Oncogenic CXCL10 signalling drives metastasis development and poor clinical outcome.

BACKGROUND: The CXCL10/CXCR3 signalling mediates paracrine interactions between tumour and stromal cells that govern leukocyte trafficking and angiogenesis. Emerging data implicate noncanonical CXCL10/CXCR3 signalling in tumourigenesis and metastasis. However, little is known regarding the role for autocrine CXCL10/CXCR3 signalling in regulating the metastatic potential of individual tumour clones. METHODS: We performed transcriptomic and cytokine profiling to characterise the functions of CXCL10 and CXCR3 in tumour cells with different metastatic abilities. We modulated the expression of the CXCL10/CXCR3 pathway using shRNA-mediated silencing in both in vitro and in vivo models of B16F1 melanoma. In addition, we examined the expression of CXCL10 and CXCR3 and their associations with clinical outcomes in clinical data sets derived from over 670 patients with melanoma and colon and renal cell carcinomas. RESULTS: We identified a critical role for autocrine CXCL10/CXCR3 signalling in promoting tumour cell growth, motility and metastasis. Analysis of publicly available clinical data sets demonstrated that coexpression of CXCL10 and CXCR3 predicted an increased metastatic potential and was associated with early metastatic disease progression and poor overall survival. CONCLUSION: These findings support the potential for CXCL10/CXCR3 coexpression as a predictor of metastatic recurrence and point towards a role for targeting of this oncogenic axis in the treatment of metastatic disease.

Hemoadsorption of high-mobility group box chromosomal protein 1 using a column for large animals.

BACKGROUND: High-mobility group box chromosomal protein 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from the systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. MATERIALS AND METHODS: In this study, we produced a new adsorbent material by chemically treating polystyrene fiber. We first determined whether the adsorbent material efficiently adsorbed HMGB1 in vitro using a bovine HMGB1 solution and a plasma sample from a swine model of acute liver failure. We then constructed a column by embedding fabric sheets of the newly developed fibers into a cartridge and tested the ability of the column to reduce plasma HMGB1 levels during a 4-hour extracorporeal hemoperfusion in a swine model of acute liver failure. RESULTS: The in vitro adsorption test of the new fiber showed high performance for HMGB1 adsorption (96% adsorption in the bovine HMGB1 solution and 94% in the acute liver failure swine plasma, 2 h incubation at 37°C; p < 0.05 vs. incubation with no adsorbent). In the in vivo study, the ratio of the HMGB1 concentration at the outlet versus the inlet of the column was significantly lower in swine hemoperfused with the newly developed column (53 and 61% at the beginning and end of perfusion, respectively) than in those animals hemoperfused with the control column (94 and 93% at the beginning and end of perfusion, respectively; p < 0.05). Moreover, the normalized plasma level of HMGB1 was significantly lower during perfusion with the new column than with the control column (p < 0.05 at 1, 2, and 3 h after initiation of perfusion). CONCLUSION: These data suggest that the newly developed column has the potential to effectively adsorb HMGB1 during hemoperfusion in swine.

Increased plasma levels of high mobility group box 1 in patients with acute liver failure.

BACKGROUND: High-mobility group box 1 (HMGB1) is a monocyte-derived late-acting inflammatory mediator, which is released in conditions such as shock, tissue injury and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in patients with acute liver failure (ALF). PATIENTS AND METHODS: We determined the plasma levels of HMGB1 and aspartate aminotransferase (AST) in 7 healthy volunteers (HVs), 40 patients with liver cirrhosis (LC), 37 patients with chronic hepatitis (CH), 18 patients with severe acute hepatitis (AH), and 14 patients with fulminant hepatitis (FH). The 14 patients with FH were divided into two subgroups depending upon the history of plasma exchange (PE) before their plasma sample collection. The hepatic levels of HMGB1 were measured in tissue samples from 3 patients with FH who underwent living-donor liver transplantation and from 3 healthy living donors. Hepatic tissue samples were also subjected to immunohistochemical examination for HMGB1. RESULTS: The plasma levels of HMGB1 (ng/ml) were higher in patients with liver diseases, especially in FH patients with no history of PE, than in HVs (0.3 ± 0.3 in HVs, 4.0 ± 2.0 in LC, 5.2 ± 2.6 in CH, 8.6 ± 4.8 in severe AH, 7.8 ± 2.7 in FH with a history of PE, and 12.5 ± 2.6 in FH with no history of PE, p < 0.05 in each comparison). There was a strong and statistically significant relationship between the mean plasma HMGB1 level and the logarithm of the mean AST level (R = 0.900, p < 0.05). The hepatic tissue levels of HMGB1 (ng/mg tissue protein) were lower in patients with FH than in healthy donors (539 ± 116 in FH vs. 874 ± 81 in healthy donors, p < 0.05). Immunohistochemical staining for HMGB1 was strong and clear in the nuclei of hepatocytes in liver sections from healthy donors, but little staining in either nuclei or cytoplasm was evident in specimens from patients with FH. CONCLUSION: We confirmed that plasma HMGB1 levels were increased in patients with ALF. Based on a comparison between HMGB1 contents in normal and ALF livers, it is very likely that HMGB1 is released from injured liver tissue.

Epstein-Barr virus-associated inflammatory pseudotumor variant of follicular dendritic cell sarcoma of the liver: a case report and review of the literature.

BACKGROUND: Follicular dendritic cell sarcoma is a rare stromal tumor with no standard treatment. However, some reports have revealed that follicular dendritic cell sarcoma has an inflammatory pseudotumor variant associated with Epstein-Barr virus infection that has a relatively good prognosis. In this report, we present a case of a resected inflammatory pseudotumor variant of follicular dendritic cell sarcoma of the liver, and have reviewed the literature on the clinicopathological, molecular, and genomic features of this tumor. CASE PRESENTATION: The inflammatory pseudotumor variant of follicular dendritic cell sarcoma originates only in the liver or spleen, causes no symptoms, and is more common in middle-aged Asian women. It has no characteristic imaging features, which partially explains why the inflammatory pseudotumor variant of follicular dendritic cell sarcoma is difficult to diagnose. Pathologically, the inflammatory pseudotumor variant of follicular dendritic cell sarcoma has spindle cells mixed with inflammatory cells and is variably positive for follicular dendritic cell markers (CD21, CD23, and CD35) and Epstein-Barr virus-encoded RNA. On genetic analysis, patients with this tumor high levels of latent membrane protein 1 gene expression and extremely low levels of host C-X-C Chemokine Receptor type 7 gene expression, indicating that the inflammatory pseudotumor variant of follicular dendritic cell sarcoma has a latent Epstein-Barr virus type 2 infection. CONCLUSIONS: The inflammatory pseudotumor variant of follicular dendritic cell sarcoma is an Epstein-Barr virus-associated tumor and a favorable prognosis by surgical resection, similar to Epstein-Barr virus-associated gastric cancer.

Decreased Incidence of Acute Cellular Rejection in Low-Muscle-Mass Recipients After Living-donor Liver Transplantation.

BACKGROUND: Sarcopenia has recently been studied as a potential risk factor for mortality and complications after liver transplantation. We investigated the impact of low muscle mass on postoperative outcomes after living-donor liver transplantation. METHODS: Our study population consisted of 100 adult recipients who underwent living-donor liver transplantation in our department between 2005 and 2017. Recipients were divided into a low-muscle-mass group (L group) and a normal-muscle-mass group (N group) based on skeletal muscle index (SMI) values, and postoperative outcomes were compared between the groups. Regarding factors that were significantly different between the groups, multivariate analyses were performed to identify predictive factors. RESULTS: Based on the SMI definition, 47 and 53 of the recipients were categorized as having low muscle mass (L group) and normal muscle mass (N group), respectively. Comparison between the groups revealed a significantly reduced incidence of rejection (10.6% in L group vs 30.2% in N group, P = .017) and increased incidences of bacterial infection (61.7% in L group vs 37.7% in N group, P = .017) in the L group compared with the N group. The survival rate did not differ significantly between the groups. Multivariate analyses indicated that muscle mass was a significant predictive factor for both rejection and bacterial infection. CONCLUSION: It is important to recognize that muscle mass has an impact not only on bacterial infection but also on rejection in recipients with low muscle mass in the postoperative course of living-donor liver transplantation.

Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase.

Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor.

Mode of action of bacterial collagenase on a synthetic substrate, (Pro-Pro-Gly)5.

Clostridial collagenase (EC 3.4.24.3) catalyzes the hydrolysis of (Pro-Pro-Gly)5 at minimum of three different rates, producing Pro-Pro, Gly-Pro-Pro and Gly-Pro-Pro-Gly, and various intermediate peptides. The intermediate and final products were separated by cation-exchange column chromatogrphy and identified, and their rates of formation were measured. Pro-Pro was released most rapidly with formation of the tridecapeptide. After the initial release of the N-terminal Pro-Pro, hexa- and heptapeptides were formed in larger amounts than tri-, tetra-, nona- and decapeptides from the tridecapeptide. The rates of disappearance of the intermediates decreased in the order trideca- greater than deca- and nona- greater than heptapeptide. The results indicate that the enzyme hydrolyzes inner linkages of the tridecapeptide having N- and C-terminal Gly residues, forming large peptides, preferentially to outer linkages, forming the tri- and tetrapeptides.

Apolar interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles.

The interaction of bovine pancreatic alpha-chymotrypsin with dimyristoyl phosphatidylcholine (PC) vesicles was measured turbimetrically. The protein interacted with the vesicles at NaCl concentrations of above 0.8 M. The turbidity reached a plateau on increase in the amount of either the protein or the vesicles in the presence of a fixed amount of the other component. The precipitates formed contained both PC and protein in ratios varying with the initial amount of each component. On mixing chymotrypsin and PC vesicles, time-dependent turbidity increase was high at below pH 2.5, but relatively small at neutral and alkaline pH values. Apolar interaction between the two components was confirmed by demonstrating an increase in fluorescence intensity of chymotrypsin in the presence of the PC in 1 M NaCl. The turbidity of a mixture of PC vesicles and bovine serum albumin (BSA) increased even in the absence of 1 M NaCl, whereas the turbidities of mixtures of the vesicles and lysozyme or alpha-lactalbumin did not change with time in the presence of 1 M NaCl at pH 8.0.

Interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles.

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.

Stimulation by phospholipid vesicles of proteolysis of egg white lysozyme by chymotrypsin.

Egg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid. Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.

Some enzymatic properties of peptidyl dipeptide hydrolase (angiotensin I-converting enzyme).

Peptidyldipeptide hydrolase [angiotensin I-converting enzyme, EC 3.4.15.1] was inhibited by inorganic and organic phosphorus compounds tested, except for beta-glycerophosphate, 5'-AMP, and 5'-ADP, at the reagent concentrations used. Orthophosphate and pyrophosphate nonspecifically inhibited the enzyme activity. The enzyme was also inhibited specifically by carboxylates. The degree of inhibition by aliphatic monocarboxylates increased in proportion to their chain length up to C14. Aromatic and omega-phenylalkylcarboxylates also inhibited the enzyme activity. The enzyme was noncompetitively inhibited by acetate, 3-phenylpropionate and laurate. The Ki's for acetate, 3-phenylpropionate, and laurate were 60, 3.3, and 2.5 mM, respectively.

Stereospecificity of peptidyl dipeptide hydrolase (angiotensin I-converting enzyme).

The stereospecificity of peptidyl dipeptide hydrolase [EC 3.4.15.1] was investigated. Six free and N-blocked alanyl peptides containing D-alanine were synthesized and tested as substrates. Their susceptibilities were determined by measuring Ala-Ala release by cation exchange column chromatography. Their Michaelis constants, Km values, and velocity maxima, Vmax values, were also determined. The enzyme showed high stereospecificity for an amino acyl residue in position 3 from C-terminus: it had an absolute requirement for the alanyl residue of the L-configuration in this position. An alanyl residue of the L-configuration in position 1 or 2 increased, but was not essential for activity. The enzyme showed little stereospecificity for an alanyl residue in position 4 from the C-terminus.

Purification of carboxypeptidase A using Sepharose 4B-bound 3-phenylpropionate.

The activity of carboxypeptidase A [EC 3.4.12.2] was inhibited by 3-phenylpropionate derivatives (p-aminocinnamate, 3-p-aminophenylpropionate and 3-p-acetylaminophenylpropionate), and to investigate its use as a ligand for affinity chromatography 3-p-aminophenylpropionate was directely and indirectly coupled to Sepharose 4B. carboxypeptidase A was adsorbed only on 3-p-aminophenylpropionate bound to the gel through p-phenylenediamine as a spacer. Carboxypeptidase A from pancreas was purified by a combination of this affinity adsorbent and ion exchange chromatography. The purified carboxypeptidase A had a homogeneity similar to that of a commercial product, as judged by disc gel electrophoresis. The carboxypeptidase activity of Pronase was slightly retarded on the gel column, but could not be separated from its caseinolytic activity. Angiotensin I-converting enzyme [peptidyl dipeptidy hydrolase, EC 3.4.15.1] obtained from hog kidney cortex was not bound to the gel.

A method to determine the affinity of heparin to thrombin and antithrombin III on equilibrium gel permeation chromatography.

Equilibrium gel permeation chromatography was employed to determine the ability of heparin to form complexes with thrombin and antithrombin III. In the eluate from a Sephacryl S-200 column, heparin caused a peak and then a trough in the fluorescence of 48 nM antithrombin III or 63 nM thrombin. The peak-heights with known amounts of heparin were used for standard curves to determine the extent of complex formation by test heparin preparations. Only heparin species with high-affinity for antithrombin III specifically formed a complex with antithrombin III under the conditions used. The ability to form a complex of heparin preparations with different anticoagulant activities for thrombin and antithrombin III could be determined satisfactorily. The heparin species with different affinities for antithrombin III did not coincide those with different affinities for thrombin. Of 4 preparations with one low-affinity and three high-affinity subfractions of heparin for antithrombin III, the species with the lowest affinity for antithrombin III had the highest affinity for thrombin. All of these observations showed that the method could be used to determine the ability to form a complex of test heparin preparations.

Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].

Nitric oxide inactivates glyoxalase I in cooperation with glutathione.

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.

Prevention of thrombin/antithrombin III reaction in the presence of protamine or polybrene.

The rate of the thrombin/antithrombin III (AT III) reaction was decreased in the presence of free polybrene or protamine. The reaction rate was also decreased in protamine-coated tubes and tubes on which polybrene was absorbed nonspecifically. The reaction was also prevented when either thrombin or AT III was transferred into noncoated tubes after instantaneous contact with protamine-coated tubes or tubes with polybrene. These facts suggest that the thrombin/AT III reaction rate is determined with concentrations of reactive protein species.

Possible contribution of solid surface for inactivation of thrombin.

Dextran sulfate and polybrene were immobilized on polypropylene tubes with plastic adhesive. Serum albumin was also immobilized on the tube by heat-denaturation. In the those tubes thrombin was protected from inactivation in the presence of 0.1 M NaCl remarkably. Cholesterol coated on the tube also protected the enzyme from inactivation. The results indicate that amounts of thrombin protected by these four coating materials corresponded to the amount of thrombin inactivated on surface of the tube. At least half of the thrombin was also inactivated within 20 sec at 37 degrees C in the presence of quartz sand, supporting a significant contribution of a solid surface to temperature-dependent inactivation of enzyme in dilute solution.

Reversible inactivation by dextran sulfate of bovine thrombin in dilute solution.

Dextran sulfate (DS)-enhanced inactivation of alpha-thrombin was faster at pH 6 than at pH 8 in the absence of NaCl. Substrate protected only partly thrombin from inactivation by DS at pH 6. Some amidolytic activity of alpha-thrombin inactivated by DS was restored time-dependently by the addition of NaCl. Moreover, beta and gamma-thrombin, and other intermediates were not formed from alpha-thrombin with DS-enhanced inactivation. All these results indicate that autolysis of alpha-thrombin in dilute solution is not responsible for enhancement of reversible inactivation of alpha-thrombin by DS.

Dissociation of the thrombin/antithrombin III reaction from amidolytic activity of the enzyme at high salt concentration.

Amidolytic activity of thrombin increased proportionally with NaCl concentrations up to 1 M NaCl, due to an increase in the turnover rate with no change in the Km of the enzyme. Neutralization of thrombin by antithrombin III (AT III) was maximum in 0.1 to 0.15 M NaCl, but completely absent at high NaCl concentrations. Moreover, ratio of rates of the amidolytic activity of thrombin to of the enzyme/AT III reaction was not constant at several pHs. The rate of the thrombin/AT III reaction appeared to be inversely correlated with the enzyme's stability. Therefore, it was concluded that some factor(s) other than the active site of the thrombin molecule was more important in its primary interaction with AT III.