Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

An automatable platform for genotoxicity testing of nanomaterials based on the fluorometric γ-H2AX assay reveals no genotoxicity of properly surface-shielded cadmium-based quantum dots.

The large number of nanomaterial-based applications emerging in the materials and life sciences and the foreseeable increasing use of these materials require methods that evaluate and characterize the toxic potential of these nanomaterials to keep safety risks to people and environment as low as possible. As nanomaterial toxicity is influenced by a variety of parameters like size, shape, chemical composition, and surface chemistry, high throughput screening (HTS) platforms are recommended for assessing cytotoxicity. Such platforms are not yet available for genotoxicity testing. Here, we present first results obtained for application-relevant nanomaterials using an automatable genotoxicity platform that relies on the quantification of the phosphorylated histone H2AX (γ-H2AX) for detecting DNA double strand breaks (DSBs) and the automated microscope system AKLIDES® for measuring integral fluorescence intensities at different excitation wavelengths. This platform is used to test the genotoxic potential of 30 nm-sized citrate-stabilized gold nanoparticles (Au-NPs) as well as micellar encapsulated iron oxide nanoparticles (FeOx-NPs) and different cadmium (Cd)-based semiconductor quantum dots (QDs), thereby also searching for positive and negative controls as reference materials. In addition, the influence of the QD shell composition on the genotoxic potential of these Cd-based QDs was studied, using CdSe cores as well as CdSe/CdS core/shell and CdSe/CdS/ZnS core/shell/shell QDs. Our results clearly revealed the genotoxicity of the Au-NPs and its absence in the FeOx-NPs. The genotoxicity of the Cd-QDs correlates with the shielding of their Cd-containing core, with the core/shell/shell architecture preventing genotoxicity risks. The fact that none of these nanomaterials showed cytotoxicity at the chosen particle concentrations in a conventional cell viability assay underlines the importance of genotoxicity studies to assess the hazardous potential of nanomaterials.

Autoimmunity in Crohn's Disease-A Putative Stratification Factor of the Clinical Phenotype.

Inflammation in inflammatory bowel diseases (IBD) has been linked to a loss of tolerance to self-antigens suggesting the existence of autoantibodies in specific disease phenotypes. However, the lack of clearly defined autoantigenic targets has slowed down research. Genome-wide association studies have identified an impressive number of immune-related susceptibility loci for IBD with no clearly discernible pattern among them. Growing evidence supports the hypothesis that innate immune responses to a low-diversity and impaired gut microbiota may be of key importance in initiating and perpetuating chronic inflammation in IBD. Increasing evidence suggests that reduced microbial diversity and microbial-mucosal epithelium interaction (including adhesion and clearance) are critically involved in IBD pathogenesis. Along these lines the discovery of autoantigenic targets in Crohn's disease (CD) has refocused research in IBD on the possible role of autoimmune responses. The identification of the major zymogen granule membrane glycoprotein 2 (GP2) as an autoantigen in CD patients and its proposed role in the sensing of the microbiota lends credence to this trend. Loss of tolerance to GP2 occurs in up to 40% of patients with CD. Corresponding autoantibodies appear to be associated with distinct disease courses (types or phenotypes) in CD. Here, we critically review autoantibodies in CD for their impact on clinical practice and future IBD research. The immunomodulatory role of GP2 in innate and adaptive intestinal immunity is also discussed.

Expression of nicotinic acetylcholine receptor subunits in HEp-2 cells for immunodetection of autoantibody specificities in sera from Myasthenia gravis patients.

Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics.

A strategy for cell-based multiplex diagnostics of Myasthenia gravis and autoimmune encephalitis by modifying the subcellular localization of cell membrane autoantigens.

Many autoimmune diseases are characterized by autoantibodies directed against cell membrane proteins. We were intrigued to develop a strategy for targeting individual cell membrane proteins to various subcellular compartments as a prerequisite for their simultaneous immunofluorescence detection. We first employed GFP and RFP reporters that were equipped with defined intracellular localization signals. Expressing these protein reporters in HEp-2 cells we found by using fluorescence microscopy that protein localization in cytoplasm or at mitochondria can be clearly discriminated from localization at Golgi, ER or lysosomes. We then tested for muscle-specific kinase, a relevant cell membrane autoantigen in Myasthenia gravis, and NMDA receptor which is relevant for autoimmune encephalitis, whether these autoantigens can be localized to the same intracellular compartments. To this end, we successfully targeted muscle-specific kinase to Golgi apparatus, mitochondria and cytoplasm. We found that its Golgi localization can be clearly distinguished from its natural cell membrane localization. The same we found for Golgi-localized NMDA receptor 1. Interestingly, cell membrane proteins kept at the Golgi system accumulated in higher amounts than their wild-type counterparts. The obtained results are the basis for the further development of multiplex assays for the immunofluorescence diagnostics of Myasthenia gravis and autoimmune encephalitis.

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.

[Use of indirect micromodification of immunoenzyme technique for serologic detection of Ixodes tick-borne borreliosis (comparative analysis)]. / Primenenie nepriamoi mikromodifikatsii immunofermentnoi reaktsii dlia serologicheskogo podtverzhdeniia iksodovykh kleshchevykh borreliozov (sravnitel'nyi analiz).

Three hundred and seventy one sera sampled in different period since the onset of the disease from 214 patients with Ixodes tick-borne borreliosdes (ITBB) caused by Borrelia afzelii and B. garinii in the Perm Region (Russia) and 299 sera from 222 patients with other diseases were simultaneously tested for IgM and IgG antibodies by using immunofluorescence assay (IFA) with standard antigen from strain Ip-21 B. afzelli by indirect micromodification of enzyme immunoassay (IMELISA) on slides and routine enzyme immunoassay (ELISA) with standard commercial test systems (Medipan Diagnostica GmbH, Germany). It is concluded that IMELISA is a technically easy and accessible test, which is close to IFA in its specificity and sensitivity, and can be widely used for serological verification of ITBB.

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.

Generation and characterization of a human monoclonal IgM antibody that recognizes a conserved epitope shared by lipopolysaccharides of different gram-negative bacteria.

A hybridoma cell line secreting a human monoclonal antibody (humab) directed to an epitope in the lipid A region of lipopolysaccharides of Gram-negative bacteria was isolated. Peripheral blood lymphocytes (PBL) obtained from a healthy volunteer were immortalized by Epstein-Barr virus (EBV) transformation. Lymphoblastoid cell lines (LCL) secreting antibodies to the lipopolysaccharides of Gram-negative bacteria were determined by an enzyme-linked immunosorbent assay (ELISA) and subsequently fused with the human-mouse heteromyeloma cell line CB-F7 by polyethylenglycol (PEG)-mediated fusion. A hybridoma line producing a humab (LPD5H4), of the IgM/lambda isotype, which strongly reacted with the lipid A portion of Salmonella and E. coli spp. in ELISA, was established. The antibody was purified by hydrophobic interaction chromatography and gel filtration. Immunoblotting experiments showed a strong reactivity of the humab LPD5H4 with the lower molecular species of different rough and smooth lipopolysaccharide (LPS) types of the bacteria species Salmonella, E. coli, Klebsiella, and Neisseria meningitidis, whereas those of Pseudomonas spp. were negative. Binding of humab LPD5H4 to solid phase bound lipid A and different rough mutants of LPS could be inhibited by the corresponding antigens in solution. Competition assays with a murine monoclonal antibody to lipid A and with polymyxin B indicate that humab LPD5H4 recognizes its epitope in this extremely conserved part of the LPS molecule. In vitro tests demonstrated that the MAb is able to partially inhibit the LPS-induced release of TNF-alpha using isolated peripheral blood mononuclear cells (PBMC).

Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity.

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.

Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene.

Recently we described the occurrence of B cells producing polyspecific natural IgM with anti-tumour specificity in the spleen of non-tumour-bearing individuals as well as in fetal organisms. Immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pI 6.0) membrane surface glycoprotein. In vitro cultivation of human cancer cell lines in the presence of the purified IgM antibodies resulted in growth inhibition and complement-mediated cell lysis. Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells. Because of this, a role for naturally occurring antibodies with anti-tumour specificity in preventing neoplasias had been suggested. We have constructed and expressed in Escherichia coli single-chain fragments (scFv: VH-linker-VL) derived from a polyspecific human monoclonal IgM autoantibody produced by a human x mouse heterohybridoma which was obtained from the spleen of an autoimmune patient. The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another polyspecific human natural antibody with no binding to tumours. Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line VH gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells. Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity. We discuss the significance of the replacement mutations in the VH gene CDRs, selected probably by B cell contact to an (auto)antigen, for generating a tumour binding capacity, not encoded by the germ-line gene.

Characterization of a B-CLL derived IgM-lambda antibody expressing typical features of a NPAB.

A human IgM-lambda hybridoma (CB-HB) was established from chronic lymphocytic leukaemia (CLL) B-cells. Immunochemical and molecular characterization of the monoclonal antibody (mab) produced by the CB-HB cells offered typical features of natural polyreactive antibodies (NPAPs) found in fetal and healthy adult organisms. In particular, the CB-HB mab reacted with different self and foreign (viral and bacterial) antigens when tested in three independent systems (solid- and fluid-phase ELISA, Western blot) showing binding constants in a range from 1.9 x 10(-7) to 7.5 x 10(-8) mol/l to four antigens chosen. In addition, the CB-HB mab binding could be inhibited by a rabbit polyclonal antiserum specific for a common idiotype (Id 102) on human polyreactive (auto)antibodies. The variable region of the CB-HB mab was found to be encoded by unmutated copies of germline genes. Interestingly, the VH-DP10 (51p1) segment, encoding for the autoantibody-associated G6-cross reactive idiotype frequently expressed on both fetal and malignant B-cells, was found to be used with a V segment (VLO11). Collectively these data imply that cells belonging to the natural polyreactive B-cell repertoire undergo malignant transformation. A stimulation by autoantigens or common foreign antigens may be involved.

Binding of natural human IgM auto-antibodies to human tumor cell lines and stimulated normal T lymphocytes.

In a recent publication we described the binding of natural IgM antibodies derived from the human fetal B cell repertoire to the cell surface of some human tumor cells including colon carcinoma, small-cell lung cancer and B lymphoma lines [1]. Further analyses showed that a similar molecule was bound by the respective monoclonal human antibodies on the cell surface of polyclonally stimulated human CD3+ T cells, but is absent from unstimulated MNC. Both CD4+ and CD8+ stimulated cells were recognized. The molecule was found to be expressed together with lymphocyte activation markers (4F2, CD72, CD25). The membrane antigen expressed on both the activated T lymphocytes and tumor cells was characterized in a 2-D electrophoresis system: molecular weight 55-60 kDa, pI-approximately 6.0. Whereas the proliferation capacity of tumor cells was detected to be decreased significantly in the presence of the binding antibodies, no influence on [3H]thymidine uptake into stimulated T cells was found, suggesting different functional consequences of binding the respective antigen on malignant and normal cells. An interesting finding is the enhanced expression of major histocompatibility complex class I molecules on tumor cells incubated with human natural antibodies.