Shengyuan Xu

Lipocalins as biochemical markers of disease

Lipocalins as biochemical markers of disease have been used extensively. The clinical indications relate to almost any field of medicine, such as inflammatory disease, cancer, lipid disorders, liver and kidney function. Some of the more well-known lipocalins that have been used as markers of disease are orosomucoid, Protein HC (alpha(1)-microglobulin), apolipoprotein D, retinol-binding protein, complement C8 gamma, prostaglandin D synthase and human tear prealbumin, and these markers will be briefly reviewed in this article. Emphasis, however, will be put on the description of another newly described lipocalin, i.e. human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), since the body fluid measurement of HNL/NGAL was shown to be a superior means to distinguish between acute viral and bacterial infections and also to accurately reflect the activity and involvement of neutrophils in a variety of other diseases.

Serum measurements of human neutrophil lipocalin (HNL) discriminate between acute bacterial and viral infections

Human neutrophil lipocalin (HNL) is a recently identified protein from human neutrophil granules. The concentrations of HNL in the circulation were measured, in a group of patients with acute infections, using a radioimmunoassay. The concentrations of HNL in patients infected by viruses and bacteria were 93.78 +/- 45.30 micrograms l-1 (SD), 404.14 +/- 355.02 micrograms l-1 (SD) in serum, and 47.81 +/- 18.18 micrograms l-1 (SD), 145.46 +/- 194.32 micrograms l-1 (SD) in plasma, respectively. The differences between the two patient groups were highly significant. There was a significant correlation between serum HNL and plasma HNL levels in bacterial infections (r = 0.73, p < 0.0001). The HNL serum levels also correlated with those of C-reactive protein (CRP) (r = 0.59, p < 0.0001). Determination of HNL in serum was more specific and sensitive than CRP in the distinction between viral and bacterial infections. At a cut-off of 155 micrograms l-1 (HNL in serum), the positive and negative predictive values for the diagnosis of bacterial infections were 92 and 96%, respectively, which were superior to the optimal predictive values of CRP. Thus, the determination of HNL level is useful in the diagnosis of acute bacterial infections.

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

BACKGROUND AND OBJECTIVES Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions. RESULTS The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL. CONCLUSIONS The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.

Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis

Background and aim: Accumulation and infiltration by neutrophil granulocytes is a prominent feature in the local inflammatory process in ulcerative colitis (UC). The present study was performed to evaluate human neutrophil lipocalin (HNL) as a specific neutrophil marker in the inflamed lesions of the colon and rectum in patients with colitis and proctitis. Methods: The activity of intestinal neutrophils with respect to release of granule proteins was studied in 18 patients with UC (10 with colitis and eight with isolated proctitis) and in 18 healthy controls using perfusion fluid and biopsies from the sigmoid colon and rectum. The released amounts of the neutrophil granule proteins HNL and myeloperoxidase (MPO) were determined by radioimmunoassays, and the location of HNL and MPO in biopsies from colonic mucosa was examined by immunohistochemistry. Results: Mucosal release of HNL and MPO was increased 10–55-fold in patients with colitis and proctitis compared with controls. Their bowel biopsies demonstrated that only neutrophils were stained with anti-HNL. We also found correlations between HNL and levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 8 (IL-8) in perfusion fluids from the sigmoidal segments of patients with proctitis, between HNL and GM-CSF in rectal segments in patients with proctitis, and in sigmoidal segments in patients with colitis. Conclusion: We conclude that the increased release of HNL and MPO in colorectal perfusion fluids indicates neutrophil involvement in the local inflammatory process, and suggest that HNL may serve as a specific marker of intestinal neutrophil activation in UC. GM-CSF, and to some extent IL-8, may play a role in neutrophil accumulation and priming in this disease.

The development of an assay for human neutrophil lipocalin (HNL)--to be used as a specific marker of neutrophil activity in vivo and vitro

Human neutrophil lipocalin (HNL) is a newly discovered protein from human neutrophil secretory granules. A double-antibody radioimmunoassay (RIA) was developed for the measurement of HNL in various body fluids and its high specificity was confirmed by the absence of cross-reaction with other granulocyte granule proteins. The RIA measures HNL within the range of 4-256 micrograms/l. The intra- and interassay coefficients of variation were less than 6% and 10%, respectively. When HNL was added to serum samples full recovery was obtained. Sera and plasma from 100 apparently healthy individuals revealed a mean level of 78.40 micrograms/l (range 37.95-190.87 micrograms/l) in serum and a mean level of 50.65 micrograms/l (range 30.51-105.8 micrograms/l) in EDTA-plasma. The distribution of HNL after gel filtration indicated that HNL exists mainly in two major forms, dimer and monomer. This, in addition to the excellent recovery, suggests that these major forms of HNL do not bind to compounds in serum or plasma that would interfere with the assay. The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the measurement of HNL in blood and other body fluids.

Human neutrophil lipocalin (HNL) as a diagnostic tool in children with acute infections: A study of the kinetics

AIM To study the changes in blood of human neutrophil lipocalin (HNL) and C-reactive protein (CRP) during the course of an acute infection in children. METHODS Children (n=92) hospitalized with symptoms and signs of acute infections were included and categorized into five groups, i.e. bacterial infection, suspected bacterial infection, viral infection, suspected viral infection and others. Blood was taken at admittance and the following 3-4 d for the measurement of CRP and HNL. RESULTS Both CRP and HNL were significantly raised at admittance in bacterial infection as compared to viral infection (p<0.001). After 25-48 h, 83% of the children with bacterial infections still had raised CRP levels in contrast to 11% having raised HNL levels. The levels of CRP, but not those of HNL, were significantly correlated to days of symptoms before admission. CONCLUSIONS HNL is a promising diagnostic tool in the distinction of acute infections caused by bacteria or virus. The differences in the kinetics of CRP and HNL make HNL a better marker for monitoring antibacterial treatment, since HNL is probably elevated only when an active bacterial infection is at hand.

Immunoassays distinguishing between HNL/NGAL released in urine from kidney epithelial cells and neutrophils

BACKGROUND The distinction between monomeric human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), secreted by injured kidney tubular cells, and dimeric HNL/NGAL, released by activated neutrophils, is important to accurately diagnose acute kidney injury (AKI). METHODS 132 urine samples from 44 intensive care unit (ICU) patients and five urine samples from non-ICU patients with urinary tract infections (UTIs) were analyzed by two monoclonal enzyme-linked immunosorbent assays (ELISA-1 and ELISA-2). The presence of monomeric and/or dimeric HNL/NGAL in each sample was visualized by Western blotting. RESULTS The ELISA-1 detected both monomeric and dimeric HNL/NGAL whereas the ELISA-2 almost exclusively detected dimeric HNL/NGAL with an area under the receiver-operating characteristics curve (AuROC) of 0.90. The ELISA-1/ELISA-2 ratio detected the monomeric form with an AuROC of 0.92. In 32 AKI patients, dimer-specific ELISA-2 levels decreased pre-AKI whereas the monomer-specific ELISA-1/ELISA-2 ratio gradually increased beyond AKI diagnosis. High ELISA-2 levels and/or low ELISA-1/ELISA-2 ratios detected a predominance of dimeric HNL/NGAL in urine from the patients with UTIs. CONCLUSIONS In combination, our two ELISAs distinguish monomeric HNL/NGAL, produced by the kidney epithelium, from dimeric HNL/NGAL, released by neutrophils during AKI development, as well as reduce the confounding effect of neutrophil involvement when bacteriuria is present.

Assays of urine levels of HNL/NGAL in patients undergoing cardiac surgery and the impact of antibody configuration on their clinical performances

BACKGROUND Acute kidney injury (AKI) is one of the most serious postoperative complications of cardiac surgery. The lack of early and powerful markers for AKI makes the morbidity and mortality still very high. HNL (Human neutrophil lipocalin)/NGAL (Neutrophil gelatinase-associated lipocalin) was recently shown as a novel biomarker for AKI after cardiac surgery. METHODS Serial urine samples from 59 patients undergoing cardiac surgery were analyzed by polyclonal antibody based radioimmunoassay (RIA), monoclonal-polyclonal antibody based enzyme-linked immunosorbent assay (ELISA). RESULTS We found 10 to 100-fold increases in urine HNL/NGAL levels in about half of the patients 2 h after termination of the operation and elevated levels in all patients 72 h post operation. The urine levels of HNL/NGAL showed a weak, but significant relation with kidney function as measured by plasma levels of cystatin C or creatinine. The 2 h-HNL/NGAL levels were positively correlated to extracorporeal circulation time (p<0001). The assays were well correlated, but had different clinical performances. CONCLUSIONS We confirmed that urine HNL/NGAL may be a useful early biomarker of postoperative kidney injury. The results indicate that the antibody configuration of the assay has an impact on the clinical performance of the assay.

The effect of granulocyte colony‐stimulating factor (G‐CSF) on the degranulation of secondary granule proteins from human neutrophils in vivo may be indirect

Granulocyte colony‐stimulating factor (G‐CSF) was administered at a dose of 7.5 or 10 μg/kg s.c. once daily for 6 d (days 1–6) to two groups consisting of eight and six healthy volunteers. The administration of G‐CSF resulted in a rapid decrease in neutrophil counts and serum levels of the secondary granule protein, human neutrophil lipocalin (HNL) after 30 min, followed by a recovery and gradual increase within 180 min. The number of circulating neutrophils and plasma and serum levels of neutrophil secondary granule proteins were dramatically elevated on day 2 (1 d after the administration of G‐CSF) and stayed so until day 7. The plasma levels of HNL and lactoferrin (LF) showed a biphasic pattern with peaks at day 2 and days 5–7, and remained highly elevated at day 12. The serum levels of HNL and LF increased rapidly (about 8‐fold and 6‐fold, respectively) on day 2 and stayed elevated until day 7, subsequently returning to baseline levels. At day 5, neutrophil release induced in vitro by f‐MLP was significantly enhanced. The cellular contents of HNL and LF were reduced to about 50% of levels before G‐CSF administration at day 5. The release of lactoferrin and HNL, but not of myeloperoxidase (MPO), was slightly enhanced after preincubation of isolated normal neutrophils with G‐CSF in vitro, but no obvious release of these proteins was observed with G‐CSF alone. The administration of G‐CSF resulted in a dramatic increase in the alkaline phosphatase (AP) activity in the plasma membrane, with maximal activity occurring at day 5. Furthermore, during administration of G‐CSF, TNF‐α in plasma increased about 25‐fold. TNF‐α started to rise at day 2 and peaked at day 6. After discontinuation of G‐CSF the levels of TNF‐α gradually decreased. The elevated levels of TNF‐α (tumour necrosis factor‐α) were temporally correlated to the other signs of neutrophil activation. GM‐CSF and IL‐8, however, were not detected in plasma. Our data suggest that G‐CSF affects the neutrophils not only directly but also indirectly by the induction of the production of other cytokines such as TNF‐α.

Granulocyte colony-stimulating factor (G-CSF) induces the production of cytokines in vivo

Granulocyte colony‐stimulating factor (G‐CSF) is a haematopoietic growth factor required for the proliferation and differentiation of haematopoietic precursors of neutrophil granulocytes and is now used to overcome congenital and acquired neutropenia. In addition to increasing the numbers of neutrophils in vivo and modulating neutrophil functions, G‐CSF may induce the production of cytokines such as tumour necrosis factor α (TNF‐α). In the present study, the plasma levels of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in six healthy volunteers given G‐CSF at 10 μg/kg once daily for 6 d were measured and found to be elevated. The elevated levels (P < 0·05) were detected on day 2, peaked on days 6–7 and returned to baseline on day 12. In vitro, G‐CSF did not enhance the secretion of TNF‐α and GM‐CSF from mononuclear cells, whole blood or endothelial cells. However, in the co‐presence of whole blood and endothelial cells, the secretion of TNF‐α was significantly enhanced by G‐CSF at low concentrations. The GM‐CSF secretion, however, was unaltered. G‐CSF pretreatment of whole blood suppressed lipopolysaccharide (LPS)‐induced secretion of TNF‐α and GM‐CSF in a dose‐dependent manner. These results together with our previous findings suggest that G‐CSF induces the production of TNF‐α and GM‐CSF in vivo, and that this production may be due to the co‐effects of endothelial cells and whole blood under the influence of G‐CSF through an as yet unknown network of cells and cytokines. Treatment of whole blood with G‐CSF suppresses LPS‐induced secretion of TNF‐α and GM‐CSF.