Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

Sensitising effects of genetically modified enzymes used in flavour, fragrance, detergence and pharmaceutical production: cross-sectional study.

OBJECTIVES: The use of genetically engineered enzymes in the synthesis of flavourings, fragrances and other applications has increased tremendously. There is, however, a paucity of data on sensitisation and/or allergy to the finished products. We aimed to review the use of genetically modified enzymes and the enormous challenges in human biomonitoring studies with suitable assays of specific IgE to a variety of modified enzyme proteins in occupational settings and measure specific IgE to modified enzymes in exposed workers. METHODS: Specific IgE antibodies against workplace-specific individual enzymes were measured by the specific fluorescence enzyme-labelled immunoassay in 813 exposed workers seen in cross-sectional surveys. RESULTS: Twenty-three per cent of all exposed workers showed type I sensitisation with IgE antibodies directed against respective workplace-specific enzymes. The highest sensitisation frequencies observed were for workers exposed enzymes derived from α-amylase (44%), followed by stainzyme (41%), pancreatinin (35%), savinase (31%), papain (31%), ovozyme (28%), phytase (16%), trypsin (15%) and lipase (4%). The highest individual antibody levels (up to 110 kU/L) were detected in workers exposed to phytase, xylanase and glucanase. In a subgroup comprising 134 workers, detailed clinical diagnostics confirmed work-related symptoms. There was a strong correlation (r=0.75, p<0.0001) between the symptoms and antibody levels. Workers with work-related respiratory symptoms showed a higher prevalence for the presence of specific IgE antibodies against workplace-specific enzymes than asymptomatic exposed workers (likelihood ratio 2.32, sensitivity 0.92, specificity 0.6). CONCLUSIONS: Our data confirm the previous findings showing that genetically engineered enzymes are potent allergens eliciting immediate-type sensitisation. Owing to lack of commercial diagnostic tests, few of those exposed receive regular surveillance including biomonitoring with relevant specific IgE assays.

The role of antibodies in enzyme treatments and therapeutic strategies.

Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs.

Self-reactive T cells: suppressing the suppressors.

The immune system is a tightly regulated and complex system. An important part of this immune regulation is the assurance of tolerance toward self-antigens to maintain immune homeostasis. However, in recent years, antigen-specific cellular immune responses toward several normal self-proteins expressed in regulatory immune cells have been reported, especially in patients with cancer. The seemingly lack of tolerance toward such proteins is interesting, as it suggests a regulatory function of self-reactive T (srT) cells, which may be important for the fine tuning of the immune system. In particular, surprising has been the description of cytotoxic srT cells that are able to eliminate normal regulatory immune cells. Such srT cells may be important as effector cells that suppress regulatory suppressor cells. The current knowledge of the nature and function of srT cells is still limited. Still, the therapeutic targeting of srT cells offers a novel approach to harness immune-regulatory networks in cancer.

Rapid downregulation of antigen processing enzymes in ex vivo generated human monocyte derived dendritic cells occur endogenously in extended cultures.

Dendritic cells, the most potent antigen presenting cells, have been shown in murine models to induce immune responses against many antigens. Their role in the initiation of antitumour immunity has received enormous attention. Their ability to process and present antigen is dependent on their state of maturation. This study examines the activity of human monocyte-derived dendritic cells at two different time points and the corresponding changes in the proteolytic enzyme activity. Dendritic cells were produced from peripheral blood mononuclear cells of normal volunteers. Plastic adherent cells were cultured for 5 or 7 days with recombinant human (rh)GM-CSF and rhIL-4. Flow cytometry showed that day 5 dendritic cells (DC) were less mature than day 7 DC as indicated by the expression of CD1a, CD11c, CD14, CD80, CD83, CD86 and MHC-II. Proteolytic activity of the enzymes cathepsin C and cathepsin G and phagocytosis of particulate antigens also showed significant differences between d5 dendritic cells and d7 dendritic cells. Allogeneic costimulatory activity of d7 dendritic cells was also significantly increased. Induction of immunity requires active presentation of antigens by antigen processing cells on their MHC-I and/or MHC-II molecules. Study of peptide carriers and peptide precursor molecules showed a significant decrease in CLIP levels in the day 7 DC, suggesting their decreased ability to process antigens but no difference in their ability to load MHC-II molecules. These findings indicate that the length of time in culture, in the absence of exogenous maturation - inducing stimuli affects dendritic cell maturation. Intracellular enzymatic activities of dendritic cells also changed rapidly with small changes in phenotype.

Selective activation of anthracycline prodrugs for use in conjunction with ADEPT.

A major limitation in the chemotherapy of cancer results from the lack of tumor specificity displayed by most anticancer drugs. In this regard, a great deal of research has been focused on the development of new chemotherapeutic agents that are able to effectively exploit the differences between neoplastic and normal tissues. Several cancerous tissues and tumors are rich in certain lysosomal enzymes as compared with those found in the normal tissues. Thus, a prodrug can be designed to selectively target such tumor cells where it can be activated to antineoplastic agent by tumor-associated antigen-targeted monoclonal antibody-enzyme conjugate (antibody directed enzyme prodrug therapy strategy) or by the action of an enzyme present at high levels in tumor tissues (prodrug monotherapy strategy). This approach protects the normal cells from the cytotoxic effects of the drug. In the last few years, a number of new MAb-based reagents has been clinically approved (Rituxan, Herceptin and Panorex), and several others are now in advanced clinical trials. This review focuses on the design of several different enzyme/prodrug combinations with an emphasis on mechanistic insight and clinical activity.

A prospective study of routine antenatal enzyme antibody screening demonstrates lack of clinical value in predicting haemolytic disease of the newborn.

A prospective study of 7065 consecutive new pregnancies identified 230 with a positive screen, of which 27% (62/230) were 'enzyme-only' antibodies. 32 of these (52%) were potentially clinically important and were all of Rh specificity: 22 anti-E, seven anti-Cw, two anti-D and one anti-c. However, only three of these enzyme-only antibodies (one anti-D, one anti-c and one anti-E) became reactive by the indirect antiglobulin test (IAT) during the course of pregnancy, and all were detected in the routine 34-36-week maternal sample. No babies were affected, and we reaffirm that routine antibody screening by enzyme techniques is unnecessary.

Engineering a regulatable enzyme for homogeneous immunoassays.

We have engineered the phage displayed TEM-1 beta-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein. Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs. Insertion points were chosen to be close enough to the active site that complex formation could affect the activity. The antibiotic resistance provided by the beta-lactamase activity was used to select the clones encoding active enzymes. Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins. Their activity could be regulated by Mab or streptavidin binding. The dissociation constants of the complexes are in the 10(-9) to 10(-6) M range. In a competitive assay, PSA could be detected at a minimal concentration of 10(-9) M. The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence. The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.

Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays.

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-alpha, -beta, or -gamma treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (alpha, beta) or Type II (gamma) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1alpha). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.

Use of commercial antibodies for detection of the primary antioxidant enzymes.

Fourteen commercial antibodies against human antioxidant enzymes were tested on whole cell lysates by Western analysis for specificity and species crossreactivity. All antibodies, except one, recognized pure protein antigen. All four catalase antibodies were of high quality, and they could also recognize the catalases from rat, mouse, dog, and hamster cells. Two CuZnSOD antibodies were very specific for CuZnSOD protein. They could also crossreact with CuZnSOD from rat, mouse, and hamster cells, but not from dog cells. All five MnSOD antibodies detected only very high levels of MnSOD. We believe that they could not properly be used in immunohistochemistry. Three GPX antibodies could not detect the specific GPX band from cell lysates. We believe that it is difficult to use these GPX antibodies in both Western blotting and immunohistochemistry.

Insights into the structure and function of membrane polypeptides carrying blood group antigens.

In recent years, advances in biochemistry and molecular genetics have contributed to establishing the structure of the genes and proteins from most of the 23 blood group systems presently known. Current investigations are focusing on genetic polymorphism analysis, tissue-specific expression, biological properties and structure-function relationships. On the basis of this information, the blood group antigens were tentatively classified into five functional categories: (i) transporters and channels, (ii) receptors for exogenous ligands, viruses, bacteria and parasites, (iii) adhesion molecules, (iv) enzymes and, (v) structural proteins. This review will focus on selected blood groups systems (RH, JK, FY, LU, LW, KEL and XK) which are representative of these classes of molecules, in order to illustrate how these studies may bring new information on common and variant phenotypes and for understanding both the mechanisms of tissue specific expression and the potential function of these antigens, particularly those expressed in nonerythroid lineage.

Tumor targeting: activation of prodrugs by enzyme-monoclonal antibody conjugates.

Selective delivery of lethal levels of drugs to tumors, without concomitant damage to normal tissues, is a major challenge in cancer chemotherapy. Prodrugs used in conjunction with enzyme-monoclonal antibody conjugates that can target tumors and convert prodrugs to their active drug forms in situ, offer exceptional promise in achieving this objective. Synthesis of prodrugs, acquisition of appropriate enzymes and monoclonal antibodies, and manufacture of conjugates afford considerable flexibility in experimental design.

Selective modification and immune evasion: a hypothesis.

A hypothesis is proposed asking why enzyme neutralization is not an effective host-response to a parasite despite the fact that some parasite housekeeping enzymes are highly immunogenic. It is hypothesized that although the structural domain can be immunogenic, the active sites of the parasite enzyme molecules have converged evolutionarily to resemble the functional part (active sites) of host's enzyme molecules, by structural modification/rearrangement (amino acid substitution/polypeptide chain folding) with the effect: (i) of functional adaptation to the host environment; and (ii) to escape detection of active sites by the host as non-self, allowing the parasite to be exposed to antiparasite enzyme antibodies, without deleterious effects on the parasite.

Parasite enzymes as a tool to investigate immune responses

Previous evidences reported by us and by other authors revealed the presence of IgG in sera of Schistosoma mansoni-infected patients to immunodominant antigens which are enzymes. Besides their immunological interest as possible inductors of protection, several of these enzume antigens might be also intersting markers of infection in antibody-detecting immunocapture assays which use the intrinsic catalytic property of these antigens. It was thus thought important to define some enzymatic and immunological characteristics of these molecules to better exploit their use as antigens. Four different enzymes from adult worms were partially characterized in their biochemical properties and susceptibility to react with antibodies of infected patients, namely alkaline phosphatase (AKP, Mg*+, pH 9.5), type I phosphodiesterase (PDE, pH 9.5), cysteine proteinase (CP, dithiothreitol, pH 5.5) and N-acetyl-ß-D-glucosaminidase (NAG, pH 5.5). The AKP and PDE are distinct tegumental membrane-bound enzymes whereas CP and NAG are soluble acid enzymes. Antibodies in infected human sera differed in their capacity to react with and to inhibit these enzyme antigens. Possibly, the specificity of the antibodies related to the extent of homology between the parasite and the host enzyme might be in part responsible for the above differences. The results are also discussed in view of the possible functional importance of these enzymes

Comparison of half-lives and cytotoxicity of N-chloroethyl-4-amino and N-mesyloxyethyl-benzoyl compounds, products of prodrugs in antibody-directed enzyme prodrug therapy (ADEPT).

The synthesis of two novel drugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoic acid (7) and 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoic acid (8) is described here. They are the active drugs of two prodrugs (9 and 10) designed for use as anti-cancer agents. The prodrugs (9, 10 and 11) were made as a series of compounds which are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to a glutamic acid residue. These relatively inactive prodrugs were designed to be activated to their corresponding alkylating agent active drugs (7, 8 and 12 respectively) at a tumour site by prior administration of a monoclonal antibody conjugated to a bacterial enzyme. This system is called antibody-directed enzyme prodrug therapy (ADEPT). The chemical half-lives of the prodrugs and their active drugs were measured in order to determine their relative reactivities. The half-lives ranged from 21 to 324 min for the active drugs and from 42 to 1158 min for the prodrugs. The viability of two different tumour cell lines was monitored with each active drug and prodrug. The IC50 values varied from 65 to 625 microM for the active drugs: no IC50 values could be obtained for the prodrugs, using a rapid incubation procedure. Each in vitro technique demonstrated the ability of the glutamic acid moiety to deactivate the drugs, forming effective prodrugs.

Application of immunochemistry to the diagnosis of human neoplasms in routine histologic sections.

The application of immunostaining techniques to the study of sections of formalin-fixed, paraffin-embedded tissues has deeply influenced the practice of surgical pathology of tumors. We have favored in our laboratory the avidin-biotin complex immunoperoxidase method and have gradually substituted monoclonal antibodies for polyclonal sera. Better results are sometimes obtained with trypsinization prior to the application of the primary antibody. The more common primary antibodies used are directed against the following categories of cellular antigens: intermediate filaments, oncofetal products, hormones or hormone-related peptides, enzymes or enzyme-related peptides, cell- or tissue-"specific" products, lymphocyte-leukocyte antigens, and immunoglobulin chains. Controls are essential to every immunohistochemical reaction. Because of the pitfalls of immunohistochemical techniques, immunohistochemistry as applied to the study of tumors should be used with utmost care and as an extension of routine surgical pathology.

Monoclonal antibodies in enzyme research: present and potential applications.

Hybridoma antibodies are powerful tools. Their impact is already apparent in many areas of basic and applied research. In contrast, their impact is just beginning to be felt in enzymology. The existing literature on monoclonal antibodies to enzymes and isozymes, reviewed in this article, is as yet largely descriptive. However, the potential applications discussed herein promise to revolutionize existing strategies of unraveling the basic biochemistry, immunochemistry, and developmental, somatic cell, and molecular genetics of enzymes and isozymes. At a clinical level, monoclonal antibodies to enzymes promise to radically improve the current modalities of diagnosis and therapy in clinical enzymology and oncology. It is becoming increasingly apparent that the future applications of hybridoma antibodies to enzymes and isozymes appear to be limited only by our imagination.