Serum KIAA1199 is an advanced-stage prognostic biomarker and metastatic oncogene in cholangiocarcinoma.

BACKGROUND: Cell proliferation and migration are the determinants of malignant tumor progression, and a better understanding of related genes will lead to the identification of new targets aimed at preventing the spread of cancer. Some studies have shown that KIAA1199 (CEMIP) is a transmembrane protein expressed in many types of noncancerous cells and cancer cells. However, the potential role of KIAA1199 in the progression of cholangiocarcinoma (CCA) remains unclear. RESULTS: Analysis of cancer-related databases showed that KIAA1199 is overexpressed in CCA. ELISA, immunohistochemistry, Western blotting and qPCR indicated high expression levels of KIAA1199 in serum, CCA tissues and CCA cell lines. In the serum (n = 41) and large sample validation (n = 177) cohorts, higher KIAA1199 expression was associated with shorter overall survival and disease-free survival times. At the cellular level, KIAA1199 overexpression (OE) promoted CCA growth and metastasis. Subcutaneous tumor xenograft experiments showed that KIAA1199 enhances CCA cell proliferation. Additionally, the expression levels of components in the EMT-related TGF-ß pathway changed significantly after KIAA1199 upregulation and silencing. CONCLUSION: KIAA1199 is a promising new diagnostic molecule and therapeutic target in CCA. The serum KIAA1199 level can be used as a promising clinical tool for predicting the overall postoperative outcomes of patients with CCA. METHODS: CCA-related KIAA1199 data were downloaded from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. To assess the prognostic impact of KIAA1199, an enzyme-linked immunosorbent assay (ELISA) was used to measure the serum level of KIAA1199 in 41 patients who underwent surgical resection. Immunohistochemical staining, Western blotting and qPCR were used to verify and retrospectively review the expression levels of KIAA1199 in cancer tissue specimens from 177 CCA patients. The effect of KIAA1199 on CCA was evaluated by cell-based functional assays and subcutaneous tumor xenograft experiments. The expression levels of proteins associated with epithelial-mesenchymal transition (EMT) and activation of relevant signaling pathways were measured via Western blotting.

The role of hyaluronic acid and hyaluronidase-1 in obstructive sleep apnoea.

Biological functions of hyaluronic acid (HA) depend on its molecular size. High-molecular weight HA (HMW-HA) is an important component of the endothelial wall and has anti-inflammatory and antioxidant properties. Under inflammation or hypoxia, HMW-HA is degraded by hyaluronidases, such as HYAL-1 resulting in pro-inflammatory low-molecular weight fragments. Obstructive sleep apnoea (OSA) is characterised by intermittent hypoxia and systemic inflammation. Our aim was to evaluate circulating HMW-HA and HYAL-1 in OSA. We recruited 68 patients with OSA and 40 control volunteers. After full-night sleep study blood samples were taken for HMW-HA and HYAL-1 measurements. HYAL-1 levels were significantly higher in patients with OSA compared to controls (0.59/0.31-0.88/ng/mL vs. 0.31/0.31-0.58/ng/mL; p = 0.005) after adjustment for gender, age, BMI and smoking. There was a trend for reduced HMW-HA concentrations in OSA (31.63/18.11-59.25/ng/mL vs. 46.83/25.41-89.95/ng/mL; p = 0.068). Significant correlation was detected between circulating HMW-HA and apnoea-hypopnoea-index (r = - 0.195, p = 0.043), HYAL-1 and apnoea-hypopnoea-index (r = 0.30, p < 0.01) as well as oxygen desaturation index (r = 0.26, p < 0.01). Our results suggest that chronic hypoxia is associated with increased plasma HYAL-1 concentration and accelerated HMW-HA degradation. Altered hyaluronan metabolism may be involved in the inflammatory cascade potentially leading to endothelial dysfunction in OSA.

HYAL-1-induced autophagy facilitates pancreatic fistula for patients who underwent pancreaticoduodenectomy.

The main mechanism of hyaluronidase 1(HYAL-1) in the development of postoperative pancreatic fistula (POPF) after pancreatoduodenectomy (PD) was unknown. In this study, a comprehensive inventory of pre-, intra-, and postoperative clinical and biological data of two cohorts (62 pancreatic cancer [PCa] and 111 pancreatic ductal adenocarcinoma [PDAC]) which could induce POPF were retrospectively analyzed. Then, a total of 7644 genes correlated with HYAL-1 was predicted in PDAC tissues and the enriched pathway, kinase targets and biological process of those correlated genes were evaluated. Finally, a mouse pancreatic fistula (PF) model was first built and in vitro studies were performed to investigate the effects of HYAL-1 on PF progression. Our data indicated that preoperative serum HYAL-1 level, pancreatic fibrosis score, and pancreatic duct size were valuable factors for detecting POPF of Grade B and C. The serum HYAL-1 level of 2.07 mg/ml and pancreatic fibrosis score of 2.5 were proposed as the cutoff values for indicating POPF. The bioinformatic analysis and in vitro and in vivo studies demonstrated that HYAL-1 facilitates pancreatic acinar cell autophagy via the dephosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and signal transducers and activators of transcription 3 (STAT3) signaling pathways, which exacerbate pancreatic secretion and inflammation. In summary, the preoperative serum HYAL-1 was a significant predictor for POPF in patients who underwent PD. Tumor-induced HYAL-1 is one of core risk in accelerating PF and then promoting pancreatic secretion and acute inflammation response through the AMPK and STAT3-induced autophagy.

Effect of Liposomal Delivery of Oxidized Dextran with Isonicotinic Acid Hydrazide on Component Profile of Pulmonary Extracellular Matrix in Mice with BCG-Induced Granulomatosis.

Intraperitoneal injections of isonicotinic acid hydrazide (INH), dextrazide (oxidized dextran+INH), or liposomes loaded with dextrazide (INH dose of 14 mg/kg) over 2 months to mice with BCG-induced granulomatosis started from postinfection day 90 induced qualitative and quantitative changes in composition of pulmonary extracellular matrix. Both dextrazide and its liposomal form decreased the levels of sulfated glycosaminoglycans and uronic acids. In contrast to INH, both preparations did not decrease the levels of total glycosaminoglycans, proteins, and galactose. This difference is explained by the fact both free and liposomal dextrazide activated MMP, but did not increase the content of TIMP-1 and TIMP-2, whereas injection of INH was followed by an increase in TIMP-2 content and a decrease in the level of free hydroxyproline, which attested to down-regulation of collagen degradation and maintenance of the conditions for pulmonary fibrosis in mice of this group.

Analyzing Hyaluronidases in Biological Fluids.

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.

Increased OGA Expression and Activity in Leukocytes from Patients with Diabetes: Correlation with Inflammation Markers.

O-linked-ß-N-Acetylglucosaminylation (O-GlcNAcylation), a reversible post-translational modification involved in diabetic complications, is regulated by only two enzymes, O-linked N-acetylglucosamine transferase (OGT) and ß-N-Acetylglucosaminidase (OGA). Increased OGA expression has been described previously in blood cells from patients with diabetes and was interpreted as an adaptative response to hyperglycemia-induced O-GlcNAcylation. OGA expression was thus proposed to have potential utility as a diagnostic marker. The present work was undertaken to determine whether determination of OGA enzymatic activity in blood cells could constitute a more rapidly accessible marker than OGA expression level measurements.Blood samples were obtained from patients with type 2 diabetes from the Department of Diabetology of the Cochin Hospital and healthy volunteers from the French blood Agency. OGA enzymatic activity and OGA mRNA expression levels were evaluated in leucocytes from patients with type 2 diabetes and from healthy donors.OGA activity was higher in leucocytes from patients with diabetes compared to control individuals. Surprisingly, OGA activity was not correlated hyperglycaemia markers (blood glucose, fructosamine, HbA1c) but was positively correlated with the inflammatory marker C-reactive protein. OGA mRNA levels were also increased in leucocytes from patients with diabetes and were correlated with mRNA coding for two pro-inflammatory proteins, TNFα and TxNIP.Therefore, OGA activity in leucocytes might be a more easily accessible biomarker than OGA expression levels. However, changes in OGA activity observed in patients with type 2 diabetes may reflect the inflammatory rather than the glycaemic status of these patients.

6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice.

BACKGROUND: Increased vascular permeability is a pathophysiological hallmark of sepsis and results in increased transcapillary leakage of plasma fluid, hypovolemia, and interstitial edema formation. 6% hydroxyethyl starch (HES 130/0.4) is commonly used to treat hypovolemia to maintain adequate organ perfusion and oxygen delivery. The present study was designed to investigate the effects of 6% HES 130/0.4 on glycocalyx integrity and vascular permeability in lipopolysaccharide (LPS)-induced pulmonary inflammation and systemic inflammation in mice. METHODS: 6% HES 130/0.4 or a balanced electrolyte solution (20 ml/kg) was administered intravenously 1 h after cecal ligation and puncture (CLP) or LPS inhalation. Sham-treated animals receiving 6% HES 130/0.4 or the electrolyte solution served as controls. The thickness of the endovascular glycocalyx was visualized by intravital microscopy in lung (LPS inhalation model) or cremaster muscle (CLP model). Syndecan-1, hyaluronic acid, and heparanase levels were measured in blood samples. Vascular permeability in the lungs, liver, kidney, and brain was measured by Evans blue extravasation. RESULTS: Both CLP induction and LPS inhalation resulted in increased vascular permeability in the lung, liver, kidney, and brain. 6% HES 130/0.4 infusion led to significantly reduced plasma levels of syndecan-1, heparanase, and hyaluronic acid, which was accompanied by a preservation of the glycocalyx thickness in postcapillary venules of the cremaster (0.78 ± 0.09 µm vs. 1.39 ± 0.10 µm) and lung capillaries (0.81 ± 0.09 µm vs. 1.49 ± 0.12 µm). CONCLUSIONS: These data suggest that 6% HES 130/0.4 exerts protective effects on glycocalyx integrity and attenuates the increase of vascular permeability during systemic inflammation.

A Polysaccharide from Amusium Pleuronectes Combined with Praziquantel Treatment Ameliorates Hepatic Fibrosis in Schistosoma Japonicum-Infected Mice.

BACKGROUND Polysaccharides from bivalves have multiple bioactivities in various aspects of biology. However, the role of a polysaccharide derived from Amusium pleuronectes on potential hepatoprotective effects remains unclear. MATERIAL AND METHODS A water-soluble polysaccharide was isolated from Amusium pleuronectes (APS-1) using ultrasound-assisted hot-water extraction. The molecular weight of APS-1 was approximately 11.7 kDa and was determined by calibration with dextran. APS-1 was analyzed by high-performance liquid chromatography (HPLC), and mainly consisted of a uniform glucose polymer. The protective effect of APS-1 on Schistosoma japonicum-induced liver fibrosis was investigated in a mouse model. RESULTS Treatment with APS-1 increased serum levels of interleukin (IL)-12 and interferon (IFN)-γ, increased superoxide dismutase (SOD) activity, and decreased levels of IL-13 and IL-5, and hyaluronidase activity. Moreover, immunohistochemical analysis revealed that the collagen content of hepatic tissue of APS-1-treated mice, including that of collagen I, II, and IV, was dramatically decreased. Furthermore, our data showed that combined treatment of APS-1 with praziquantel had more pronounced effects than treatment with either APS-1 or praziquantel alone. CONCLUSIONS Our findings suggest that the treatment using APS-1 in combination with praziquantel attenuated S. japonicum egg-induced hepatic fibrosis, and possessed potent hepatoprotective activity.

Phase 1 trials of PEGylated recombinant human hyaluronidase PH20 in patients with advanced solid tumours.

BACKGROUND: Hyaluronan accumulation in tumour stroma is associated with reduced survival in preclinical cancer models. PEGPH20 degrades hyaluronan to facilitate tumour access for cancer therapies. Our objective was to assess safety and antitumour activity of PEGPH20 in patients with advanced solid tumours. METHODS: In HALO-109-101 (N=14), PEGPH20 was administered intravenously once or twice weekly (0.5 or 50 µg kg-1) or once every 3 weeks (0.5-1.5 µg kg-1). In HALO-109-102 (N=27), PEGPH20 was administered once or twice weekly (0.5-5.0 µg kg-1), with dexamethasone predose and postdose. RESULTS: Dose-limiting toxicities included grade ⩾3 myalgia, arthralgia, and muscle spasms; the maximum tolerated dose was 3.0 µg kg-1 twice weekly. Plasma hyaluronan increased in a dose-dependent manner, achieving steady state by Day 8 in multidose studies. A decrease in tumour hyaluronan level was observed in 5 of the 6 patients with pretreatment and posttreatment tumour biopsies. Exploratory imaging showed changes in tumour perfusion and decreased tumour metabolic activity, consistent with observations in animal models. CONCLUSIONS: The tumour stroma has emerging importance in the development of cancer therapeutics. PEGPH20 3.0 µg kg-1 administered twice weekly is feasible in patients with advanced cancers; exploratory analyses indicate antitumour activity supporting further evaluation of PEGPH20 in solid tumours.

An upconversion luminescence nanoprobe for the ultrasensitive detection of hyaluronidase.

A new upconversion luminescence nanoprobe for the detection of hyaluronidase has been developed by coupling the hyaluronic acid-bearing upconversion fluorescence nanoparticles (HA-UCNPs) with poly(m-phenylenediamine) (PMPD) nanospheres via covalent linkage. The nanoprobe alone exhibits an extremely low background signal owing to the effective fluorescence quenching by electron-rich PMPD and the near-infrared excitation characteristic (λex = 980 nm) of HA-UCNPs; upon reaction with hyaluronidase, however, a more than 31-fold fluorescence enhancement is produced. Compared with the corresponding nanosystem assembled via physical adsorption, the prepared nanoprobe shows a largely increased stability and a much higher signal-to-background ratio, which offers an ultrasensitive assay for hyaluronidase, with a detection limit of 0.6 ng/mL. The nanoprobe has been successfully used to determine hyaluronidase in human serum samples from both colorectal cancer patients and healthy people, disclosing that the serum hyaluronidase level in colorectal cancer patients is roughly 3 times higher than that in healthy people. Furthermore, the nanoprobe has also been employed to study the activity change of hyaluronidase affected by different concentrations of arsenate (a potential carcinogen), and the results show that even a low dosage of arsenate (50 µg/L) can raise the activity of hyaluronidase by about one-third, revealing the relationship between arsenate and the enzyme. The proposed method is not only simple but also highly sensitive, making it useful to assay hyaluronidase in relevant clinical samples.

DNA methylation array analyses identified breast cancer-associated HYAL2 methylation in peripheral blood.

Breast cancer (BC) is the leading cause of cancer-related mortality in women worldwide. Changes in DNA methylation in peripheral blood could be associated with malignancy at early stage. However, the BC-associated DNA methylation signatures in peripheral blood were largely unknown. Here, we performed a genome-wide methylation screening and identified a BC-associated differentially methylated CpG site cg27091787 in the hyaluronoglucosaminidase 2 gene (HYAL2) (discovery round with 72 BC case and 24 controls: p = 2.61 × 10(-9) adjusted for cell-type proportions). The substantially decreased methylation of cg27091787 in BC cases was confirmed in two validation rounds (first validation round with 338 BC case and 507 controls: p < 0.0001; second validation round with 189 BC case and 189 controls: p < 0.0001). In addition to cg27091787, the decreased methylation of a 650-bp CpG island shore of HYAL2 was also associated with increased risk of BC. Moreover, the expression and methylation of HYAL2 were inversely correlated with a p-value of 0.006. To note, the BC-associated decreased HYAL2 methylation was replicated in the T-cell fraction (p = 0.034). The cg27091787 methylation level enabled a powerful discrimination of early-stage BC cases (stages 0 and I) from healthy controls [area under curve (AUC) = 0.89], and was robust for the detection of BC in younger women as well (age < 50, AUC = 0.87). Our study reveals a strong association between decreased HYAL2 methylation in peripheral blood and BC, and provides a promising blood-based marker for the detection of early BC.

Ratiometric fluorescent biosensor for hyaluronidase with hyaluronan as both nanoparticle scaffold and substrate for enzymatic reaction.

Hyaluronidases (HAase) are involved in various physiological and pathological processes and have been reported as urinary marker for bladder cancer. In this study, a novel ratiometric fluorescent sensing system based on both aggregation-induced emission (AIE) and aggregation-induced quenching (ACQ) was developed to quantitatively assess hyaluronidase level. First, a tetraphenylethylene derivative with positive charges (TPE-2N(+), typical AIE molecule) at both ends and an anthracene derivative with positive charge at one end (AN-N(+), typical ACQ molecule) was synthesized. These two positively charged compounds were then mixed with a negatively charged hyaluronan (HA), which induced the aggregation of the compounds as well as the nanoparticles formation as a result of electrostatic complexation, with TPE-2N(+) acting as cross-linking agent. The aggregation also caused the efficient quenching of the emission of AN-N(+) due to ACQ effect, as well as the fluorescence enhancement of TPE-2N(+) due to AIE effect. In the presence of HAase, the enzymatic reaction led to the degradation of HA and triggered disassembly of the nanoparticles; as a result, the emission of AN-N(+) was restored and that of TPE-2N(+) was suppressed. This fluorescence variation affords the system a robust ratiometric biosensor for HAase, and the ratio of fluorescence intensity for AN-N(+) (I414) to that for TPE-2N(+) (I474) can be used as the sensing signal for detecting HAase activity. In this system, hyaluronan serves not only as the scaffold for nanoparticle formation but also as the substrate for enzymatic reaction. This assay system is operable in aqueous media with very low detection limit of 0.0017 U/mL and is capable of detecting HAase in biological fluids such as serum and urine. This strategy may provide a new and effective approach for developing other enzyme assays.

Hyaluronidases and their inhibitors in the serum of colorectal carcinoma patients.

Colorectal cancer is the third most commonly diagnosed type of cancer. Hyaluronan is involved in this malignancy. Moreover, hyaluronidases - its degrading enzymes - display controversial roles regarding their involvement in tumor development. HYAL-1 is the major tumor derived hyaluronidase. The aim of the study was the determination and evaluation of hyaluronidase levels in serum of colorectal cancer patients, before and after surgery, with a view to assessing its potential role as a tumor marker for recurrence. By zymography and Western blotting, it was confirmed that HYAL-1 was the only hyaluronidase present in samples. Quantification of its activity indicated a statistically significant decrease in samples seven days postoperatively, compared with the respective ones before surgery. HYAL-1 levels before surgery were significantly reduced in comparison with healthy samples and samples one year postoperatively. Hyaluronidase inhibitor activity was demonstrated under mild alkaline conditions via reverse zymography. A statistically significant increase was observed in samples seven days postoperatively, when compared with samples before surgery. HYAL-1 levels in sera of colorectal cancer patients were lower than those of healthy population, possibly because of the local accumulation of the enzyme in tumor microenvironment. A gradual elevation up to one year postoperatively to reach healthy levels might indicate a role of HYAL-1 levels in cancer.

Role of hyaluronic acid, its degrading enzymes, degradation products, and ferritin in the assessment of fibrosis stage in Egyptian patients with chronic hepatitis C.

BACKGROUND/AIMS: Liver biopsy is considered a gold standard for fibrosis staging, but it has a high risk of morbidity. Therefore, there is an interest in developing noninvasive markers for the prediction of liver fibrosis stages. METHODS: Hyaluronic acid, ferritin, N-acetyl-ß-D-glucosaminidase, ß-glucuronidase, glucosamine, aspartate transaminase, and alanine transaminase were assayed in 210 individuals with chronic hepatitis C infection. Statistical analysis was carried out by logistic regression and receiver-operating characteristic curves. RESULTS: The best linear combination of only significant blood markers was used for the determination of the fibrosis discriminant score; score=[1.64 (numerical constant)-0.002×hyaluronic acid (pg/l)-2.68×ß-glucuronidase (µmol/ml/min)-0.026×glucosamine (µg/dl)-0.001×ferritin-0.033 (ng/ml)×aspartate transaminase/alanine transaminase]. The selected fibrosis discriminant score function correctly classified 81% of patients with severe liver fibrosis at a discriminant cut-off score=0.55 (i.e. less than 0.55 indicated mild liver fibrosis and greater than 0.55 indicated severe liver fibrosis), with a sensitivity of 100% and a specificity of 73%. CONCLUSION: A simple fibrosis index can be useful to select hepatitis C virus-infected patients with a very low risk of significant fibrosis in whom the protocol of liver biopsies may be avoided.

Damage of the endothelial glycocalyx in dialysis patients.

Damage to the endothelial glycocalyx, which helps maintain vascular homeostasis, heightens the sensitivity of the vasculature to atherogenic stimuli. Patients with renal failure have endothelial dysfunction and increased risk for cardiovascular morbidity and mortality, but the state of the endothelial glycocalyx in these patients is unknown. Here, we used Sidestream Darkfield imaging to detect changes in glycocalyx dimension in dialysis patients and healthy controls from in vivo recordings of the sublingual microcirculation. Dialysis patients had increased perfused boundary region and perfused diameters, consistent with deeper penetration of erythrocytes into glycocalyx, indicating a loss of glycocalyx barrier properties. These patients also had higher serum levels of the glycocalyx constituents hyaluronan and syndecan-1 and increased hyaluronidase activity, suggesting the shedding of these components. Loss of residual renal function had no influence on the imaging parameters but did associate with greater shedding of hyaluronan in blood. Furthermore, patients with higher levels of inflammation had more significant damage to the glycocalyx barrier. In conclusion, these data suggest that dialysis patients have an impaired glycocalyx barrier and shed its constituents into blood, likely contributing to the sustained endothelial cell activation observed in ESRD.

A dietary colorant crocin mitigates arthritis and associated secondary complications by modulating cartilage deteriorating enzymes, inflammatory mediators and antioxidant status.

Articular cartilage degeneration and inflammation are the hallmark of progressive arthritis and is the leading cause of disability in 10-15% of middle aged individuals across the world. Cartilage and synovium are mainly degraded by either enzymatic or non-enzymatic ways. Matrix metalloproteinases (MMPs), hyaluronidases (HAases) and aggrecanases are the enzymatic mediators and inflammatory cytokines and reactive oxygen species being non-enzymatic mediators. In addition, MMPs and HAases generated end-products act as inflammation inducers via CD44 and TLR-4 receptors involved NF-κB pathway. Although several drugs have been used to treat arthritis, numerous reports describe the side effects of these drugs that may turn fatal. On this account several medicinal plants and their isolated molecules have been involved in modern medicine strategies to fight against arthritis. In view of this, the present study investigated the antiarthritic potentiality of Crocin, a dietary colorant carotenoid isolated from stigma of Crocus sativus. Crocin effectively neutralized the augmented serum levels of enzymatic (MMP-13, MMP-3 and MMP-9 and HAases) and non-enzymatic (TNF-α, IL-1ß, NF-κB, IL-6, COX-2, PGE(2) and ROS) inflammatory mediators. Further, Crocin re-established the arthritis altered antioxidant status of the system (GSH, SOD, CAT and GST). It also protected the bone resorption by inhibiting the elevated levels of bone joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Taken together, Crocin revitalized the arthritis induced cartilage and bone deterioration along with inflammation and oxidative damage that could be accredited to its antioxidant nature. Thus, Crocin could be an effective antiarthritic agent which can equally nullify the arthritis associated secondary complication.

Discriminant function based on parameters of hyaluronic acid metabolism and nitric oxide to differentiate metastatic from non-metastatic colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide. Because there is currently no useful serological marker for metastatic colorectal cancer, the search for simple biomarkers for colorectal cancer diagnosis and prognosis is needed. Hyaluronic acid level was determined by ELISA; in addition to its degrading enzymes, degradation products and nitric oxide were determined by standard techniques in 185 CRC patients with and without metastases. Statistical analyses were performed by logistic regression and receiver-operating characteristic (ROC) curves. The multivariate discriminate analysis (MDA) selects a function based on absolute values of six biochemical markers; score = [-0.62 (numerical constant) + hyaluronic acid (pg/l) × 0.002 + hyaluronidase (mg N-acetyl glucosamine/ml/18 h) × 0.009-ß-glucuronidase (µmol/ml/min) × 0.07 + N-acetyl-ß-D-glucosaminidase (µmol/ml/min) × 0.019-glucuronic acid (µg/dl) × 0.001 + nitric oxide (µmol/l) × 0.01]. The selected MDA function correctly classified 92% of the metastatic CRC patients at a discriminate cut-off score = 0.24 (i.e., less than 0.24 indicated patients with non-metastatic colon cancer, and greater than 0.24 indicated patients with metastatic colon cancer with high degrees of sensitivity (100%) and specificity (93%)). The positive predictive and negative predictive values were also high (81% and 85%, respectively). Colorectal cancer patients can be simply and efficiently classified into metastatic or non-metastatic using their MDA score.

Role of urinary biomarkers of N,N-dimethylformamide in the early detection of hepatic injury among occupational exposed workers.

AIM: To determine the sensitive and convenient biomarkers for the early detection of hepatic injury in N,N-dimethylformamide (DMF) exposed workers. METHODS: Seventy-nine individuals in a synthetic leather factory were investigated with questionnaire survey. The air samples, urine samples, and blood samples were collected at the specific time point. Airborne DMF and the urine metabolites of DMF were measured by gas chromatography (GC), high-performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Traditional liver function tests and hepatic fibrosis parameters were performed by auto-chemistry analyzer and ELISA methods. RESULTS: The urine concentration of N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC), one of the metabolites of DMF, was positively correlated with activities of liver function enzymes. About 60% subjects with urine AMCC concentration above 40 mg/g creatinine showed raised liver enzymes activities. In terms of hepatic fibrosis parameters, we found 4 of 5 abnormal total serum bile acid (SBA) and 4 of 4 abnormal serum hyaluronidase (HA) among workers with higher amount of urine AMCC. CONCLUSION: Workers exposed to DMF with higher urine AMCC levels were more likely to develop liver diseases. In addition, SBA and HA have the potential to act as early indicators of toxic hepatic fibrosis activities for occupational health surveillance.

Skeletal and hematological anomalies in HYAL2-deficient mice: a second type of mucopolysaccharidosis IX?

The metabolism of hyaluronan (HA) relies on HA synthases and hyaluronidases, among which hyaluronidase-1 (HYAL1) and -2 (HYAL2) have been proposed as key actors. Congenital HYAL1 deficiency leads to mucopolysaccharidosis IX (MPS IX), a rare lysosomal storage disorder characterized by joint abnormalities. Knowledge of HYAL2 is limited. This protein displays weak in vitro hyaluronidase activity and acts as a receptor for oncogenic ovine retroviruses. We have generated HYAL2-deficient mice through a conditional Cre-lox system. Hyal2(-/-) mice are viable and fertile. They exhibit localized congenital defects in frontonasal and vertebral bone formation and suffer from mild thrombocytopenia and chronic, possibly intravascular, hemolysis. In addition, Hyal2(-/-) mice display 10-fold increases in plasma levels of HA and 2-fold increases in plasma hyaluronidase activity. Globally, there is no HA accumulation in tissues, including bones, but liver sinusoidal cells seem overloaded with undigested HA. Taken together, these elements demonstrate for the first time that murine HYAL2 has a physiological activity in vivo that is relevant for craniovertebral bone formation, maintenance of plasma HA concentrations, and erythrocyte and platelet homeostasis. In addition, the viability of HYAL2-deficient mice raises the possibility that a similar defect, defining a new MPS disorder, exists in humans.

Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis.

AIMS/HYPOTHESIS: Cardiovascular disease contributes to mortality in type 1 diabetes mellitus, but the specific pathophysiological mechanisms remain to be established. We recently showed that the endothelial glycocalyx, a protective layer of proteoglycans covering the endothelium, is severely perturbed in type 1 diabetes, with concomitantly increased plasma levels of hyaluronan and hyaluronidase. In the present study, we evaluated the relationship between hyaluronan and hyaluronidase with carotid intima-media thickness (cIMT), an established surrogate marker for cardiovascular disease. SUBJECTS AND METHODS: Non-smoking type 1 diabetes patients without micro- or macrovascular complications and matched controls were recruited and cIMT of both carotid arteries was measured. To evaluate the relationship between cIMT and hyaluronan and hyaluronidase as well as other parameters, uni- or multivariate regression analyses were performed. RESULTS: We included 99 type 1 diabetes patients (age 10-72 years) and 99 age- and sex-matched controls. Mean cIMT, HbA(1c), high sensitivity C-reactive protein, hyaluronan and hyaluronidase were significantly increased in type 1 diabetes vs controls. Plasma hyaluronan and hyaluronidase were correlated in type 1 diabetes. In univariate regression analyses, mean IMT was associated with plasma hyaluronan, age and male sex, whereas after multivariate analysis only age and sex remained statistically significant. CONCLUSIONS/INTERPRETATION: We conclude that type 1 diabetes patients show structural changes of the arterial wall associated with increased hyaluronan metabolism. These data may lend further support to altered glycosaminoglycan metabolism in type 1 diabetes as a potential mechanism involved in accelerated atherogenesis.