key: cord-0008537-q72zp2o4
authors: Grubb, Anders O.
title: Cystatin C-Properties and use as diagnostic marker
date: 2006-11-06
journal: Adv Clin Chem
DOI: 10.1016/s0065-2423(01)35015-1
sha: 0c2c9843d300150c32c42c751ae5832267bdc5e1
doc_id: 8537
cord_uid: q72zp2o4

This chapter focuses on the most well characterized inhibitors—cystatin C—and provide some information on its structure, biochemical properties, its role in normal and abnormal physiological processes, as well as on its use as a diagnostic marker. A major part of the cysteine proteases are evolutionary related to the structurally well–defined cysteine protease papain and are called papain–like cysteine proteases. The biological roles and the cystatin superfamily inhibitors of papain–like cystein proteases are also discussed. The aminoacid sequence and schematic structure of human cystatin C is also presented. The evolutionary relationships among all known inhibitory active human cystatins and kininogen cystatin domains are diagrammatically represented. The distribution of cystatins in body fluids and additional functions attributed to cystatin C are described. The serum or plasma cystatin C is used as a marker for glomerular filtration rate (GFR). The urine cystatin C is used as a marker for proximal tubular damage. The two types of brain hemorrhage associated with Cystatin C amyloid deposits are also demonstrated. The conditions connected with deposition of amyloid β–protein in cystatin C and cerebral hemorrhage is also provided.

isolated fractions thereof (G8) inhibit papain. In retrospect, it might be concluded that Grob detected the inhibitory capacities of the plasma proteins cystatin C, lowand high-molecular-weight kininogens, and ~2-macroglobulin. However, it was not until the 1980s and 1990s that the majority of the multitude of presently identified cysteine protease inhibitors were isolated and structurally and functionally characterized (A1, B 1, B5, F2, F3, I2, N3, N4, R6, R7, T9) . The most comprehensive class of cysteine protease inhibitors is the cystatin superfamily of inhibitors. The name "cystatin" was originally given to a low-molecular-weight inhibitor of papain isolated from chicken egg white (B5, B8) , since this inhibitor was a cvsteine protease inhibitor. The middle section, stat, of cystatin might also be considered as alluding to the capacity of cystatins to arrest the activity of cysteine proteases, since stat-is the supine stern of the Latin verb sisto, meaning "to arrest." In 1986 it was agreed that "cystatins" should be used to denote the entire superfamily of cysteine protease inhibitors structurally and functionally related to chicken cystatin (B3) .

Cystatins have been demonstrated not only in higher organisms but also in a large number of plants and lower organisms including rice seeds, Drosophila, and Candida albicans (D3, K6, T8) .

The amino acid sequence of human cystatin C (Fig. l) was determined in 1981 (G11) and, since it did not display any significant homology with the sequences of any protein of the superfamilies known then, it was evident that it belonged to a new protein superfamily. Retrospectively, it can be seen that the amino acid sequence of cystatin C was the first sequence ofa cystatin to be determined (B5) . The function of cystatin C as an inhibitor of cysteine proteases was identified about 2 years later, when the sequence of chicken cystatin was determined, showing that the two proteins had a sequence identity of 44% (B2, B25, $2. T10) . Studies during the two last decades have identified 10 further human cysteine protease inhibitors, which display strong sequence homologies to cystatin C and chicken cystatin and, consequently, belong to the human cystatin superfamily. The human cystatin family therefore presently comprises 11 identified proteins ( Table 1 ) . Two of these, cystatins A and B, form the family 1 cystatins and are mainly, or exclusively, intracellular proteins, while cystatins C, D, E, F, S, SA, and SN are mainly extracellular and/or transcellular proteins and constitute the family 2 cystatins. The family 3 cystatins, high-and low-molecular-weight kininogen, contain three cystatin domains each (S 1) and are mainly intravascular proteins, which, in addition to being inhibitors of cysteine proteases, are precursor molecules for the production of the vasoactive kinins, bradykinin and kallidin (M9) . High-molecularweight kininogen also participates in the contact-phase activation of the endogenous blood coagulation cascade (M9) . The three cystatin domains (called DI-D3) 120 FIG. I. Amino acid sequence and schematic structure of human cystatin C. The shaded area marks the inhibitory site tbr papain-like cysteine proteases, which does not overlap with the inhibitory site for mammalian legumains comprising, inter alia, the Asn 39 residue. The arrow indicates the Leu 68 residue, which is replaced with a Gin residue in the cerebral hemorrhage producing cystatin C variant. The asterisk marks the Pro 3 residue, which is partly hydroxylated. of the kininogens inhibit not only papain-like cysteine proteases (D2 and D3) but also calpains (D2) . Figure 2 displays a schematic illustration of the evolutionary relationships among all known inhibitory active cystatins and kininogen cystatin domains.

All human cystatins are assumed to have major biological roles as inhibitors of one or more target proteases of human and/or nonhuman origin. Identification of target proteases of biomedical relevance is, however, difficult. Only few examples of clear-cut identifications of target proteases for inhibitors are known, and these identifications usually rely on experiments by Nature and not by Man. Two examples are the identifications of granulocyte elastase as a target enzyme for cq-antitrypsin revealed by the pathophysiology of OL 1-antitrypsin deficiency (emphysema and liver cirrhosis) (03) and of thrombin as a target enzyme lbr antithrombin revealed by the pathophysiology of antithrombin deficiency (thromboembolism) (C3) . No serious disease states in which the major pathophysiological events can be ascribed to the lack of the specific inhibitory capacity of a cystatin have so far been described. Individuals with complete deficiencies of high-and low-molecular-weight kininogens have been described, but they do not seem to suffer from any serious pathophysiological abnormality (C7) . Mutations in the genes for cystatins B and C produce progressive myoclonus epilepsy (B12, LI, L2, P2, P3) and hereditary cerebral hemorrhage (A6, G2, P1), respectively, but the present knowledge of the pathophysiology of these disorders does not allow the identification of target proteases for any of these cystatins. Although therefore no unequivocal identification of target proteases for human cystatins has been feasible yet, it is possible to select, or exclude, candidate target proteases for the different cystatins based upon theoretical considerations (B 13) as well as upon comparisons with known examples of target proteases for, inter alia, those serine protease inhibitors mentioned above. One requirement that must be fulfilled by a cystatin to render it a biomedical significant inhibitor of a potential target enzyme is that the molar concentration of the cystatin must be higher than that of the active protease at the site in the body where the protease is released. A second requirement is that the equilibrium constant for dissociation of a protease-cystatin complex must be low to secure a negligible amount of free proteolytic activity at equilibrium. Finally, the association rate constant for the formation of the protease-cystatin complex Fic. 2. Schematic diagram of the evolutionary relationships among all known inhibitory active human cystatins and kininogen cystatin domains. The phylogenetic tree was constructed using "Growtree," included in the GCG software package (version 8.1; Genetic Computer Group, Inc., Madison. WI). The phylogenic distances were obtained according to the method of Kimura (K4) . The reconstruction of the tree was done by the unweighted-pair group method using arithmetic averages. must be high so that any free protease activity is rapidly quenched. Since all human cystatins display a unique set of equilibrium and association rate constants when tested against a limited collection of cysteine proteases, each cystatin has a unique inhibitory spectrum (Table 2) . However, these inhibitory spectra are usually overlapping, and it is quite probable that some cystatins might share target proteases.

The distribution in body fluids of the different cystatins is remarkably different (Fig. 3) . For example, while cystatin C is present in appreciable amounts in all investigated body fluids, cystatins S, SN, and SA are virtually confined to saliva, tears, and seminal plasma (A1). Cystatin D is present only in saliva and tear fluid (AI, F3). In some body compartments, e.g., spinal fluid, cystatin C represents more than 90% of the total molar concentration of cysteine protease 

Data from (A9, LII, N3, N4, S1).

inhibitors, while in other compartments, e.g., blood plasma, it only represents a few percent of the total cysteine protease inhibitory capacity (A1). Moreover, the total cysteine protease-inhibiting capacity varies also considerably among different body compartments. For example, the total papain-inhibiting capacity of blood plasma is about 12 ixmol/L, while that of cerebrospinal fluid is less than 1 Ixmol/L (A1). Since each body fluid displays a unique set of cystatins, it is also clear that the different body fluids display unique cysteine protease inhibitory spectra, although these partially overlap, like the inhibitory spectra of the individual cystatins. Table 3 shows that different cysteine proteases with different catalytic properties are controlled by separate cystatins in the various body fluids.

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C 13, with human legumain as a typical enzyme (C6) . Human legumain has, like cathepsin S, been proposed to be involved in the class II MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases ( Fig. 1 ) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A10). Cystatin C has also been suggested to possess biological functions, presumably unrelated to its protease-inhibiting potential. For example, human cystatin C has been described to play a regulatory role in inflammatory processes, imer alia, by down-regulation of the phagocytosis-associated respiratory burst reaction displayed by polymorphonuclear neutrophils as well as by down-regulation of their chemotactic response (L6, L7). Chicken cystatin, and thus probably human cystatin C, has also been shown to up-regulate nitric oxide release from peritoneal macrophages (V1). However, these suggested additional functions of cystatin C remain to be confirmed.

The protein now generally designated with the functional name cystatin C was first discovered in 1961 (B26, C8, M1) and its function as a cysteine protease inhibitor was thus unidentified for more than 20 years. As a consequence, several trivial names were used for the same protein, and it is important to know these for complete retrieval of data on cystatin C by bibliographic studies. The following trivial names can be found in the literature: y -trace, post--,/-globulin, gamma-CSF, post-gamma protein, ",/c-globulin, 8 aT, and high alkaline fraction (HAF) (B26, C1, C8, H9, HI0, K5, L3, M2, M4, Sll).

The complete amino acid sequence of the single polypeptide chain of human cystatin C was determined in 1981 (G1 1) and later corroborated by identification and sequencing of the corresponding cDNA ( Fig. 4) (A3) and gene (AS, A7).

The three-dimensional structure of cystatin C is not yet determined, although some crystallographic data are available (K8), but it can be presumed that it is similar to that described for the homologous protein chicken cystatin (B 19 ) and this has, at least partially, been confirmed by NMR studies (El) . A schematic structure for human cystatin C is given in Fig. 1 .

Studies of truncated forms of cystatin C (A8) and of cystatin C variants produced by site-directed mutagenesis (B 18, H4, HS, H6, L 12, M7), as well as identification of sequence similarities between all cystatins, have indicated that the inhibitory center of cystatin C for papain-like cysteine proteases comprises three peptide segments, Arg8-Leu9-Vall°-Gly 11, Gln55-Ile56-ValS7-AlaSS-Gly59, and Prol°S-Trpl°6. Peptidyl derivatives, structurally based upon the aminoterminal segment of the inhibitory center, have been synthesized and shown to be efficient inhibitors of cysteine proteases (G9, H3) . Some of these peptidyl derivatives have also displayed antibacterial and antiviral properties (B 16, B17). Table 4 displays some of the physicochemical properties of cystatin C as well as its normal concentration in body fluids.

No investigations have demonstrated that the diagnostic usefulness of the serum level of cystatin C is different from that of the plasma level of cystatin C. The term Numbering of the nucleotide sequence starts at the first nucleotide and proceeds in the 5' to 3' direction. Amino acid numbering begins with residue l of the mature protein (G11 ) and the putative hydrophobic signal sequence thus comprises residues -26 to 1. The Kozak initiation consensus and the polyadenylation signal are underlined. "serum cystatin C" will therefore in the following paragraphs, unless otherwise indicated, also refer to the plasma level of cystatin C.

Determination of the structure of the human cystatin C gene and its promoter has demonstrated that the gene is of the housekeeping type, which indicates a stable production rate of cystatin C by most nucleated cell types (A7) . The presence of a hydrophobic leader sequence in precystatin C (Fig. 4) strongly indicates that the protein normally is secreted (A3, A7). Indeed, immunochemical and Northern blot studies of human tissues and cell lines have shown that cystatin C and/or its mRNA is present in virtually all investigated cell types (A7, J1, L9, L10, $7). Likewise, investigations of the production of cystatin C by human cell lines in culture have displayed that nearly all cell lines investigated secrete cystatin C (B 14, C4, N4) (Abrahamson, M., personal communication). Studies of the serum level of cystatin C in large patient cohorts have failed to correlate the serum level to any pathophysiological state besides those affecting the glomerular filtration rate, which also is compatible with a stable secretion of cystatin C from most human tissues (G12, K9, N2, $6) . However, some reports have described that stimulation of macrophages in vitro down-regulates their secretion of cystatin C (C4, W l), but inflammatory conditions are not generally associated with decreased serum levels of cystatin C.

Blood plasma proteins with molecular masses below 15-25 kDa are generally almost freely filtered through the normal glomerular membrane and then almost completely reabsorbed and degraded by the normal proximal tubular cells. This should consequently also be true for cystatin C with a molecular mass of 13 kDa and with a probable ellipsoid shape with axes of about 30 and 45 A (B19). Indeed, studies of the handling of human cystatin C in the rat have shown that the plasma renal clearance of cystatin C is 94% of that of the generally used GFRmarker 51Cr-EDTA and that cystatin C thus is practically freely filtered in the glomeruli (T4) . At least 99% of the filtered cystatin C was found to be degraded in the tubular cells. Figure 5 shows the rat plasma concentration of intact human 125I-cystatin C and 51Cr-EDTA relative to the initial concentrations after intravenous injection. Figure 6 displays the plasma disappearance of cystatin C in normal and nephrectomized rats and indicates that the renal plasma clearance of cystatin C is about 85% of the total plasma clearance (renal + extrarenal). When the GFR of a set of rats was variably lowered by constricting their aortas above the renal arteries, the renal plasma clearance of cystatin C correlated strongly with that of 51Cr-EDTA (Fig. 7) , with a linear regression coefficient of 0.99 and with the r intercept not being statistically different from 0 (T4). This observation clearly implied an insignificant peritubular uptake of cystatin C. Immunohistochemical and Northern blot studies of human kidneys have also strongly indicated that human cystatin C normally is degraded by proximal tubular cells after its passage through the glomerular membrane (J 1).

The knowledge that most human tissues produce cystatin C and that it, being a low-molecular-mass protein, is removed from plasma by glomerular filtration, suggested that its plasma, or serum, level might be a potentially good marker for GFR. Early investigations demonstrated that serum cystatin C, indeed, was a marker for GFR, at least as good as serum creatinine in the populations investigated (G12, $6) . These studies also showed that the serum cystatin C level was a better GFR marker than the serum levels of the other low-molecular-mass proteins investigated, [32-microglobulin, retinol-binding protein, and complement factor D (GI 2, $6). However, in these early studies the cystatin C concentration was determined by enzyme amplified single radial immunodiffusion (L 15). This procedure is slow, requiring at least 10-20 h, and has a relatively high coefficient of variation (about 10%), which decrease the usefulness of the obtained serum cystatin C value as a GFR marker in the clinical routine. The development, about 10 years later, of automated particle-enhanced immunoturbidimetric methods, which were rapid as well as more precise, therefore significantly improved the possibility of using serum cystatin C as a GFR marker in clinical routine work (K9, N2) . So did the introduction of a sandwich enzyme immunoassay for the determination of serum cystatin C (P4). Since the automated particle-enhanced immunoturbidimetric procedure for determination of serum cystatin C was introduced in 1994, the vast majority of all studies of the use of serum cystatin C as a GFR marker have relied upon the commercially available version of this procedure. A commercially available automated particle-enhanced immunonephelometric method has also recently been described (E2, F1, M10) .

Serum creatinine is ubiquitously used as an indicator for GFR despite the knowledge that a substantial proportion of patients with reduced GFR display serum creatinine levels within the normal range and that even a 50c~ reduction of GFR not infrequently is associated with a normal concentration of serum creatinine (LS, P5, $3). The usefulness of serum creatinine as a marker for GFR is limited by, inter alia, the influence of an individual's muscle mass on the production rate of creatinine (H8, P5, $5), by the tubular secretion and reabsorption of creatinine, by the dietary intake of creatine and creatinine, and by analytical difficulties (P5) . These significant limitations in the use of serum creatinine as an indicator for GFR has made it of interest to search for better indicators for GFR. Several recent studies have compared the use of serum cystatin C and creatinine as markers for GFR as determined by "golden standard" procedures based upon determinations of the plasma clearance of injected low-molecular-mass substances such as CrSI-EDTA, 99mTc-DTPA, and iohexol. These studies have indicated either that serum cystatin C is a better GFR marker than serum creatinine, particularly for individuals with small to moderate decreases in GFR, in the so-called creatinine-blind GFR range, or that the two parameters are of equal value as GFR indicators (B20, B22, H7, J4, K9, N2, N6, P4, P7, R2, R3, S12, S13, T6, V2). Figures 8 and 9 illustrate one Fl~. 8. Correlation between glomerular filtration rate and (top) reciprocal serum cystatin C (mg/liter) or (bottom) reciprocal serum creatinine (~zmol/liter) in 27 male and 24 female patients; .... is the lower reference limit for glomerular fltration rate. The difference in the diagnostic capacity of serum cystatin C and serum creatinine to identify patients in the "creatinine-blind'" area with a glomerular filtration rate of 60-80 ml/min x 1.73 m 2 is obvious in this investigation of a population of patients with various renal conditions. Diagnostic specificity FIG. 9 . Nonparametric ROC plots for the diagnostic accuracy of serum cystatin C and creatinme in distinguishing between normal and reduced glomerular filtration rate (> and < 80 ml/minx 1.73 in 2, respectively) in 51 patients with various renal conditions (the same population as the one displayed in Fig. 8 ).

study indicating the usefulness of serum cystatin C in the creatinine-blind GFR range. Nearly all investigations have emphasized that serum cystatin C, in contrast to serum creatinine, is uninfluenced by gender and muscle mass. Several studies indicate that virtually the same reference values might be used for serum cystatin C for males and females from 1 year of age, up to 50 years of age, when the age-related decline in GFR becomes significant (B20, B22, H7, LI4, N6, R3).

Serum cystatin C has also been described to be a better predictor than serum creatinine of fasting total homocysteine serum levels, probably because of its closer correlation to GFR (B23, N7). solution by quantitative amino acid analysis and spectrophotometric analysis and then diluting it with cystatin C-free human serum to physiological concentrations (K9) . Based upon the use of such a cystatin C calibrator and a commercially available automated particle-enhanced immunoturbidimetric method, several studies of reference values for serum cystatin C, comprising populations of both adults and children have been published (B20, B22, H7, K9, L14, N6) . The results for adults have generally shown that there is no sex differences for any age group and that the well-known decrease in GFR with age is mirrored by an increase in the cystatin C level with age (Fig. 10) . However, the decrease of GFR with age is slow before 50 years of age, and it has been suggested (N6) that it is sufficient for most practical purposes to use separate reference values only for the age groups 20-50 and above 50 years of age (Table 5) .

The results for children (Fig. 11 ) have demonstrated that the cystatin C level, in contrast to the creatinine level, was constant for children beyond the first year and with no difference between the sexes (B20, B22, H7, R3) . The recommended reference values for children beyond 1 year of age were virtually identical to those recommended for adults 20-50 years of age, so it might be justifiable to use the same values for both age groups (Table 5 ). It should be emphasized, however, that efficient international use of serum cystatin C as a GFR marker requires the establishment of a generally available international cystatin C calibrator. The relatively large variation in the reference values for serum cystatin C suggested so far (R1, R3) is most probably due to the use of different, often ill-described, cystatin C calibrators. FIc. 12. Serum 132-microglobulin in relation to age in a population of 121 healthy women and 121 healthy men, 20-89 years old. No sex difference was foun& but the influence of age on glomerular filtration rate and thus on serum 132-microglobulin is evident.

Since all plasma proteins with molecular masses below 15-25 kDa are almost freely filtered through the normal glomerular membrane, their serum concentrations in a person will be strongly influenced not only by their production rates, but also, and often to at least the same extent, by the person's GFR. Thus, when the production rates of such low-molecular-mass proteins are of interest for evaluation of biomedical processes, the diagnostic specificity of the ratios between the serum concentrations of such proteins and cystatin C can be expected to be higher than that of the serum concentration of each specific protein. For example, the influence of age on GFR and thus on the serum [32-microglobulin level (Fig. 12) is not seen for the ratio between the serum levels of [32-microglobulin and cystatin C (Fig. 13 ) (N6). This ratio might thus be a more specific marker for cell proliferation than the isolated serum [32-microglobulin level.

When the first automated particle-enhanced immunoturbidimetric method was introduced, it was claimed to be undisturbed by hypertriglyceridemia (K9) . However, widespread clinical use of it since then has demonstrated that the results are influenced by sample turbidity caused by, inter alia, chylomicronemia, which might produce both falsely low and high values for serum cystatin C (Nilsson-Ehle, R, submitted manuscript; Grubb, A., unpublished results) . The influence of chylomicronemia on the analytical procedure might partly explain the relatively large biological variation reported for serum cystatin C (K3) as well as the outcome of some studies which have failed to show any advantage of serum cystatin C over serum creatinine as a GFR marker. Studies of the biological variation of serum ....................................................................................................................................................... FIG. 13. Serum 132-microglobulin/cystatin C ratio in relation to age in a population of 121 healthy women and 121 healthy men, 20-89 years old. The serum 132-microglobulin/cystatin C ratio is, in contrast to the serum levels of each separate protein, not influenced by the age-dependent decrease in glomerutar filtration rate.

cystatin C, using nonturbid samples, indicate that it is comparable to that of serum creatinine (N5) (Nilsson-Ehle, R, submitted manuscript).

It has also been observed that some, but not all, rheumatoid factors interfere in some of the presently used particle-enhanced immunoturbidimetric methods and produce erroneously high results (Grubb, A., unpublished results) .

It should also be emphasized that although the precision of the automated particle-enhanced immunometric methods is higher than that of the enzymeamplified single radial immunodiffusion first used to quantitate serum cystatin C, it is still lower than that for most methods for determination of serum creatinine. Moreover, the data for the intraindividual variation of serum cystatin C strongly indicate that a higher precision of the method would markedly improve the clinical usefulness of serum cystatin C determinations according to the criterion of Cotlove (N6).

Available evidence indicates that serum cystatin C is a better marker for GFR than serum creatinine, particularly for the identification of an initial small decrease in GFR, i.e., in the so-called creatinine-blind GFR range. The most efficient use of this knowledge in clinical practice requires that quantitative methods of good precision, undisturbed by sample turbidity, are used. At least some of the presently available particle-enhanced immunometric methods seem to fulfil the first criterion of acceptable precision, but nonturbid fasting samples should preferably be used until new methods undisturbed by turbidity are developed.

Combined use of serum cystatin C and creatinine produces the best possible information on GFR in situations where more accurate, but invasive and more expensive, clearance determinations cannot be performed for biomedical or economic reasons. If both serum cystatin C and creatinine are within the relevant reference limits, the risk of missing a decrease in GFR will be minimal.

In situations when the GFR has been determined by accurate invasive clearance methods, either serum cystatin C or creatinine might be used to follow changes in GFR. However, as long as the precision of the methods for creatinine determination is higher than that of the methods for cystatin C determination, there are presently no valid reasons to use serum cystatin C for this purpose, particularly since creatinine determinations generally are cheaper than cystatin C determinations.

It should be observed that there is a clear possibility that some renal disease processes might differently affect the filtration of cystatin C, a positively charged 13,000-Da molecule, and the filtration of creatinine, an uncharged 113-Da molecule. Indeed, although serum cystatin C has been described to be a better GFR marker than serum creatinine for patients with renal transplants (R8), transplanted and untransplanted patients with the same reduction in inulin clearance have been reported to display different levels of serum cystatin C (B21). Future studies might therefore show that the use of several GFR markers, differing in physicochemical properties, might be optimal for the noninvasive monitoring of kidney function.

Since cystatin C is a low-molecular-mass protein, it is almost freely filtered through the normal glomerular membrane and then nearly completely reabsorbed and degraded by the normal proximal tubular cells. The urine level of cystatin C is therefore low in healthy individuals. The mean urine cystatin C in an adult healthy population has been estimated to 0.095 rag/liter or 8.0 mg/mol creatinine with a range of 0.033-0.29 mg/liter or 5.2-13.3 mg/mol creatinine (L15). Proximal tubular dysfunction results in impaired reabsorption of low-molecular-mass proteins and increased urinary excretion of cystatin C can therefore be used as a sensitive marker for disease processes affecting proximal tubular cells (A1, C I0, K1, K2, L15, L16, T5) . However, the practical use of the urine level of cystatin C as a marker for tubular dysfunction is hampered by two facts. First, the upper reference limit for the urine concentration of cystatin C is so low (L15) that presently available rapid and cheap immunochemical methods cannot be used to demonstrate the small increases in the urinary levels of cystatin C which signal initial, and often reversible, stages of proximal tubular dysfunction. Second, cystatin C is proteolytically degraded in a significant proportion of native urine samples (G 13, T7) . Although these two difficulties can be overcome by the use of sensitive, more sophisticated quantitative methods and the addition of preservatives to urine samples (A1, T2), respectively, this state of affairs argues against the practical use of urine cystatin C as a marker for proximal tubular dysfunction (GI3) . The urine level of free protein HC (alias eq-microglobulin) seems to be a more practical marker for proximal tubular dysfunction as the upper reference limit is high (about 8 rag/liter or 700 mg/mol creatinine) and since protein HC is stable in most native urine samples (G13, T2, T3) . But it cannot be excluded that future studies will display that the diagnostic potentials of urine cystatin C and urine protein HC might differ in some disease states, since cystatin C is positively charged at the pH range of urine, while protein HC is negatively charged.

Cystatin C amyloid deposits have been demonstrated to be associated with two types of brain hemorrhage. One type is the dominantly inherited form of cerebral hemorrhage, which is caused by a mutation in the cystatin C gene and displays amyloid deposition of the cystatin C variant but no co-deposition of amyloid [3-protein. The other type comprises the cerebral hemorrhage conditions, which are connected with cerebral deposition of amyloid [3-protein and display co-deposition of wildtype cystatin C in the amyloid.

The Icelandic physician Arni Arnason described in 1935 several families suffering from high incidences of cerebral hemorrhage affecting young adults and showed that the disease displayed autosomal dominant inheritance (Fig. 14) (A 12). Afflicted individuals generally suffered from their first cerebral bleeding before 40 years of age and subsequently from recurrent multifocal cerebral hemorrhages. It was not possible to identify carriers of the trait in the affected families, but Gudmundsson et al. showed in 1972 that the disease was associated with amyloid deposits mainly located in the media of the walls of cerebral medium-sized arteries and suggested "hereditary cerebral hemorrhage with amyloidosis (HCHWA)'" as a suitable name for the syndrome (G 14) . The N-terminal sequence of a main component of the amyloid fibril was determined in 1983 and found to be identical to the sequence of cystatin C starting at residue 11 (C9) . Subsequent immunohistochemical studies corroborated that cystatin C was a major component of the amyloid deposits (L13) and showed that also vessels and other tissues outside the central nervous system contained cystatin C amyloid deposits (B9, L13) . In 1984 immunochemical quantitation of the level of cystatin C in cerebrospinal fluid demonstrated that virtually all carriers of the trait for the disease, independent of whether they had suffered from their first cerebral hemorrhage or not, could be identified by their low cystatin C concentration (G10, J3). Not only did this observation allow the identification of healthy carriers of the disease trait, but also suggested that the pivotal pathophysiological process was an abnormal metabolism of cystatin C. Subsequent studies showed that the complete sequence of the cystatin C polypeptide chain deposited as amyloid in one individual with brain hemorrhage differed in one position from the sequence previously determined for cystatin C isolated from a Swedish patient without cerebral hemorrhage (G2). It was therefore suggested that a point mutation responsible for this amino acid substitution, a glutamine residue replacing a leucine residue at position 68 of the cystatin C polypeptide chain, could constitute the genetic background to the disease, although it also might represent a disease-unrelated polymorphism (G2). The potential disease-causing mutation would, according to the sequence later determined for a full-length cDNA encoding cystatin C, destroy an Alu I-cleavage site in the cystatin C gene (A3). This observation allowed the identification of a restriction fragment length polymorphism permitting identification of carriers of the mutation (P1) as well as the construction of a simple and rapid polymerase chain reaction (PCR)-based procedure for the same purpose (A6) . When individuals from seven afflicted families were studied, it was observed that all individuals suffering from early-onset cerebral hemorrhage carried one mutated allele, while most healthy relatives and all unrelated persons did not (A6) . The results of these studies therefore permitted the conclusion that the investigated mutation causes the disease and that all patients are heterozygous (A6, P1).

Two designations for the disease originally described by Arnason are presently used, viz., hereditary cystatin C amyloid angiopathy (HCCAA) and "hereditary cerebral hemorrhage with amyloidosis, Icelandic" (HCHWA-I). The first mentioned might be the preferred one, since it indicates the amyloid-forming protein and agrees with the recent observations that the amyloid depositions are neither confined to the cerebral vasculture (B9, L13) , nor to Icelandic patients (G5) . It is also an appropriate designation for the condition before the first cerebral hemorrhage has occurred.

The disease should be considered when a normotensive, previously healthy individual below 40 years of age is hit by a severe cerebral hemorrhage and particularly if young relatives previously have suffered from brain hemorrhage. Recurrent cerebral hemorrhages generally occur and result in increasing motor disability and gradual loss of mental functions, but in some individuals the disease does not progress for several years and a few carriers of the mutated allele attain a normal life span (J2, O1) . In a few cases the presenting symptom may be dementia rather than brain hemorrhage (J3) . The disease is not uncommon in the Icelandic population, with more than 150 cases described during the last 70 years, but it is rare (G5) or not described in other populations (G4, M8) .

When HCCAA is suspected because of clinical observations and/or family history, the diagnosis can easily be verified by demonstrating a low cerebrospinal fluid level of cystatin C (G10, J3) or the presence of the mutated cystatin C allele producing the Leu 68--+Gln cystatin C variant (A6, P1). The first-mentioned procedure requires lumbar puncture and, to secure a stable level of cystatin C in the sample, the addition of a serine protease inhibitor, e.g., benzamidinium chloride, directly when the sample is drawn (Grubb, A., unpublished results) . The PCRbased procedure is more robust, does not require lumbar puncture, and can be used for prenatal diagnosis and is therefore presently the preferable diagnostic method.

It has been possible to produce the disease-causing Leu68--~Gln cystatin C variant in an Escherichia coli expression system (A4) . Parallel studies of Leu68--~ Gln cystatin C and the wild-type protein have revealed that although both are efficient inhibitors of cysteine proteases, they differ considerably in their tendency to dimerize and form aggregates. While wild-type cystatin C is monomeric and functionally active even after prolonged storage at elevated temperatures, Leu68--+Gln cystatin C starts to dimerize and lose inhibitory capacity immediately after its isolation. The dimerization of Leu68--~Gln cystatin C is highly temperaturedependent, with a rise in incubation temperature from 37 to 4ffC resulting in a 150% increase in dimerization rate and a considerable concomitant rise in the formation of larger aggregates (Fig. 15) a pathophysiological process in which Leu68---~Gln cystatin C, due to its tendency to spontaneously aggregate, is partly trapped intracellularly and not secreted from the cell as efficiently as the wild-type protein. Continuous intracellular accumulation of Leu68--+Gln cystatin C would, in combination with its specific physicochemical properties, lead to amyloid formation, cell damage, and death (A4) . Recent in vitro studies have indeed shown that the intracellular processing of Leu68---~Gln cystatin C differs from that of wild-type cystatin C (B15) and results in the formation of stable dimers that are partially retained in the endoplasmic reticulum (B 11). It has also been asserted that no Leu68---,Gln cystatin C is secreted from the cells of HCCAA patients (A13) , but this has been questioned (B 11). The temperature dependence of the aggregation of Leu68--+ Gin cystatin C might be of clinical relevance, since medical intervention to abort febrile episodes might reduce the in vivo formation of aggregates in carriers of the disease trait and thus possibly delay the point of time for their initial brain hemorrhage (A4).

Cerebral amyloid angiopathy with wild-type, or a variant of~ amyloid [~-protein as the major amyloid constituent, is a condition with a high prevalence in the elderly and is also commonly found in patients with Alzhemier's disease or Down's syndrome (G6, H1, I3, M5, V3) . The condition is associated with cerebral hemorrhage and may account for more than 10% of the brain hemorrhage cases in the elderly (I3). Immunohistochemical investigations of the amyloid deposits have demonstrated that all, or a considerable portion of them, display cystatin C immunoreactivity in addition to their amyloid [3-protein immunoreactivity (B 10~ HI, I3, M5, M6, V3) . Quantitative estimations have generally indicated that cystatin C is a minor constituent of the deposits, however (I3, M5, M6, V3) . Efforts to demonstrate the presence of cystatin C variants, e.g., the one producing HCCAA, in the amyloid deposits have so far been unfruitful (A 11, I4, M6, N 1). It has been reported that the cerebrospinal fluid level of cystatin C is low in some of these conditions ($4), but if this observation can be used for diagnostic purposes is still uncertain. It is evident that the pathophysiological significance of the occurrence of cystatin C as a minor constituent in the amyloid deposits of these conditions remains to be determined.

The author's work described in this discourse was supported by the Swedish Medical Research Council (project 5196) and by A. Osterlund's, A. Pfihlsson's, and G. and J. Kock's Foundations.

Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids

Efficient production of native, biologically active human cystatin C by Escherichia coli

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C

Increased body temperature accelerates aggregation of the Leu-68-+Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopatby

The human cystatin C gene (CST3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20

Hereditary cystatin C amyloid angiopathy: Identification of the disease-causing mutation and specific diagnosis by polymerase chain reaction based analysis

Structure and expression of the human cystatin C gene

Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site

Giant cell arteritis in association with cerebral amyloid angiopathy: Immunohistochemical and molecular studies

Apoplexie und ihre Vererbung

Hereditary cystatin C amyloid angiopathy: Monitoring the presence of the Leu-68---~Gln cystatin C variant in cerebrospinal fluids and monocyte cultures by MS

Human Protein Data

The place of human ",/-trace (cystatin C) amongst the cysteine proteinase inhibitors

Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin

Cysteine proteinase inhibitors of the cystatin superfamily, hz

Handbook of Proteolytic Enzymes

Types and families of endopeptidases

Cystatin, the egg white inhibitor of cysteine proteinases

Skin deposits in hereditary cystatin C amyloidosis

Dementia with nonhereditary cystatin C angiopathy

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Novel cystatin B mutation and diagnostic PCR assay in an Unverricht-Lundborg progressive myoclonus epilepsy patient

Pathophysiological interpretation of kinetic constants of protease inhibitors

Promoter-mediated, dexamethasone-induced increase in cystatin C production by HeLa cells

Intracellular accumulation of the amyloidogenic L68Q variant of cystatin C in NIH/3T3 cells

Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor

Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus

The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine prnteinases

The 2.0 ,~ X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases

Reference values for cystatin C serum concentrations in children

Cystatin C serum concentrations underestimate glomerular filtration rate in renal transplant recipients

Cystatin C--A new marker of glomerular filtration rate in children independent of age and height

Serum cystatin C as a determinant of fasting total homocysteine levels in renal transplant recipients with a normal serum creatinine

Cloning expression, and sequencing of a protease gene (tpr) from Porphyromonas gingi~'alis W83 in Escherichia coli

Human cystatin, a new protein inhibitor of cysteine proteinases

The occurrence of post-gamma protein in urine: A new abnormality

Post-'y-globulin: Isolation and physicochemical characterization

A comparison of the enzymatic properties of the major cysteine proteinases from Trypanosoma congolense and To'panosoma cruz, i. Mol

VCH Verlagsgesellschaft mbH

Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro

Expression of cathepsin L in human tumors

Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase

Genetic basis of total kininogen deficiency in Williams' trait

Proteins in normal cerebrospinal fluid not found in serum

Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein, gamma trace

Direct radioimmunoassay of rat cystatin C: Increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy

High-level expression of active human cystatin C in Escherichia coli

In vivo and in vitro evidence for the involvement of cysteine proteinases in bone resorption

Sequence analysis, and chromosomal localization of a gene encoding a cystatin-like protein from Drosophila melanogastel

but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts

Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L

NMR structural studies of human cystatin C dimers and monomers

Evaluation of the Dade Behring N Latex Cystatin C assay on the Dade Behring Nephelometer lI System. Scand

Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems (BNA, BN II)

Structure and expression of the gene encoding cystatin D, a novel human cysteine proteinase inhibitor

Human cystatin D. eDNA cloning, characterization of the Escherichia colt expressed inhibitor, and identification of the native protein in saliva

Pycnody~nstosis. a I~sosomal disease caused by cathepsin K deficiency

Amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of Icelandic type is a variant of'y-trace basic protein (cystatin C)

The endo/lysosomal protease cathepsin B is able to process conalbumin fragments for presentation to T cells, hmnunology 74(3 I

A molecular genetic ~;tud 5 of intracerebral hemorrhage

Cystatin C mutation in an elderly man with spuradic amyloid angiopathy and intracerebral hemorrhage

Cerebral amyloid angiopathy: Prospects for clinical diagnosis and treatment

Proteolytic enzymes. 1. The control of their activity

Proteolytic enzymes. 111. Further studies on protein, polypeptide and other inhibitor,', of serum proteinase, leucoproteinase, trypsin and papain

Synthesis of cysteine proteinase inhibitors structurally based on the proteinase interacting N-terminal region of human cystatin C

Abnormal metabolism of'T-trace alkaline microprotein. The basic defect in hereditary cerebral hemorrhage with amyloidosis

Human 'y-trace, a basic microprotein: Amino acid sequence and presence in the adenohypophysis

Serum concentration of cystatin C, factor D and [32-microglobulin as a measure of glomerular filtration rate

Diagnostic value of analysis of cystatin C and protein HC in binh)gical fluids

Hereditary cerebral hemorrhage with amyloidosis

Co-localization of [3/A4 and cystatin C in cortical blood vessels in Dutch, but not in Icelandic hereditary cerebral hemorrhage with amyloidosis

Leukocystatin, a new class 11 cystatin expressed selectively by hematopoietic cells

Cystatin C based peptidyl diazomethanes as cysteine proteinase inhibitors: lnflucnce of the peptidyl chain length

hnportance of the evolutionarily con served glycine residue in the N-terminal region of human cystatin C (Gly-I 1) for cysteine endopeptidase inhibition

Structural basis for different inhibitory specificities of human cystatins C and D

Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases

Serum cystatin C as a determinant of glomerular filtration rate in children

Measurement of muscle mass in humans: Validity of the 24-hour urinary creatinine method

Trace proteins in biological fluids. IV. Physicochemical properties and sites of formation of 3'-trace and [3-trace proteins

Use of an antiserum against cerebrospinal fluid in demonstration of trace proteins in biological fluids

Biosynthesis and transport of lysosomal enzymes in human monocytes and macrophages. Effects of ammonium chloride, zymosan and tunicamycin

Cerebral amyloid angiopathy: A significant cause of cerebellar as well as lobar cerebral hemorrhage in the elderly

Cerebral amyloid angiopathy in the elderly: The clinicopathological features, pathogenesis, and risk factors

Transthyretin and cystatin C are catabolized in proximal tubular epithelial cells and the proteins are not useful as markers for renal cell carcinomas

Hereditary cystatin C (3'-trace) amyloid angiopathy of the CNS causing cerebral hemorrhage

Hereditary cystatin C (3'-trace) amyloid angiopathy of the central nervous system causing cerebral hemorrhage

Cystatin C: A promising marker of glomerular filtration rate to replace creatinine

Low molecular weight proteinuria in Chine se herbs nephropathy

Low molecular weight proteinuria in human immunodeficiency virus-infected patients

Biological variation of cystatin C: Implications for the assessment of glomerular filtration rate

Estimation of evolutionary distances between homologous nucleotide sequences

Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia

Two distinct cystatin species in rice seeds with different specificifies against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin--II

Interactions between a viral protease and cystatins

Expression of a selenomethionyl derivative and preliminary crystallographic studies of hmnan cystatin C

Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate

Unstable insertion in the 5' flanking region of the cystatin B gene is the most common mutation in progressive myoclonus epilepsy type 1, EPMI

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPMI)

Immunological comparison of some proteins found in cerebrospinal fluid, urine and extracts from brain and kidney

The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase

Human cystatin C, a cysteine proteinase inhibitor, inhibits bone resorption in vitro stimulated by parathyroid hormone and parathyroid hormone-related pcptide of malignancy

Modulation of phagocytosis-associated respiratory burst by human cystatin C: Role of the N-ternfinal tet.apeptide Lys-Pro-Pro-Arg

Neutrophil cbemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteases, ln

Serum creatinine and renal function

Cystatin C and transthyretin expression in normal and neoplastic tissues of the human brain and pituitary

Cystatin C in the human pancreas and gut: An immuuohistochemical study of normal and neoplastic tissues

Characterization by rapidkinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actindin

Evidence for the interaction of valine-10 in cystatin C with the $2 subsite of cathepsin B

hnmunohistochemical characterization of the amyloid deposits and quantitation of pertinent cerebrospinal fluid proteins in hereditary cerebral hemorrhage with amyloidosis. Stroke 18f 2 I

The cerebrospinal fluid and plasma concentrations of ~/-trace and 132-microglobulin at various ages and in neurological disorders

Quantitation of ",/-trace in human biological fluids: Indications for production in the central nervous system. Scand

Human brain cortical neurons contain 3,-trace. Rapid isolation, immunohistochemical and physicochemical characterization of human 'y-trace

Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolvtica

Immunochemical evidence for a gamma globulin peculiar to cerebrospinal fluid

Purification of the gamma globulin characteristic of cerebrospinal fluid

An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation

Les proteines du liquid cephalorachidien

lmmunohistochemical characterization of cerebrovascular amyloid in 46 autopsied cases using antibodies to 13 protein and cystatin C

Characterization of amyloid fibril protein from a case of cerebral amyloid angiopathy showing immunohistochemical reactivity for both 13 protein and cystatin C

Amino acid substitutions in the N-terminal segment of cystatin C create selective protein infiibitors of lysosomal cysteine proteinases

Absence of cystatin C mutation in sporadic cerebral amyloid angiopathy-related hemorrhage

Kininogens. bl "'Human Protein Data

Quantitative automated particleenhanced immunonephelometric assay for the routinary measurement of human cystatin C

No mutations in cystatin C gene in cerebral amyloid angiopathy with cystatin C deposition

Serum cystatin C measured by automated immunoassay: a more sensitive marker of changes in GFR than serum creatinine

Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins

Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor

Reference intervals for the glomerular filtration rate and cell-proliferation markers: Serum cystatin C and serum [32-microglobulin/cystatin C-ratio. Stand

The increase of plasma homocysteine concentrations with age is partly due to tile deterioration of renal function as determined by plasma cystatin C

The characteristics ofcysteine proteinases of parasitic protozoa

The molectdar pathology of hereditary cy,qatin C amyloid angiopathy causing brain hen]orrhage

Cysteine proteases and their inhibitors

Alpha-l-proteinase inhibitor, ht "Human Protein Data

Mutation in cystatin C gone causes hereditary brain haemorrhage

Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis it] cystatin B deficient mice

eta& Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM 1 )

Sandwich enzyme immunoassay of cystatin C in serum with commercially available antibodies

Serum creatinine as an index of renal function: New insights into old concepts

Developmental regulation of invariant chain proteolysis controls MHC class 1I trafficking in mouse dendritic cells

Is serum cystatin C a sensitive marker of glomerular filtration rate (GFR)? A preliminary study on renal transplant patients

Differences in ~,ecretion of the proteinase cathepsin B at the edges of human breast carcinomas and fibroadenomas

Serum cystatin C as an endogenous marker of the renal function--A review

Sermn cystatin C as a marker of the renal function. Stand

Reference interxal for serum cystatin C in children

MEROPS: The peptidase database

is and processing of cathepsin L, an elastase, by human alveolar macrophages. Bloc'hem

Human Protein Data

Human Protein Data

Rapid and accurate assessment of glomerular filtration rate in patients with renal transplants using serum cystatin C

Inhibition of a Plasmodium vinckei cysteine proteinase cures murine malaria

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodiumfalciparnm

Human low-Mr Kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases

Amino acid sequence and possible secondary structure

Limitations of creatinine as a filtration marker in glomerulopathic patients

Diagnosis of cerebral amyloid angiopathy by enzyme-linked immunosorbent assay of cystatin C in cerebrospinal fluid

Rapid evaluation of creatinine clearance

The blood serum concentration of cystatin C (7-trace) as a measure of the glomerular filtration rate. Scand

lmmunolocalization of cystatin C. Distribution in normal human tissues (in Swedish)

Association with plasma membrane in metastatic tumors

Cathepsin B and cystatins: evidence for a role in cancer progression

Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer

The HAF enigma: Origin and clinical consequences of the appearance of high alkaline fractions on isoelectric focusing patterns of cerebrospinal fluid

Improved prediction of decreased creatinine clearance by serum cystatin C: use in cancer patients before and during chemotherapy

Correlation of plasma concentrations of cystatin C and creatinine to inulin clearance in a pediatric population

Coexistence of renin and cathepsin B in epithelioid cell secretory granules

Stability of albumin, protein HC, immunoglobulin G, K-and ~.-chain immunoreactivity, orosomucoid and c~l-antitrypsin in urine stored at various conditions. Scand

Analysis of proteinuria: Reference limits for urine excretion of albumin, protein HC, immunoglobulin G, K-and X-immunoreactivity, orosomucoid and ~l-antitrypsin. Scand

Renal handling of radiolabelled human cystatin C in the rat. Stand

Competition bet~een albumin and 1o~ molecular-weight proteins for renal tubular uptake in experimental nephropathies. NephroH 66/4 ~

Cystatin C measurement and its practical use in patients with various renal diseases

The different electrophoretic forms of post-',/-globulin, their antigenic identity and their structural variability

Candi&l albicans produces a cystatin-type cysteine proteinase inhibitor

The cystatins: Protein inhibitors of cysteine proteinases

Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin 1tom chicken egg white

Cystatins up-regulate nitric oxide release from interferon-y-activated mouse peritoneal macrophages

Relationships among serum cystatin C, serum creatinine, lean tissue mass and glomerular filtration rate in healthy adults

lmmunnreactive A4 and -`/-trace peptide colocalization in amyloidotic arteriolar lesions in brains of patients with Alzheimer's disease

Constitutive secretion of cystatin C (-`/-trace) by monocytes and macrophages and its downregulation alter stimulation