Kenneth Lau

Vascular endothelial growth factor: A new member of the platelet-derived growth factor gene family

Using applications of the polymerase chain reaction (PCR) technique, cDNA clones have been isolated encoding bovine vascular endothelial growth factor (VEGF), a mitogen with specificity for vascular endothelial cells. Analysis of the clones indicates that VEGF can exist in two forms, probably due to alternative RNA splicing. The amino acid sequences predicted from the clones also show that VEGF shares homologies of about 21% and 24% respectively with the A and B chains of human platelet-derived growth factor (PDGF), and has complete conservation of the eight cysteine residues found in both mature PDGF chains. The homology is not reflected in function, however, since the cell types responsive to VEGF are distinct from those responsive to homo- and heterodimers of the PDGF chains.

Integrative Urinary Peptidomics in Renal Transplantation Identifies Biomarkers for Acute Rejection

Noninvasive methods to diagnose rejection of renal allografts are unavailable. Mass spectrometry followed by multiple-reaction monitoring provides a unique approach to identify disease-specific urine peptide biomarkers. Here, we performed urine peptidomic analysis of 70 unique samples from 50 renal transplant patients and 20 controls (n = 20), identifying a specific panel of 40 peptides for acute rejection (AR). Peptide sequencing revealed suggestive mechanisms of graft injury with roles for proteolytic degradation of uromodulin (UMOD) and several collagens, including COL1A2 and COL3A1. The 40-peptide panel discriminated AR in training (n = 46) and test (n = 24) sets (area under ROC curve >0.96). Integrative analysis of transcriptional signals from paired renal transplant biopsies, matched with the urine samples, revealed coordinated transcriptional changes for the corresponding genes in addition to dysregulation of extracellular matrix proteins in AR (MMP-7, SERPING1, and TIMP1). Quantitative PCR on an independent set of 34 transplant biopsies with and without AR validated coordinated changes in expression for the corresponding genes in rejection tissue. A six-gene biomarker panel (COL1A2, COL3A1, UMOD, MMP-7, SERPING1, TIMP1) classified AR with high specificity and sensitivity (area under ROC curve = 0.98). These data suggest that changes in collagen remodeling characterize AR and that detection of the corresponding proteolytic degradation products in urine provides a noninvasive diagnostic approach.

A diagnostic algorithm combining clinical and molecular data distinguishes Kawasaki disease from other febrile illnesses

BackgroundKawasaki disease is an acute vasculitis of infants and young children that is recognized through a constellation of clinical signs that can mimic other benign conditions of childhood. The etiology remains unknown and there is no specific laboratory-based test to identify patients with Kawasaki disease. Treatment to prevent the complication of coronary artery aneurysms is most effective if administered early in the course of the illness. We sought to develop a diagnostic algorithm to help clinicians distinguish Kawasaki disease patients from febrile controls to allow timely initiation of treatment.MethodsUrine peptidome profiling and whole blood cell type-specific gene expression analyses were integrated with clinical multivariate analysis to improve differentiation of Kawasaki disease subjects from febrile controls.ResultsComparative analyses of multidimensional protein identification using 23 pooled Kawasaki disease and 23 pooled febrile control urine peptide samples revealed 139 candidate markers, of which 13 were confirmed (area under the receiver operating characteristic curve (ROC AUC 0.919)) in an independent cohort of 30 Kawasaki disease and 30 febrile control urine peptidomes. Cell type-specific analysis of microarrays (csSAM) on 26 Kawasaki disease and 13 febrile control whole blood samples revealed a 32-lymphocyte-specific-gene panel (ROC AUC 0.969). The integration of the urine/blood based biomarker panels and a multivariate analysis of 7 clinical parameters (ROC AUC 0.803) effectively stratified 441 Kawasaki disease and 342 febrile control subjects to diagnose Kawasaki disease.ConclusionsA hybrid approach using a multi-step diagnostic algorithm integrating both clinical and molecular findings was successful in differentiating children with acute Kawasaki disease from febrile controls.

Urine Peptidomic and Targeted Plasma Protein Analyses in the Diagnosis and Monitoring of Systemic Juvenile Idiopathic Arthritis

PurposeSystemic juvenile idiopathic arthritis is a chronic pediatric disease. The initial clinical presentation can mimic other pediatric inflammatory conditions, which often leads to significant delays in diagnosis and appropriate therapy. SJIA biomarker development is an unmet diagnostic/prognostic need to prevent disease complications.Experimental DesignWe profiled the urine peptidome to analyze a set of 102 urine samples, from patients with SJIA, Kawasaki disease (KD), febrile illnesses (FI), and healthy controls. A set of 91 plasma samples, from SJIA flare and quiescent patients, were profiled using a customized antibody array against 43 proteins known to be involved in inflammatory and protein catabolic processes.ResultsWe identified a 17-urine-peptide biomarker panel that could effectively discriminate SJIA patients at active, quiescent, and remission disease states, and patients with active SJIA from confounding conditions including KD and FI. Targeted sequencing of these peptides revealed that they fall into several tight clusters from seven different proteins, suggesting disease-specific proteolytic activities. The antibody array plasma profiling identified an SJIA plasma flare signature consisting of tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin (IL)-18, regulated upon activation, normal T cell expressed and secreted (RANTES), P-Selectin, MMP9, and L-Selectin.Conclusions and Clinical RelevanceThe urine peptidomic and plasma protein analyses have the potential to improve SJIA care and suggest that SJIA urine peptide biomarkers may be an outcome of inflammation-driven effects on catabolic pathways operating at multiple sites.

Plasma profiles in active systemic juvenile idiopathic arthritis: Biomarkers and biological implications.

Systemic juvenile idiopathic arthritis (SJIA) is a chronic arthritis of children characterized by a combination of arthritis and systemic inflammation. There is usually non‐specific laboratory evidence of inflammation at diagnosis but no diagnostic test. Normalized volumes from 89/889 2‐D protein spots representing 26 proteins revealed a plasma pattern that distinguishes SJIA flare from quiescence. Highly discriminating spots derived from 15 proteins constitute a robust SJIA flare signature and show specificity for SJIA flare in comparison to active polyarticular juvenile idiopathic arthritis or acute febrile illness. We used 7 available ELISA assays, including one to the complex of S100A8/S100A9, to measure levels of 8 of the15 proteins. Validating our DIGE results, this ELISA panel correctly classified independent SJIA flare samples, and distinguished them from acute febrile illness. Notably, data using the panel suggest its ability to improve on erythrocyte sedimentation rate or C‐reactive protein or S100A8/S100A9, either alone or in combination in SJIA F/Q discriminations. Our results also support the panels potential clinical utility as a predictor of incipient flare (within 9 wk) in SJIA subjects with clinically inactive disease. Pathway analyses of the 15 proteins in the SJIA flare versus quiescence signature corroborate growing evidence for a key role for IL‐1 at disease flare.

“Toxic memory” via chaperone modification is a potential mechanism for rapid mallory‐denk body reinduction

The cytoplasmic hepatocyte inclusions, Mallory‐Denk bodies (MDBs), are characteristic of several liver disorders, including alcoholic and nonalcoholic steatohepatitis. In mice, MDBs can be induced by long‐term feeding with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 3 to 4 months or rapidly reformed in DDC‐induced then recovered mice by DDC refeeding or exposure to a wide range of toxins for only 5 to 7 days. The molecular basis for such a rapid reinduction of MDBs is unknown. We hypothesized that protein changes retained after DDC priming contribute to the rapid MDB reappearance and associate with MDB formation in general terms. Two‐dimensional differential‐in‐gel‐electrophoresis coupled with mass spectrometry were used to characterize protein changes in livers from the various treatment groups. The alterations were assessed by real‐time reverse‐transcription polymerase chain reaction and confirmed by immunoblotting. DDC treatment led to pronounced charged isoform changes in several chaperone families, including Hsp25, 60, 70, GRP58, GRP75, and GRP78, which lasted at least for 1 month after discontinuation of DDC feeding, whereas changes in other proteins normalized during recovery. DDC feeding also resulted in altered expression of Hsp72, GRP75, and Hsp25 and in functional impairment of Hsp60 and Hsp70 as determined using a protein complex formation and release assay. The priming toward rapid MDB reinduction lasts for at least 3 months after DDC discontinuation, but becomes weaker after prolonged recovery. MDB reinduction parallels the rapid increase in p62 and Hsp25 levels as well as keratin 8 cross‐linking that is normally associated with MDB formation. Conclusion: Persistent posttranslational modifications in chaperone proteins, coupled with protein cross‐linking and altered chaperone expression and function likely contribute to the “toxic memory” of DDC‐primed mice. We hypothesize that similar changes are important contributors to inclusion body formation in several diseases. (HEPATOLOGY 2008.)

A Potential Biomarker in the Cord Blood of Preterm Infants Who Develop Retinopathy of Prematurity

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP ≥stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of ≥stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15–16 kD and pI 4–5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7–9 were less abundant in these patients. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as α-, β-, and γ-globin chains. Partial deamidation of Asn139 in β-globin chains was observed only in the pI 4–5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.

The Exosome Total Isolation Chip

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the devices broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

Masking of a cathepsin G cleavage site in vivo contributes to the proteolytic resistance of major histocompatibility complex class II molecules

The expression of major histocompatibility complex class II (MHC II) molecules is post‐translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II‐degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent‐solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)‐DR isolated from both HLA‐DM‐expressing and DM‐null cells. Even following CatG cleavage, peptide binding was retained by pre‐loaded, soluble recombinant HLA‐DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane‐proximal β2 domain. All allelic variants of HLA‐DR tested and murine I‐Ag7 class II molecules were susceptible, whereas murine I‐Ek and HLA‐DM were not, consistent with their altered sequence at the P1’ position of the CatG cleavage site. CatG effects were reduced on HLA‐DR molecules with DRB mutations in the region implicated in interaction with HLA‐DM. In contrast, addition of CatG to intact B‐lymphoblastoid cell lines (B‐LCLs) did not cause degradation of membrane‐bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen‐presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context‐dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.

Microfluidic Device for Coupling Capillary Electrophoresis and Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry

We have designed and fabricated a polydimethylsiloxane (PDMS) microfluidic device for coupling capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The coupling is advantageous in biological research because CE has the power of separating analytes in a sample based on mobility difference and MALDI-MS provides accurate and sensitive mass analysis of the analytes. The goal is realized by fractionating the separated analytes inside the microfluidic device and pushing the analyte fractions into open reservoirs. Each analyte fraction is then mixed with a matrix solution and deposited on a MALDI target for MALDI-MS. Therefore, a two-step analysis of analytes in the form of CE-MALDI-MS is achieved by using the microfluidic device.