Serum autotaxin is increased in pruritus of cholestasis, but not of other origin, and responds to therapeutic interventions.

UNLABELLED: Pruritus is a seriously disabling symptom accompanying many cholestatic liver disorders. Recent experimental evidence implicated the lysophospholipase, autotaxin (ATX), and its product, lysophosphatidic acid (LPA), as potential mediators of cholestatic pruritus. In this study, we highlight that increased serum ATX levels are specific for pruritus of cholestasis, but not pruritus of uremia, Hodgkin's disease, or atopic dermatitis. Treatment of patients with cholestasis with the bile salt sequestrant, colesevelam, but not placebo, effectively reduced total serum bile salts and fibroblast growth factor 19 levels, but only marginally altered pruritus intensity and ATX activity. Rifampicin (RMP) significantly reduced itch intensity and ATX activity in patients with pruritus not responding to bile salt sequestrants. In vitro, RMP inhibited ATX expression in human HepG2 hepatoma cells and hepatoma cells overexpressing the pregnane X receptor (PXR), but not in hepatoma cells in which PXR was knocked down. Treatment of severe, refractory pruritus by the molecular adsorbents recirculation system or nasobiliary drainage improved itch intensity, which, again, correlated with the reduction of ATX levels. Upon reoccurrence of pruritus, ATX activity returned to pretreatment values. CONCLUSION: Serum ATX activity is specifically increased in patients with cholestatic, but not other forms of, systemic pruritus and closely correlates with the effectiveness of therapeutic interventions. The beneficial antipruritic action of RMP may be explained, at least partly, by the PXR-dependent transcriptional inhibition of ATX expression. Thus, ATX likely represents a novel therapeutic target for pruritus of cholestasis.

Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei.

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.

Peripheral Blood RNA Levels of QSOX1 and PLBD1 Are New Independent Predictors of Left Ventricular Dysfunction After Acute Myocardial Infarction.

BACKGROUND: The identification of patients with acute myocardial infarction (MI) at risk of subsequent left ventricular (LV) dysfunction remains challenging, but it is important to optimize therapies. The aim of this study was to determine the unbiased RNA profile in peripheral blood of patients with acute MI and to identify and validate new prognostic markers of LV dysfunction. METHODS: We prospectively enrolled a discovery cohort with acute MI (n=143) and performed whole-blood RNA profiling at different time points. We then selected transcripts on admission that related to LV dysfunction at follow-up and validated them by quantitative polymerase chain reaction in the discovery cohort, in an external validation cohort (n=449), and in a representative porcine MI model with cardiac magnetic resonance-based measurements of infarct size and postmortem myocardial pathology (n=33). RESULTS: RNA profiling in the discovery cohort showed upregulation of genes involved in chemotaxis, IL (interleukin)-6, and NF-κB (nuclear factor-κB) signaling in the acute phase of MI. Expression levels of the majority of these transcripts paralleled the rise in cardiac troponin T and decayed at 30 days. RNA levels of QSOX1, PLBD1, and S100A8 on admission with MI correlated with LV dysfunction at follow-up. Using quantitative polymerase chain reaction, we confirmed that QSOX1 and PLBD1 predicted LV dysfunction (odds ratio, 2.6 [95% CI, 1.1-6.1] and 3.2 [95% CI, 1.4-7.4]), whereas S100A8 did not. In the external validation cohort, we confirmed QSOX1 and PLBD1 as new independent markers of LV dysfunction (odds ratio, 1.41 [95% CI, 1.06-1.88] and 1.43 [95% CI, 1.08-1.89]). QSOX1 had an incremental predictive value in a model consisting of clinical variables and cardiac biomarkers (including NT-proBNP [N-terminal pro-B-type natriuretic peptide]). In the porcine MI model, whole-blood levels of QSOX1 and PLBD1 related to neutrophil infiltration in the ischemic myocardium in an infarct size-independent manner. CONCLUSIONS: Peripheral blood QSOX1 and PLBD1 in acute MI are new independent markers of LV dysfunction post-MI.

Serum autotaxin measurement in haematological malignancies: a promising marker for follicular lymphoma.

Autotaxin (ATX) is a tumour cell motility-stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B-cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients' clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin-2 receptor and beta2-microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.

"Proteotyping": population proteomics of human leukocytes using top down mass spectrometry.

Characterizing combinations of coding polymorphisms (cSNPs), alternative splicing and post-translational modifications (PTMs) on a single protein by standard peptide-based proteomics is challenging owing to <100% sequence coverage and the uncoupling effect of proteolysis on such variations >10-20 residues apart. Because top down MS measures the whole protein, combinations of all the variations affecting primary sequence can be detected as they occur in combination. The protein form generated by all types of variation is here termed the "proteotype", akin to a haplotype at the DNA level. Analysis of proteins from human primary leukocytes harvested from leukoreduction filters using a dual on-line/off-line top down MS strategy produced >600 unique intact masses, 133 of which were identified from 67 unique genes. Utilizing a two-dimensional platform, termed multidimensional protein characterization by automated top down (MudCAT), 108 of the above protein forms were subsequently identified in the absence of MS/MS in 4 days. Additionally, MudCAT enables the quantitation of allele ratios for heterozygotes and PTM occupancies for phosphorylated species. The diversity of the human proteome is embodied in the fact that 32 of the identified proteins harbored cSNPs, PTMs, or were detected as proteolysis products. Among the information were three partially phosphorylated proteins and three proteins heterozygous at known cSNP loci, with evidence for non-1:1 expression ratios obtained for different alleles.

Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material.

Host cell proteins are manufacturing process-related impurities that may co-purify with the product despite extensive efforts to optimize the purification process. The risks associated with these impurities can vary and may be patient and/or therapeutic dependent. Therefore, it is critical to monitor and control the levels of these impurities in products and their potential impact on safety and efficacy. Lebrikizumab is a humanized immunoglobulin G4 monoclonal antibody (mAb) that binds specifically to soluble interleukin 13. This mAb is currently in phase III clinical development for the treatment of asthma. Following initial phase III studies, the material used in lebrikizumab clinical trials was found to have a process-related impurity identified as Chinese hamster ovary phospholipase B-like 2 (PLBL2) which co-purified with lebrikizumab. The immunogenic potential of PLBL2 and its potential impact on the immunogenicity of lebrikizumab in clinical studies were therefore evaluated. Data from the clinical studies demonstrated that ∼90% of subjects developed a specific and measurable immune response to PLBL2. Given the high incidence of antibodies to PLBL2 as well as the comparable safety profile observed between placebo- and drug-treated subjects, no correlation between safety events and anti-PLBL2 antibodies could be made. Additionally, no impact on the incidence of anti-lebrikizumab antibodies was observed, suggesting the lack of an adjuvant effect from PLBL2. Interim analysis from ongoing phase III studies using material with substantially reduced levels of PLBL2 with patients having had longer exposure shows significantly less and dose-dependent frequency of immune responses to PLBL2.

Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR) both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease. RESULTS: Among the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27) and the highly downregulated gene Charcot-Leyden crystal protein (CLC) to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray. CONCLUSION: The gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

Ca2+ effect on ATP-independent lysophospholipid transacylases is indissociable from Ca2+ effect on phospholipase A2 activity in rat platelet sonicates.

CoA-dependent transacylation and phospholipid hydrolysis were studied in parallel experiments using rat platelet sonicates. The decrease observed in palmitoyllyso-sn-glycero-3-phosphocholine (palmitoyllyso-GPC) transcylation as a function of Ca2+ concentration was found to be correlated with appearance of endogenous lysoderivatives. We also demonstrated that endogenously produced acyllyso-sn-glycero-3-phosphoethanolamine (acyllyso-GPE) induced CoA-dependent arachidonate transfer from diacyl-GPC. These results further argue for a two-step arachidonate release from diacyl-GPC when platelets are stimulated with thrombin.

Murine erythrocytes contain high levels of lysophospholipase activity.

Murine erythrocytes were found to be unique in the high levels of lysophospholipase activity in the cytosol of these cells. The specific activity of the enzyme in the cytosol of the murine cells is 10-times higher than in the cytosol of rabbit erythrocytes and approximately three orders of magnitude higher than those in the red cells of rat, man, pig and ox.

Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.

A vasoactive lipid present in rat plasma that had been incubated at 37 degrees C for 48 h was identified as lysophosphatidic acid with the following fatty acid composition: palmitic acid (25.2%), stearic acid (8.4%), oleic acid (7.0%), linoleic acid (44.4%) and arachidonic acid (11.9%). The involvement of lysophospholipase D in the production of lysophosphatidic acid was suggested on the basis of extensive analyses of phospholipids in the plasma. Results indicated that lysophospholipase D hydrolyzed polyunsaturated lysophosphatidylcholines preferentially to the saturated species. Exogenously added platelet activating factor was found to be first converted to its lyso derivative by an acetylhydrolase in rat plasma and then degraded by lysophospholipase D to 1-alkyllysophosphatidic acid to a similar extent with exogenously added lyso derivative of platelet activating factor; their percent conversions to lysophosphatidic acid were higher than those of saturated 1-acyllysophosphatidylcholines, but lower than those of polyunsaturated 1-acyllysophosphatidylcholines.

Selective release of phospholipase A2 and lysophosphatidylserine-specific lysophospholipase from rat platelets.

Rat platelets released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of phospholipases was energy-dependent and was not in parallel with that of a known lysosomal marker enzyme, N-acetyl-beta-D-glucosaminidase. The phospholipases are derived from other granules (dense granules or alpha-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated platelet reaction mixture was recovered in the supernatant after centrifugation at 105,000 X g. The degree of hydrolysis of phospholipids by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) greater than phosphatidylserine (PS) greater than phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (greater than pH 7.0) and absolutely requires Ca2+. Lysophospholipase was specific to lysophosphatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor lysophosphatidylcholine (lysoPC) was hydrolyzed appreciably. Both 1-acyl- and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60 degrees C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Ca2+ to the mixtures restores the full activity.

Quantitative studies on translocation and metabolic conversion of lysophosphatidylcholine incorporated into the membrane of intact human erythrocytes from the medium.

The fate of palmitoyl-lysophosphatidylcholine (lysoPC) incorporated into the membrane of intact human erythrocytes from a medium was investigated under nonhemolytic conditions at 37 degrees C by means of 14C-labeled tracers. The lysoPC was first incorporated into the outer half of the membrane lipid bilayer and then gradually translocated into the inner half during the incubation. At the same time it was metabolically converted into phosphatidylcholine (PC) and free fatty acid (FFA) plus glycerophosphorylcholine by the actions of acyltransferase and lysophospholipase, respectively. The half times of the conversion were about 14 h, while the value of 0.5 h was obtained when the half time was measured with the hemolysate of the lysoPC-loaded erythrocytes. Chymotrypsin treatment of unsealed ghosts caused a definite decrease in lysophospholipase activity, while similar treatment of resealed ghosts did not. This together with other evidence already reported in the literature suggests that both lysophospholipase and acyltransferase may be located in the inner surface of the membrane. The above findings strongly suggest that the most of the lysoPC loaded to the membrane is gradually translocated from the outer to the inner half of the bilayer and soon converted to either PC or FFA.

Phospholipid degradation in rat calcium ionophore-activated platelets is catalyzed mainly by two discrete secretory phospholipase As.

An "A1 type" phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.

Pathologic states associated with activation of eosinophils and with eosinophilia.

Eosinophils are involved in cytotoxicity against helminths and tumor cells and effect both tissue damage and tissue protection in hypersensitivity reactions. Their migratory patterns and oxidative mechanisms are most similar to those of neutrophils, but their tissue longevity and functional variation are more similar to those of monocytes and macrophages. Their enzyme components and behaviors are only now beginning to be defined, and their biologic functions in pathologic states remain a topic of considerable discussion.

Metabolism of lysophosphatidylcholine by swine platelets.

Incubation of intact platelets from Sinclair(S-1) miniature swine with 32P-labeled lysophosphatidylcholine (lyso PC) indicated the presence of an active lysophospholipase with a pH optimum of 8.0 for hydrolysis of the substrate. However, lyso PC was incorporated into the membrane phosphatidylcholines by the acyltransferase pathway upon addition of ATP, Mg++ and CoA to the platelet suspension. These results suggest that intact platelets are able to resist the cytotoxic effects of lyso PC in plasma, and the phospholipids in platelet membranes are not readily affected by the lipid environment of the plasma. The acyltransfer reaction apparently is saturated with endogenous free fatty acids since arachidonic acid added exogenously did not further enhance the incorporation activity. Neither the acyltransferase nor the lysophospholipase activity was affected by Ca++, but divalent metal ions such as Zn++ inhibited the lysophospholipase activity. Cholesterol but not cholesteryl esters elicited a biphasic effect on both enzymes, stimulating at low concentration but inhibiting at a cholesterol to lyso PC ratio greater than 1. Serum albumin inhibited the lysophospholipase but gave a small biphasic effect to the acyltransferase.

Studies on the selectivity of enzymes involved in platelet-activating factor formation in stimulated cells.

The present studies were undertaken to obtain further insight into the selectivities of the enzymes, i.e., phospholipase A2 and acetyltransferase, involved in platelet-activating factor (PAF) production upon stimulation of human polymorphonuclear leukocytes (PMN) and platelets. After appropriate stimulation of the cells in the presence of [3H]acetate the total PAF and analogs, i.e., 1-alkyl-2-acetyl-, 1-alkenyl-2-acetyl-, and 1-acyl-2-acetyl-glycero-3- phosphocholine were isolated by high performance liquid chromatography. The isolated mixture was subjected to treatment with phospholipase A1 to differentiate acetate incorporation into 1-ether linked and 1-ester linked species. The ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs amounted to 13.8 +/- 1.0 and 1.3 +/- 0.1 for PMN and platelets, respectively. When compared to the ratio of 1-ether linked and 1-ester linked species in the diradylglycerophosphocholine precursors in each cell type, i.e., 1.13 for PMN and 0.22 for platelets, these data suggested a pronounced selectivity for the phospholipase A2 and/or acetyltransferase in the process of PAF production. When the experiments were repeated with cells that had been pretreated with phenylmethanesulfonylfluoride (PMSF) to block the acetylhydrolase, the most dramatic effects were observed on acetate incorporation into 1-acyl-2-acetyl-glycero-3-phosphocholine, which increased much more than that into 1-alk(en)yl-2-acetyl-glycero-3-phosphocholine. Under these conditions, the ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs became 1.4 +/- 0.2 and 0.17 +/- 0.02 for PMN and platelets, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated levels of phospholipase B in mice infected and challenged with Plasmodium yoelii.

Marked increases in phospholipase B activity were measured in spleen homogenates and sera of CD-1 mice given a primary infection of Plasmodium yoelii. The rise in enzyme activity paralleled parasitemia and splenomegaly, all reaching maximum values 14 days after infection. Maximum enzyme activity and splenomegaly were noted 5 days after challenge in mice given a secondary infection indicating an anamnesticlike response following a subsequent contact with the infecting parasite. These observations represent a new finding of this enzyme activity in a protozoan infection.

Enhancement of lysophospholipase activity with Trichinella spiralis antigen: evidence for cell cooperation.

Murine lymphocytes, neutrophils, macrophages and eosinophils were assayed for lysophospholipase in order to determine the cellular source of the enzyme. The eosinophil was the only cell that demonstrated a positive reaction for the enzyme. The role of other cells and/or antigen in production of the enzyme by the eosinophil was also investigated. Results demonstrated that eosinophils cultured with both Trichinella spiralis antigen and other leukocytes (lymphocytes and/or macrophages) yielded enzyme activities significantly greater than did eosinophils cultured alone or with only antigen. More specific experiments showed that T-lymphocytes were the cells responsible for influencing the eosinophils' production of lysophospholipase in the presence of antigen, and that their influence was enhanced by the presence of macrophages. These results suggest that increased lysophospholipase activity present in parasitized tissue is not only due to an increased number of eosinophils infiltrating parasitized tissues, but is also due to each eosinophil synthesizing more of the enzyme for release. The necessity for antigen and other cells suggests a need for cell cooperation in the production of the enzyme, specifically T-lymphocytes and macrophage interaction with the eosinophil.

Suppression of Nippostrongylus brasiliensis (Nematoda)-induced lysophospholipase activity and peripheral eosinophilia by Eimeria nieschulzi (Apicomplexa).

Interspecific interactions between Nippostrongylus brasiliensis and Eimeria nieschulzi were studied by measuring fecal lysophospholipase (LYPH) activity and relative numbers of peripheral eosinophils in rats singly or concurrently infected with one or both parasite species. Three groups of 10 rats each were inoculated with 2 X 10(3) N. brasiliensis L3 larvae and/or 5 X 10(5) E. nieschulzi sporulated oocysts. Groups 1 and 2 were infected with E. nieschulzi or N. brasiliensis, respectively. Group 3 rats were infected first with N. brasiliensis, followed on day 8 postinoculation (PI) with E. nieschulzi. Each rat served as its own control. Results revealed LYPH levels rose steadily in Group 2 rats, reaching significant peaks on days 10 and 12 PI before decreasing to control levels. Lysophospholipase activity in Groups 1 and 3, however, did not differ from control values. Group 2 rats also demonstrated peripheral eosinophilia, with peak values occurring on days 10, 12, 14, and 16 PI, while rats in Groups 1 and 3 exhibited no eosinophilia. These results demonstrate that E. nieschulzi suppressed intestinal LYPH activity and relative peripheral eosinophilia and demonstrate that a host's immune response to a single parasite may be significantly altered when a second parasite species is present.

Gene expression profiling of high altitude polycythemia in Han Chinese migrating to the Qinghai-Tibetan plateau.

Chronic mountain sickness (CMS) is a condition in which the hematocrit is increased above the normal level in residents at high altitude. High altitude polycythemia (HAPC) is the most characteristic sign of CMS. However, the pathogenesis of HAPC is poorly understood. The present study aimed to investigate the gene expression profile of HAPC in Han Chinese migrating to the Qinghai-Tibetan Plateau and to identify the pathogenetic mechanisms. A total of 9 differentially expressed genes were identified in HAPC patients using microarrays: 5 were up-regulated and 4 were down-regulated. Functional analysis of the array data revealed that cell division cycle 42 (CDC42) and the human immune response may be key features underlying the mechanism and development of HAPC.