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.

Altered Macrophage Function Associated with Crystalline Lung Inflammation in Acid Sphingomyelinase Deficiency.

Deficiency of ASM (acid sphingomyelinase) causes the lysosomal storage Niemann-Pick disease (NPD). Patients with NPD type B may develop progressive interstitial lung disease with frequent respiratory infections. Although several investigations using the ASM-deficient (ASMKO) mouse NPD model revealed inflammation and foamy macrophages, there is little insight into the pathogenesis of NPD-associated lung disease. Using ASMKO mice, we report that ASM deficiency is associated with a complex inflammatory phenotype characterized by marked accumulation of monocyte-derived CD11b+ macrophages and expansion of airspace/alveolar CD11c+ CD11b- macrophages, both with increased size, granularity, and foaminess. Both the alternative and classical pathways were activated, with decreased in situ phagocytosis of opsonized (Fc-coated) targets, preserved clearance of apoptotic cells (efferocytosis), secretion of Th2 cytokines, increased CD11c+/CD11b+ cells, and more than a twofold increase in lung and plasma proinflammatory cytokines. Macrophages, neutrophils, eosinophils, and noninflammatory lung cells of ASMKO lungs also exhibited marked accumulation of chitinase-like protein Ym1/2, which formed large eosinophilic polygonal Charcot-Leyden-like crystals. In addition to providing insight into novel features of lung inflammation that may be associated with NPD, our report provides a novel connection between ASM and the development of crystal-associated lung inflammation with alterations in macrophage biology.

Associations Between Inflammatory Endotypes and Clinical Presentations in Chronic Rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by mucosal inflammation in the nose and paranasal sinuses. Inflammation in CRS is also heterogeneous and is mainly characterized by type 2 (T2) inflammation, but subsets of patients show type 1 (T1) and type 3 (T3) inflammation. Whether inflammatory endotypes are associated with clinical phenotypes has yet to be explored in detail. OBJECTIVE: To identify associations between inflammatory endotypes and clinical presentations in CRS. METHODS: We compared 121 patients with nonpolypoid CRS (CRSsNP) and 134 patients with polypoid CRS (CRSwNP) and identified inflammatory endotypes using markers including IFN-γ (T1), eosinophil cationic protein (T2), Charcot-Leyden crystal galectin (T2), and IL-17A (T3). We collected clinical parameters from medical and surgical records and examined whether there were any associations between endotype and clinical features. RESULTS: The presence of nasal polyps, asthma comorbidity, smell loss, and allergic mucin was significantly associated with the presence of T2 endotype in all patients with CRS. The T1 endotype was significantly more common in females, and the presence of pus was significantly associated with T3 endotype in all patients with CRS. We further analyzed these associations in CRSsNP and CRSwNP separately and found that smell loss was still associated with T2 endotype and pus with the T3 endotype in both CRSsNP and CRSwNP. Importantly, patients with CRS with T2 and T3 mixed endotype tended to have clinical presentations shared by both T2 and T3 endotypes. CONCLUSIONS: Clinical presentations are directly associated with inflammatory endotypes in CRS. Identification of inflammatory endotypes may allow for more precise and personalized medical treatments in CRS.

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy.

We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody-antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody-antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA. Graphical Abstract ᅟ.

Identification of lysophospholipase protein from Spiroplasma eriocheiris and verification of its function.

Spiroplasma eriocheiris is known to cause tremor disease in the Chinese mitten crab Eriocheir sinensis; however, the molecular characterization of this pathogen is still unclear. S. eriocheiris has the ability to invade and survive within mouse 3T6 cells. The invasion process may require causing damage to the host cell membrane by chemical, physical or enzymatic means. In this study, we systematically characterized a novel lysophospholipase (lysoPL) of S. eriocheiris TDA-040725-5T. The gene that encodes lysoPL in S. eriocheiris (SE-LysoPL) was cloned, sequenced and expressed in Escherichia coli BL21 (DE3). Enzymatic assays revealed that the purified recombinant SE-LysoPL hydrolysed long-chain acyl esterases at pH 7 and 30 °C. SE-LysoPL was detected in the membrane and cytoplasmic protein fractions using the SE-LysoPL antibody in Western blot. The virulence ability of S. eriocheiris was effectively reduced at the early stage of infection (m.o.i.=100) by the SE-LysoPL antibody neutralization test. To the best of our knowledge, this is the first study to identify and characterize a gene from S. eriocheiris encoding a protein exhibiting lysoPL and esterase activities. Our findings indicate that SE-LysoPL plays important roles in the pathogenicity of S. eriocheiris.

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.

Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB-P).

Human phospholipase B-precursor (PLB-P) is a newly identified and purified protein from human neutrophils. The precise function of PLB-P in vivo is not yet known. Its existence in neutrophils and the enzymatic activity against phospholipids imply a role in the defence against invading microorganisms and in the generation of lipid mediators of inflammation. We describe here the generation of specific antibodies against PLB-P, the tissue localizations of PLB-P and the establishment of an accurate, specific, and reproducible radioimmunoassay (RIA). A survey of normal and malignant tissues showed strong immunostaining of PLB-P in neuronal and myeloid cells and in adrenal glands. Elevated levels were found in sera of patients with influenza A infection i.e. >1 microg/L and in gut fluids of patients with inflammatory bowel disease i.e. >20 microg/L. The levels correlated to markers of neutrophil activation, suggesting a neutrophil origin of PLB-P in these conditions. The antibodies and the assay will be useful in the future basic and clinical investigations of PLB-P.

Phospholipase A2 and phospholipase B activities in fungi.

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.

Detection of antibodies to phospholipase B in patients infected with Cryptococcus neoformans by enzyme-linked immunosorbent assay (ELISA).

Secreted phospholipases are virulence factors of several fungi, including Cryptococcus neoformans. We describe for the first time the detection by ELISA of antibodies to cryptococcal phospholipase B in the serum of patients infected with C. neoformans or C. gattii. Sixty-nine sera from 25 patients with cryptococcosis, 23 patients with Candida infections and 26 with bacterial or viral infections were tested. The sensitivity of the ELISA in patients with cryptococcosis was 100% in immunocompetent hosts and 64.3% in immunosuppressed patients with cryptococcal meningitis. Absorbance readings were significantly higher in immunocompetent patients (P<0.001). Titres remained positive for up to 2 years. Positive results were noted in 10 episodes of invasive candidiasis, three patients colonised with Candida, and three cases of bacterial infection. Mean absorbance readings were significantly lower in patients with bacterial infection (P <0.001). We conclude that phospholipase B is produced in vivo during cryptococcal infection and that serum phospholipase B antibodies are a sensitive marker of present or past infection. Cross-reactivity of the ELISA with sera from patients with candidiasis indicates that common epitopes are present on cryptococcal and candidal phospholipase B, hence fungal phospholipase B may constitute a new therapeutic target.

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG.

Recent studies in mice have indicated that the long-lasting specific antibody responses seen after vaccination are probably due to the existence of long-lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long-lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production.

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Purification of a lysophospholipase from bovine brain that selectively deacylates arachidonoyl-substituted lysophosphatidylcholine.

A high activity lysophospholipase A (lysoPLA) was purified from the soluble fraction of bovine brain. The separation included sequential DEAE-Sephacel, phenyl-Sepharose FF, heparin-Sepharose CL-6B, and Q-Sepharose FF column chromatography. Mono Q, Sephacryl S300HR, and hydroxylapatite column chromatography in the presence of the detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) and glycerol further purified the activity to 17,000-fold. The enzyme was purified to homogeneity by polyacrylamide gel electrophoresis using nondenaturing conditions. The pure enzyme migrated as a single polypeptide of 95 kDa mass by SDS-polyacrylamide gel electrophoresis and deacylated arachidonoyl-lysophosphatidylcholine (ara-lysoPC) at rate of 70 micromol/(min mg). The enzyme showed selectivity for arachidonoyl-substituted lysoPC, since palmitoyl-lysoPC was deacylated at a much lower rate (7 micromol/(min mg)). LysoPLA activity was maximal at pH 7.4-8.0 and was increased 1.3-fold by MgCl2 (5 mM). By including MgCl2, however, the range of optimal activity was expanded to pH values up to 9.0. The 95-kDa protein also deacylated arachidonoyl groups from 1-O-hexadecyl-2-arachidonoyl-PC (PLA2 activity) at a rate of 15 micromol/(min mg). Moreover, the deacylation of arachidonoyl groups from diacylPC was greatly increased by including purified bovine brain PLA1 in the reaction mixture. Thus, the same 95-kDa polypeptide catalyzed both lysoPLA and PLA2 activities, but the rate of arachidonoyl group deacylation was increased by prior sn-1 deacylation. Finally, the 95-kDa polypeptide cross-reacted with antibodies raised against a human recombinant cPLA2, implying that the 95-kDa protein is structurally similar to cPLA2. Additionally, these data suggest that the combined actions of PLA1 and the 95-kDa protein generate significant amounts of free arachidonic acid in the brain.

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.

Is intestinal villus phospholipase A2/lysophospholipase bound pancreatic carboxylester lipase?

Similarities in substrate specificity, localization and molecular weight between villus membrane phospholipase A2/lysophospholipase and carboxylester lipase of pancreatic origin suggested their possible identity. To test this, a preparation of the phospholipase A2/lysophospholipase released from brush border vesicles by papain was compared to authentic, pancreatic carboxylester lipase. Susceptibility of both activities to the inhibitor, diisopropylfluorophosphate, was consistent with their identity, but inconclusive. It also indicated that two populations of phospholipase A2 species may be present in the papain-released preparation. However, comparison of binding of the activities to Sepharose-coupled, anti-carboxylester-lipase IgG indicates that they are immunologically distinct.

Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells.

Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.

Phospholipase B from the plasma membrane of Saccharomyces cerevisiae. Separation of two forms with different carbohydrate content.

Two forms of phospholipase B could be solubilized from the plasma membrane of Saccharomyces cerevisiae, separated by gel filtration with Sephacryl S-300 and identified by SDS-polyacrylamide gel electrophoresis as glycoproteins of the apparent molecular weights of about 220 000 (phospholipase B1) and 145 000 (phospholipase B2). The enzymes are very similar in respect to their catalytic properties. Both forms converted lysophosphatidylcholine to diacylphosphatidylcholine and unesterified fatty acids. The carbohydrate content of the glycoproteins could be reduced by treatment with endoglycosidase H and HF. By incubation of phospholipase B1 and phospholipase B2 with endoglycosidase H from Streptomyces griseus, one main protein with an apparent Mr of 67 000 and the same residual carbohydrate content was obtained. Treatment with HF reduced phospholipase B1 and phospholipase B2 to proteins with an apparent Mr of 52 000 and 67 000, respectively. These results could indicate that the two forms are similar in respect to their protein moieties. An antiserum raised in mice against phospholipase B2 showed no crossreactivity with phospholipase B1 as detected by immunoblot analysis. The reactivity of phospholipase B2 was diminished or abolished by progressive removal of carbohydrate. These results were taken as indications for differences in the carbohydrate component of the two enzyme forms.

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques.

1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. Microsomal lysophospholipase activity was completely inhibited by [3H]diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrohporesis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [125I]iodosulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.