Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn's disease.

BACKGROUND AND AIMS: The aetiopathogenesis of Crohn's disease, an inflammatory bowel disease (IBD), is not yet fully understood. Autoimmune mechanisms are thought to play a role in the development of Crohn's disease, but the target antigens and the underlying pathways have not been sufficiently identified. METHODS: Based on data from immunoblotting and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry, the major antigenic target of pancreatic autoantibodies (PABs), which are specific for Crohn's disease, was identified. Specificity of autoantibody reactivity was confirmed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) using purified rat and human recombinant GP2 synthesised in transiently transfected mammalian HEK 293 cells. Real-time polymerase chain reaction (rt-PCR) and IIF were used to detect mRNA and antigen localisation in human colon biopsies. RESULTS: The major zymogen granule membrane glycoprotein 2 (GP2) was identified as the autoantigen of PABs in Crohn's disease. PAB-positive sera from patients with Crohn's disease (n = 42) displayed significantly higher IgG reactivity to rat GP2 in ELISA than either PAB-negative sera (n = 31), or sera from patients with ulcerative colitis (n = 49), or sera from blood donors (n = 69) (p<0.0001, respectively). Twenty-eight (66%) and 18 (43%) of 42 PAB-positive sera demonstrated IgG and IgA reactivity to human recombinant GP2 in IIF, respectively. Patients with PAB-negative Crohn's disease (n = 31) were not reactive. GP2 mRNA transcription was significantly higher in colon biopsies from patients with Crohn's disease (n = 4) compared to patients with ulcerative colitis (n = 4) (p = 0.0286). Immunochemical staining confirmed GP2 expression in human colon biopsies from patients with Crohn's disease. CONCLUSION: Anti-GP2 autoantibodies constitute novel Crohn's disease-specific markers, the quantification of which could significantly improve the serological diagnosis of IBD. The expression of GP2 in human enterocytes suggests an important role for anti-GP2 response in the pathogenesis of Crohn's disease.

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.

Cu/Zn-SOD in human pancreatic tissue and pancreatic juice.

CONCLUSION: Cu/Zn-SOD is present in pancreatic juice and tissue. Immunohistochemical studies reveal a localization of this enzyme in islet, duct, and centroacinar cells, but to a much lower extent in pancreatic acinar cells. BACKGROUND: It is generally accepted that oxygen radicals are involved in the pathogenesis of acute and chronic pancreatitis. An imbalance of radical-generating and radical-scavenging processes is thought to lead to the damage of pancreatic acinar cells that initiate the autodigestion of the whole organ. METHODS: We investigated the distribution pattern of the cytosolic radical-scavenging enzyme, copper/zinc-superoxide dismutase (Cu/Zn-SOD), in pancreatic juice and tissue. In patients with chronic pancreatitis or pancreatic malignancies, Cu-Zn-SOD was quantitated in different fractions of pancreatic juice by means of an enzyme immunoassay using two Cu/Zn-SOD-specific monoclonal antibodies. Cryostat or paraffin sections of pancreatic tissue were analyzed by immunohistochemical techniques. RESULTS: We found this enzyme to be present in the first secretin-triggered fraction of endoscopically obtained pancreatic juice in concentrations similar to serum. In contrast, after cholecystokinin stimulation, only low levels could be found in pancreatic juice, indicating that this enzyme is not actively secreted. Interestingly, pancreatic juice of patients with chronic pancreatitis or pancreas tumor contained higher levels (25-29 ng/mL) of Cu/Zn-SOD than juice of controls without pancreatic diseases (15 ng/mL). Immunohistochemical studies of Cu/Zn-SOD in pancreatic tissue revealed a more intense staining of duct cells, islet cells, and centroacinar cells, whereas acinar cells showed almost no staining for Cu/Zn-SOD.

Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS).

Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria. The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS). Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS. Immunoblotting has become a common method to verify the specificity of antigen antibody interactions. Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems. These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties. In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs. The polyreactive monoclonal human antibody CB03 used was screened for lipid A/LPS reactivity in ELISA experiments. The binding was confirmed in the described blot system and depends on the membranes and blocking agents used. The use of nitrocellulose versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining.

Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Cu,Zn SOD in German families with ALS.

We studied the gene for Cu,Zn SOD in 15 German patients with familial ALS and did not find any mutation. Activity of the enzyme and its expression at the protein level was also normal in each patient and in 18 patients suffering from the sporadic form of ALS.

Tetrasomy 21 pter-->q22.1 and Down syndrome: molecular definition of the region.

Down syndrome is usually caused by complete trisomy 21. Rarely, it is due to partial trisomy of the segment 21q22. We report on a 33-month-old girl with tetrasomy 21 pter-->q22.1 resulting from an extra chromosome idic(21)(q22.1). She has craniofacial traits typical of Down syndrome, including brachycephaly, third fontanel, upward slanting palpebral fissures, round face, and protruding tongue. Speech development is quite delayed whereas motor development is only mildly retarded. The molecular content of the extra isodicentric chromosome was defined by molecular genetic investigations using 13 single copy probes unique to chromosome 21, and SOD1 expression studies. The child was found to have 4 copies of the region defined by D21S16 (21cen) through D21S93 on 21q22.1 and two copies of the remaining region defined by SOD1-->D21S55-->D21S123. In view of the recent assignment of Down syndrome facial characters to the 21q22 region, defined in part by D21S55, it is significant that this child shows a subset of Down syndrome facial manifestations, without duplication of this region. These results suggest that genes contributing to the facial and some of the hand manifestations of Down syndrome also exist in the chromosomal region proximal to D21S55 in band 21q22.1.

A recombinant human immunodeficiency virus type-1 capsid protein (rp24): its expression, purification and physico-chemical characterization.

An expression system has been established in Escherichia coli to facilitate the preparation of the HIV-1 capsid protein in amounts sufficient for structural analysis. A plasmid vector pTCA5, containing the gene for the recombinant HIV-1 capsid protein rp24 under the control of the lambda-PR-promoter, was constructed which gave an expression product that spanned 234 amino acid residues. It differs at the N-terminus from the authentic sequence in that the residues Pro-Ile- are replaced by Met-Asn-Ser-Ala-Met-. Recombinant p24 was produced, as inclusion bodies in E. coli LE392 containing pTCA5, at a level of approximately 15% of the total cellular protein. After dissolution of the inclusion bodies in the acidic urea system, the protein was easily reconstituted in a soluble state by dialysis. The yield of reconstituted and purified protein was 12 mg per liter in rich medium. Recombinant rp24 consists of about 40% alpha-helix and 10% beta-sheet from circular dichroism measurements and the two cysteine residues, within the rp24 sequence, are bridged by a disulfide bond.

Real-time biospecific interaction analysis of a natural human polyreactive monoclonal IgM antibody and its Fab and scFv fragments with several antigens.

Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single-chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed. Interaction of scFv with kappa casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody. Apparent binding constants of the complete antibody to immobilized kappa casein (9.2 x 10(7) M-1) and to human myoglobin (1.6 x 10(7) M-1) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.

The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Intracellular Cu/Zn superoxide dismutase levels in T and non-T cells from normal aged subjects.

The decreased immune response associated with aging may, in part, reflect intrinsic age-related biochemical alteration in lymphocytes from older subjects. The 'reactive oxygen species hypothesis' of aging postulates that these molecules are involved in the modifications leading to cellular senescence. Superoxide dismutase (SOD), and in particular the Cu/Zn-dependent intracellular form, plays a critical role in the defense against these species, but it is controversial whether this function declines in lymphocytes in old age. We utilized two different methods to evaluate Cu/Zn SOD levels in T and non-T cells (CD3+, CD3-, CD4+, CD8+, CD16+) from young and old individuals: a specific and sensitive enzyme immunoassay performed on extracts of sorted cells, and a flow cytometry double fluorescence technique with monoclonal antibodies against Cu/Zn SOD and the different lymphocyte subsets. The Cu/Zn SOD cell content was assayed both in basal conditions and after peripheral blood lymphocyte stimulation with Concanavalin A, anti-CD3 monoclonal antibody and phorbol myristate acetate. In basal conditions, and considering the various subsets, no differences were found between young and old individuals, although data analysis revealed high and low responders in both groups. Taking all the subjects together, higher levels of this enzyme were found in CD3+ T lymphocytes, in particular in the CD4+ cells. After peripheral blood lymphocyte stimulation, Cu/Zn SOD concentration was higher than levels in unstimulated cells, both in young and old individuals, and particularly using Concanavalin A with respect to anti-CD3 and phorbol myristate acetate. In conclusion, the synthesis of Cu/Zn SOD does not seem to be affected by aging in proliferating cells. The highest levels of Cu/Zn SOD present in CD4+ cells, both from young and old individuals, may prevent the oxidant stress of these elements which play a major role in the inflammation sites.

Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody.

In vitro and in vivo experiments to explain the function of natural polyreactive antibodies, usually of the IgM isotype, require large amounts of purified antibodies. We have developed a two-step purification procedure using a human natural polyreactive monoclonal IgM antibody (CB03). This combines hydrophobic interaction chromatography on phenyl-Superose and gel filtration over Superose 12 and readily permits scaling-up to isolate mg to g amounts of antibody. Retention of the CB03 antibody during gel filtration by precipitation and interaction with the gel matrix was overcome by the addition of 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The yield of purified antibody was 34% and Fab fragments were obtained from the purified CB03 antibody by hot tryptic digestion (yield, 68% of theoretical amount). In an enzyme-linked immunosorbent assay, Fab and complete antibody had similar reaction patterns with different antigens.

Quantitative determination of CD4/CD8 molecules by a cell marker ELISA.

Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P < 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P < 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.

Elevated serum superoxide dismutase levels correlate with disease severity and neutrophil degranulation in idiopathic pulmonary fibrosis.

1. Tissue damage in idiopathic pulmonary fibrosis is due in part to oxidant-antioxidant imbalance. 2. We evaluated the serum levels of the antioxidant enzyme Cu/Zn superoxide dismutase (EC 1.15.1.1) in 25 patients with idiopathic pulmonary fibrosis, 34 patients with sarcoidosis and 40 healthy control subjects by an enzyme immunometric assay. 3. We found that patients with idiopathic pulmonary fibrosis have higher serum Cu/Zn superoxide dismutase levels than control subjects and patients with sarcoidosis. In addition, serum Cu/Zn superoxide dismutase levels correlate with disease severity indexes in patients with idiopathic pulmonary fibrosis. 4. The increase in serum Cu/Zn superoxide dismutase level in idiopathic pulmonary fibrosis could depend on degranulation of activated neutrophils or release from damaged cells. To elucidate the contribution of neutrophil degranulation we determined the polymorphonuclear cell elastase level in the same specimens. We found a strong correlation between serum Cu/Zn superoxide dismutase and polymorphonuclear cell elastase activities, and, in patients with idiopathic pulmonary fibrosis, we observed higher levels of polymorphonuclear cell elastase than in control subjects and patients with sarcoidosis, which correlated positively with disease severity indexes. 5. Cu/Zn superoxide dismutase can catalyse the dismutation of O2 into H2O2 and generate OH.. These oxygen radicals are probably the major factors responsible for tissue damage (in particular, alveolar and endothelial cells) and fibrosis in experimental lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-colour combination enzyme-linked immunosorbent assay for the simultaneous detection of HBV and HIV infection.

To reduce the cost, time and waste in screening for HIV and HBV infections a combined assay for HIV-1 and -2 antibodies and HBsAg has been developed. Monoclonal anti-HBs antibodies were co-immobilized with synthetic peptides representing immunodominant regions of HIV-1 and -2. The presence of anti-HIV antibodies in the samples was detected with alkaline phosphatase-labelled anti-human IgG and of HBsAg with horseradish peroxidase-labelled monoclonal anti-HBs antibodies by a sequential substrate reaction. In this assay, HBsAg was detectable in a concentration range between 0.25 and 0.30 U/ml and the results were available within 3 h. The specificity, tested on 5000 serum samples from blood donors after confirmation, was 99.8% for HBsAg and 99.5% for anti-HIV antibody detection. All serum samples taken from 600 HIV-1- and 115 HIV-2-infected individuals were correctly classified as reactive. The two-colour HBsAg-anti-HIV-1/-2 combination ELISA meets all the requirements of single parameter assays with regard to precision, stability and robustness.

Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies.

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.

Enzyme immunoassay techniques. An overview.

In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups 'analyte-observed' and 'reagent-observed' assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee 'one step' assays of only a few minutes. Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10-1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2-10. Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzyme complex to analyte-containing immune complexes are discussed. Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules. Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.