanced immunoassays. Non-parametric Kruskal-Wallis and Mann-Whitney U tests were used to test differences between the groups. RESULTS: Plasma levels of C-reactive protein and ceruloplasmin were increased in pregnant women with and without preeclampsia compared to non-pregnant women. Plasma levels of serum amyloid A protein and C-reactive protein were not elevated in women with preeclampsia compared to women with normal pregnancy. CONCLUSION: The description of preeclampsia as a systemic inflammatory state was not reflected in the plasma levels of serum amyloid A protein and C-reactive protein.

Standardization of Cystatin C: development of primary and secondary reference preparations.

A Primary Reference Preparation has been produced using pure, recombinant, Cystatin C in a solvent of 0.1 mol/L KCl. Dry mass determination of the Primary Reference Preparation resulted in a Cystatin C concentration of 5.20 g/L. Agarose-electrophoresis and SDS-electrophoresis, as well as N-terminal sequencing, verified the purity, homogeneity and identity of Cystatin C in the Primary Reference Preparation. For the Secondary Reference Preparation, a serum pool was collected and stabilized. A pilot batch was made to verify the selected procedure and spiking with the pure, recombinant Cystatin C. The final Secondary Reference Preparation is now produced (4468 vials) and ready for value assignment and further characterization.

Inaccuracy of GFR predictions by plasma cystatin C in patients without kidney dysfunction and in advanced kidney disease.

BACKGROUND: In clinical practice there is need for a simple and reliable test for determination of impaired renal function. With reductions in GFR, the plasma cystatin C concentration (C, mg/l) will increase earlier than serum creatinine, and it is generally agreed that plasma cystatin C is only little affected by body weight, age or sex. However, some reports indicate that cystatin C may be influenced not only by GFR, but also by malignancy, inflammation and high doses of corticosteroids. The aim of the present study was to investigate how plasma cystatin C predicts GFR in distinct subcategories of patients with various disorders as well as in organ transplant patients. METHODS: Plasma cystatin C was measured in 536 patients (age range 0.3-96 years, 262 females, 274 males), consecutively referred to our hospital for determination of GFR by iohexol clearance. Correlations of log GFR vs. log cystatin C were used to compare plasma cystatin C and measured GFR for the following categories: individuals with no known kidney disease (No-KD), malignant patients with (mostly) normal GFR, solid organ-transplanted patients, and patients with native chronic kidney disease (CKD). RESULTS: In patients with normal kidney function and cystatin C level GFR>30 ml/min(-1) (1.73 m2)(-1)) or solid organ transplantation (GFR=84.55 C(1.7666) and GFR=83.95(C-1.5968), respectively). CONCLUSION: Therefore, for these categories, a common equation for all patients with increased cystatin C, irrespective of cause of renal impairment, could be used, namely that presented by Grubb et al. [2005] (GFR=83.93(C-1.676)). However, at marked reductions of renal function (GFR<30 or cystatin C>2), i.e. for CKD Stages 4 and 5, the Grubb prediction equation is less accurate. Based on our data, we suggest the equation GFR=50.52 C(-1.26) for this category of patients.

Increased plasma levels of beta2-microglobulin, cystatin C and beta-trace protein in term pregnancy are not due to utero-placental production.

OBJECTIVE: To study concentration gradients of the low molecular mass proteins, beta2-microglobulin, cystatin C and beta-trace protein, between the uterine and ante-cubital veins, the umbilical artery and vein and in the amniotic fluid compartment. MATERIALS AND METHODS: The study comprised 27 healthy women with uncomplicated pregnancies undergoing caesarean section at term. Samples were collected simultaneously and paired t-tests were used to compare mean plasma concentrations. RESULTS: There was no significant concentration gradient in the plasma levels of beta2-microglobulin, cystatin C or beta-trace protein between the uterine and antecubital veins. There were no correlations between the protein levels in the compartments. CONCLUSION: The utero-placental unit does not contribute significantly to the maternal levels of beta2-microglobulin, cystatin C and beta-trace protein in normal pregnancy, and the proteins are not likely to be transferred across the placental barrier.

Prediction of relative glomerular filtration rate in adults: new improved equations based on Swedish Caucasians and standardized plasma-creatinine assays.

OBJECTIVE: To evaluate newly developed equations predicting relative glomerular filtration rate(GFR) in adult Swedish Caucasians and to compare with the Modification of Diet in Renal Disease(MDRD) and Mayo Clinic equations using enzymatic and zero-calibrated plasma creatinine assays. MATERIAL AND METHODS: GFR was measured with iohexol clearance adjusted to 1.73 m(2). One population sample (n=436/Lund) was used to derive an equation based on plasma-creatinine/age/gender, and a second with the addition of lean body mass (LBM). Both equations were validated in a separate sample (n=414/Malmö). The coefficients of the equations were eventually fine-tuned using all 850 patients and yielding Lund-Malmö equations without (LM) and with LBM-term (LM(LBM)). Their performance was compared with the MDRD(CC) (conventional creatinine calibration), MDRD(IDMS) (isotope dilution mass spectroscopy traceable calibration) and Mayo Clinic equations. RESULTS: The Lund equations performed similarly in both samples. In the combined set, the Mayo Clinic/MDRD(CC) resulted in +19.0/+10.2 % median bias, while bias for the other equations was < 10 %. LM(LBM) had the highest accuracy (86 % of estimates within 30 % of measured GFR), significantly (p < 0.001) better than for MDRD(IDMS) (80 %). In men with BMI < 20 kg/m(2), MDRD(IDMS)/LM had +46 %/+19 % median bias. MDRD(IDMS) also overestimated GFR by 22 %/14 % in men/women above 80 years of age. The LM(LBM) equation had < 10 % bias irrespective of BMI, age or GFR except for a 15 % negative bias at GFR > 90 mL/min/1.73 m(2). CONCLUSION: The newly developed Lund-Malmö equations for GFR estimation performed better than the MDRD(IDMS) and Mayo Clinic equations in a Swedish Caucasian sample. Inclusion of an LBM term improved performance markedly in certain subgroups.

Temporal changes of the plasma levels of cystatin C, beta-trace protein, beta2-microglobulin, urate and creatinine during pregnancy indicate continuous alterations in the renal filtration process.

OBJECTIVE: To determine the plasma levels of the renal functional markers creatinine, urate, cystatin C, beta2-microglobulin and beta-trace protein in samples from the first, second, early third and late third trimesters of 398 healthy women with uncomplicated singleton pregnancies. MATERIAL AND METHODS: Plasma samples from 58 healthy non-pregnant women served as controls. The creatinine levels were significantly lower at all time-points in pregnancy, whereas the urate levels were lower during the first and second trimesters but increased in the late third trimester. The cystatin C, beta2-microglobulin and beta-trace protein levels displayed similar changes with increased levels in the third trimester but unaltered levels during the first and second trimesters. RESULTS: The results indicate an increased filtration of low-molecular weight molecules during pregnancy, particularly during the first and second trimesters, whereas filtration of 10-30 kDa molecules is decreased in the third but unaltered in the first and second trimesters. The levels of albumin and alph2-macroglobulin were measured in the same samples. CONCLUSIONS: The albumin levels decreased in the second and third trimesters, whereas the levels of chi2-macroglobulin were unchanged, which is compatible with a virtually unaltered transfer of chi2-macroglobulin between the intra- and extravascular space during pregnancy and a significantly increased extravascular fraction of albumin.

Cystatin C, beta-2-microglobulin and beta-trace protein in pre-eclampsia.

BACKGROUND: An altered renal function is an essential component of the patho-physiology of pre-eclampsia. The plasma levels of low molecular mass proteins, e.g. beta-trace protein, beta-2-microglobulin and cystatin C, are increased in the third trimester of normal pregnancy. The plasma levels of cystatin C and beta-2-microglobulin are further increased in pre-eclampsia, and the cystatin C level has been reported to be a reliable marker for the disease. The aim of this investigation was to study the plasma levels of beta-trace protein, beta-2-microglobulin and cystatin C in pre-eclampsia, and to determine the diagnostic performance of these proteins compared to that of urate and creatinine. METHODS: A case-control study of 57 women diagnosed with pre-eclampsia, and 218 healthy women with uncomplicated singleton pregnancies in the third trimester. Women in the catchment area of Lund, Sweden, were included during an 18-month period from October 2003 to April 2005. Venous blood samples were drawn upon inclusion when diagnosis was made. The maternal plasma concentrations of the 3 proteins were analysed by automated particle-enhanced immunoturbidimetric assays. RESULTS: The plasma levels of the 3 proteins were significantly higher in the third trimester of pre-eclamptic patients compared to healthy pregnant women in the third trimester. The upper reference limits (parametric 97.5 percentile) were 2.57 mg/l for beta-2-microglobulin, 0.72 mg/l for beta-trace protein and 1.37 mg/l for cystatin C. ROC analysis showed similar diagnostic performance for the 3 proteins, with beta-trace protein displaying the best diagnostic performance of all the analytes. CONCLUSIONS: In this study, the maternal plasma levels of beta2-microglobulin, beta-trace protein and cystatin C were all significantly elevated in pre-eclampsia compared to those of healthy pregnant women, and displayed similar diagnostic performance for diagnosing pre-eclampsia. The results indicate that low molecular mass proteins are useful as markers of renal impairment in pre-eclampsia.

SAXS studies of human protein HC (alpha1-microglobulin).

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (lambda=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration R(G)= 2.20 nm and D(max)=6.3 nm and the dimer by R(G)=2.99 nm and D(max)=9.5 nm.

Cystatin C reduces the in vitro formation of soluble Abeta1-42 oligomers and protofibrils.

There are an increasing number of genetic and neuropathological observations to suggest that cystatin C, an extracellular protein produced by all nucleated cells, might play a role in the pathophysiology of sporadic Alzheimer's disease (AD). Recent observations indicate that small and large soluble oligomers of the beta-amyloid protein (Abeta) impair synaptic plasticity and induce neurotoxicity in AD. The objective of the present study was to investigate the influence of cystatin C on the production of such oligomers in vitro. Co-incubation of cystatin C with monomeric Abeta1-42 significantly attenuated the in vitro formation of Abeta oligomers and protofibrils, as determined using electron microscopy (EM), dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, thioflavin T (ThT) spectrofluorimetry and gel chromatography. However, cystatin C did not dissolve preformed Abeta oligomers. Direct binding of cystatin C to Abeta was demonstrated with the formation of an initial 1:1 molar high-affinity complex. These observations suggest that cystatin C might be a regulating element in the transformation of monomeric Abeta to larger and perhaps more toxic molecular species in vivo.

Standardization of p-creatinine assays and use of lean body mass allow improved prediction of calculated glomerular filtration rate in adults: a new equation.

OBJECTIVE: To evaluate the Cockcroft-Gault (CG) equation, using various body weight expressions, and the Sawyer equation in predicting glomerular filtration rate (GFR) using an enzymatic and zero-calibrated Jaffe plasma-creatinine assay, and to derive a new robust equation in adults. MATERIAL AND METHODS: The CG weight measures included total, ideal and adjusted body weight (ABW; lowest of total and ideal) and two lean body mass (LBM) expressions, while the Sawyer equation is based primarily on LBM. Iohexol clearance was used to measure GFR. One derivation set (n = 436; enzymatic assay) was used to evaluate and bias-adjust existing equations when indicated, and to derive a new equation based on plasma-creatinine, age, gender and the body weight measure yielding the best adjusted R2. All equations were then validated in a separate set (n = 414; Jaffe assay). RESULTS: The existing equations all performed similarly in both sets. Prediction errors of equations based on LBM showed no correlation with BMI. The CGABW and Sawyer equations performed best. The new equation with LBM yielded the highest adjusted R2. In the combined set (n = 850), its accuracy (86 %/98 % of estimates within 30 %/50 % of measured GFR) was significantly better than for the CGABW (79 %/95 %) and Sawyer equations (79 %/93 %) (p<0.001) for each 30 mL/min GFR subgroup within +/-30 % and +/-50 %, except within +/-30 % >120 mL/min. Prediction error did not correlate with BMI, age or gender. CONCLUSION: A new creatinine-based equation derived in a mainly Caucasian patient sample is a better predictor of GFR than CG-type equations irrespective of the body weight measure used or, if bias-adjusted, when using zero-calibrated creatinine assays.

Glycaemic and nonglycaemic effects of pioglitazone in triple oral therapy of patients with type 2 diabetes.

OBJECTIVES: To examine pioglitazone as add-on to metformin and insulin secretagogues in patients with type 2 diabetes and inadequate glycaemic control and its effect on glycaemic control, surrogate measures of insulin sensitivity (adiponectin) and beta-cell function (proinsulin/insulin) and fluid retention. DESIGN AND SETTING: Prospective open-label study of 54 patients with type 2 diabetes and HbA1c>or=6.5% admitted to outpatient unit at Malmö University Hospital. The patients received 30-45 mg pioglitazone daily during 26 weeks in addition to their existing antidiabetic medication. After 26 weeks, one-third of patients were followed for 3 months without pioglitazone. RESULTS: HbA1c decreased (7.8+/-0.9-6.3+/-0.9%, P<0.001) with 61% of patients achieving levels<6.5%. However, in the group followed for another 3 months HbA1c increased (6.1+/-0.73-7.1+/-0.9, n=18, P<0.001) after pioglitazone withdrawal. Adiponectin increased (6.1+/-2.8-13.2+/-5.8 microg mL-1, P<0.001) and the proinsulin to insulin ratio decreased (0.89+/-0.66-0.66+/-0.53, P<0.001). Nt-proBNP increased from 487.3+/-252.2 to 657.8+/-392.1 pmol L-1 (P<0.001). CONCLUSIONS: Pioglitazone is effective in achieving glycaemic targets and reducing risk factors involved in atherosclerosis and improving beta-cell function when used as part of triple oral therapy in patients with type 2 diabetes and secondary drug failure. Nt-proBNP increase with concomitant decrease in haemoglobin suggests a subclinical sign of fluid retention.

A cystatin C-based formula without anthropometric variables estimates glomerular filtration rate better than creatinine clearance using the Cockcroft-Gault formula.

t In 1976, Cockcroft and Gault introduced a widely used formula comprising several anthropometric variables to compensate for the inadequacies of creatinine level as a marker of glomerular filtration rate (GFR). The present work investigates the possibility of introducing cystatin C-based formulas without anthropometric variables to predict GFR, determined by an invasive "gold standard" procedure (iohexol clearance), and to compare the diagnostic efficiency of such formulas with that of Cockcroft and Gault. All 451 adult patients referred to the University Hospital for determination of GFR by iohexol clearance measurements during a period of 6 months were included in the study. Calculations of bias (median percent error), correlation (adjusted R2), and accuracy (percentage of estimates within 30 and 50% of iohexol clearance) were used in the comparison. The cystatin C-based formula GFR (ml/min)=89.12 x cystatin C(-1.675) had lower bias and higher accuracy in predicting GFR than the Cockcroft-Gault formula. If a cystatin C-based formula including gender was constructed: GFR (ml/min)=99.19 x cystatin C(-1.713) x (0.823 for women), an even lower bias and higher accuracy were obtained. It is suggested that measurement of cystatin C should be used for the initial prediction of GFR of a patient.

Different cysteine proteinases involved in bone resorption and osteoclast formation.

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.

Nt-proANP in plasma, a marker of salt sensitivity, is reduced in type 2 diabetes patients.

OBJECTIVE: We recently showed that plasma concentration of N-terminal atrial natriuretic peptide (Nt-proANP) is strongly directly related to salt sensitivity. The aims of the present study were to test (i) whether plasma concentration of N-terminal brain natriuretic peptide (Nt-proBNP) is related to salt sensitivity and (ii) whether Nt-proANP, as a marker of salt sensitivity, differs between type 2 diabetes patients and nondiabetic subjects without a history of coronary heart disease. METHODS: Nt-proBNP was determined in 30 Swedish normal subjects with heredity for primary hypertension and salt sensitivity was defined as the difference between mean arterial blood pressure after 1 week on a high-salt diet (240 mmol day(-1)) and 1 week on a low-salt diet (10 mmol day(-1)). Nt-proANP was measured in 253 patients with type 2 diabetes and in 230 nondiabetic subjects aged 40-70 years, all without a history of coronary heart disease. RESULTS: Amongst the 30 subjects, in whom salt sensitivity was directly measured, Nt-proBNP was not correlated with salt sensitivity (R=-0.18, P=0.35). Nt-proANP (median, interquartile range) was lower in patients with type 2 diabetes (505, 387-661 pmol L(-1)) than in nondiabetic subjects (536, 421-696 pmol L(-1)) (P=0.02). In a multiple regression analysis heart rate (P <0.00001), diastolic blood pressure (P=0.02) and diabetes status (P=0.02) were inversely related whereas age (P <0.00001), cystatin C (P=0.0006), hypertension treatment (P=0.002) and female sex (P=0.006) were directly related to ln(Nt-proANP). CONCLUSION: In contrast to Nt-proANP, Nt-proBNP is not related to salt sensitivity. Salt sensitivity, as estimated by Nt-proANP, seems to be reduced in type 2 diabetes.

Serum cystatin C advantageous compared with serum creatinine in the detection of mild but not severe diabetic nephropathy.

OBJECTIVE: To determine whether serum cystatin C is more accurate than serum creatinine in the detection of diabetic nephropathy, also after adjustment for age. METHODS: Forty-one patients with type 1 and 82 patients with type 2 diabetes were evaluated with serum creatinine, serum cystatin C, and (51)Cr-EDTA clearance (reference method). Cystatin C was measured by a particle-enhanced turbidimetric method and creatinine by an enzymatic method. Statistical estimations were performed both without and with age adjustment created by z-scores for (51)Cr-EDTA clearance, creatinine, and cystatin C. The cut-off levels for glomerular filtration rate (GFR) ((51)Cr-EDTA clearance) were 60 and 80 mL min(-1) 1.73 m(-2), respectively, in absolute values and 80, 90 and 95% CIs, respectively, in age-adjusted values (z-scores). RESULTS: Estimations without age adjustment showed significantly (P = 0.0132) closer correlation for cystatin C (r = 0.817) versus (51)Cr-EDTA clearance as compared with creatinine (r = 0.678). However, when using age-adjusted values, the correlation for cystatin C and creatinine, respectively, versus (51)Cr-EDTA clearance did not differ. When comparing the diagnostic utilities for serum cystatin C versus serum creatinine in manifest renal impairment (GFR < 60 mL min(-1) 1.73 m(-2) or z-scores <-1.28 SD), there were no significant differences between the two markers whether age adjusted or not. However, for diagnosing mild nephropathy (GFR < 80 mL min(-1) 1.73 m(-2) or z-score -0.84 SD), serum cystatin C is significantly more useful. CONCLUSIONS: Serum cystatin C performed better compared with serum creatinine even when measured enzymatically, to detect mild diabetic nephropathy. However, serum creatinine was as efficient as serum cystatin C to detect advanced diabetic nephropathy.

Gene deletion of cystatin C aggravates brain damage following focal ischemia but mitigates the neuronal injury after global ischemia in the mouse.

Cystatin C is distributed in all human tissues and fluids with a particular abundance in the cerebrospinal fluid. Cystatin C is a strong endogenous inhibitor of lysosomal cysteine proteases, such as cathepsin B, L, H and S, that are involved in various biological processes such as degradation of cellular proteins and regulation of enzymes, as well as in pathological processes. Pharmacological inhibition of cathepsins has been shown to reduce neuronal damage after brain ischemia, suggesting that cystatin C is an endogenous neuroprotectant. Cystatin C has also amyloidogenic properties and is co-localized with beta-amyloid in degenerated neurons in Alzheimer's disease, suggesting a role in neuronal degeneration. To test the hypothesis that endogenous cystatin C is neuroprotective during brain ischemia, global and focal brain ischemia was induced in mice with the cystatin C gene knocked out. Following focal ischemia, larger brain infarcts were found in cystatin C knockout mice, probably due to a reduced inhibition of the cathepsins during ischemia. In contrast, brain damage after global ischemia was diminished in cystatin C knockout mice, suggesting that cystatin C has an aggravating effect on selective neuronal damage after global ischemia.