T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens' syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Differential miRNA-Expression as an Adjunctive Diagnostic Tool in Neuroendocrine Tumors of the Lung.

Pulmonary malignancies with neuroendocrine differentiation represent a rare subclass of lung carcinomas, which vary in the extent of differentiation and grade of biological aggressiveness. In particular, neuroendocrine tumors are classified into well differentiated typical and atypical carcinoids as well as poorly differentiated large cell neuroendocrine and small cell lung carcinomas. Tiny MicroRNAs have been identified as reliable classifiers in distinct cancer types and seem to play important roles in cellular processes like regulation of cell growth, differentiation and apoptosis. In the present study, two different microRNAs (miR-21 and miR-34a) were explored for their involvements in pathogenesis of subtypes and finally in differential diagnosis of pulmonary neuroendocrine tumors. miR-21 was upregulated in poorly differentiated neuroendocrine tumors (mean rank: 26.8; 28.75) as compared to carcinoids (mean rank: 12.33; 12.07) with a significance of 0.00033. High-expression levels of miR-34a were associated with atypical carcinoids (p = 0.010). A close association is implicated between the elevated miR-21 values in high-grade and miR-34a patterns in low-grade atypical neuroendocrine lung carcinomas, which could potentially be exploited as practical supportive markers for differential lung cancer diagnosis in routine. However, some additional extended research and validation studies are needed to utilize them as routine markers or potential molecular targets for personalized medicine.

Use of Nonradioactive Detection Method for North- and South-Western Blot.

Many proteins bind to nucleic acids. For the first characterization of novel proteins, a fast and simple technique for testing their nucleic acid binding capabilities is desirable. Here we describe the use of a North-western and South-western blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

Immunoblotting Using Radiolabeled Reagents for Detection.

Development of immunoblots is commonly performed using enzyme-labeled antibodies which convert soluble substrates into insoluble colored products. A simple, rapid, and sensitive alternative method which produces low background and allows a rapid quantitative evaluation is the use of radiolabeled antibodies or protein A conjugates. Here we describe the use of iodinated secondary antibodies for immunodetection of an autoantigen during HPLC purification.

Sequential Use of Immunoblots for Characterization of Autoantibody Specificities.

Sera of patients with systemic autoimmune diseases frequently contain autoantibodies to nuclear autoantigens. Immunoblotting of recombinant and native autoantigens is a commonly used technique for the identification and characterization of autoantibody specificities. Here we describe an easy procedure which facilitates the comparison of antibody specificities by reusing the same immunoblot for at least three times in order to detect an abundantly expressed autoantigen in total cellular extracts.

A miniaturized blotting system for simultaneous detection of different autoantibodies.

Sera of tumor patients frequently contain autoantibodies to tumor associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.

Signature of microsatellite instability, KRAS and BRAF gene mutations in German patients with locally advanced rectal adenocarcinoma before and after neoadjuvant 5-FU radiochemotherapy.

BACKGROUND: Multiple activating mutations of the signal- and repair pathway, such as BRAF-, KRAS-mutations and microsatellite instabilities are involved in colorectal cancer pathogenesis. Molecular characterization of specifically locally advanced rectal cancers is scarce. Therefore the retrospective study addresses the intratumoral status of KRAS, BRAF and microsatellites loci with respect to tumor response and patients' antecedent including nicotine abusus, familial history, and health care to further molecularly identify rectal cancer patients. METHODS: The study assesses the molecular status of 50 rectal cancer samples (25 before and 25 after neoadjuvant 5-FU radiochemotherapy). KRAS and BRAF mutations were examined through two independent analytical methods (sequencing and SNaPshot) to ensure efficient mutation detection. The microsatellite analysis was conducted using a fluorescent multiplex PCR-based method. RESULTS: KRAS mutations were found in 9 of 25 (36%) rectal cancer patients and were not significantly associated with the response to therapy (P=0.577), age (P=0.249) or sex of the patient (P=0.566). No link exists between KRAS mutation status and nodal (P=0.371) or metastatic stage (P=0.216). For two patients, KRAS mutation status changed after application of neoadjuvant 5-FU radiochemotherapy. All tumor samples were diagnosed BRAF-negative. Two rectal cancer patients exhibited a MSI-H phenotype and showed no tumor response. CONCLUSIONS: So one can conclude that (I) KRAS mutations status may change after neoadjuvant 5-FU radiochemotherapy relevant for further therapeutic decisions; (II) MSI-H patients do not respond to neoadjuvant 5-FU radiochemotherapy. Further prospective studies are needed to validate these results.

Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC): results of a multi-centre ALK-testing.

BACKGROUND: The reliable identification of non-small cell lung cancers (NSCLC) with chromosomal breaks in the gene of the anaplastic lymphoma kinase (ALK) is crucial for the induction of therapy with ALK-inhibitors. In order to ensure a reliable detection of ALK-breaks by means of fluorescence in situ hybridization (FISH) testing, round robin tests are essential. In preparation of a nation (German)-wide round robin test we initiated a pre-testing phase involving 8 experts in FISH-diagnostics to identify NSCLC cases (n = 10) with a pre-tested ALK-status. In addition, ALK immunohistochemistry (IHC) was performed to assess ALK protein expression. MATERIAL AND METHODS: Sections derived from a tissue microarray, each consisting of 3 cores from 10 NSCLC cases, were independently tested for ALK protein expression by IHC and genomic ALK-breaks by FISH involving 8 institutes of pathology. Based on a pre-screening, 5 cases were identified to be clearly ALK-break negative, whereas the remaining 5 cases were ALK-break positive including one case with low percentage (20%) of positive cells. The latter had been additionally tested by RT-PCR. RESULTS: The 5 unequivocal ALK-break negative NSCLC were almost consistently scored negative by means of FISH and IHC by all 8 experts. Interestingly, 4 of the 5 cases with pre-defined ALK-breaks revealed homogenous FISH results whereas IHC for the detection of ALK protein expression showed heterogeneous results. The remaining case (low number of ALK-break positive cells) was scored negative by 3 experts and positive by the other 5. RT-PCR revealed the expression of an EML4-ALK fusion gene variant 1. CONCLUSION: ALK-break negative NSCLC cases revealed concordant homogeneous results by means of FISH and IHC (score 0-1) by all 8 experts. Discordant FISH results were raised in one ALK-break positive case with a low number of affected tumor cells. The remaining 4 ALK-break positive cases revealed concordant FISH data whereas the ALK-IHC revealed very diverse results. The cases with concordant FISH results provide an excellent basis for round robin ALK-FISH testing. As long as standardized ALK-IHC protocols are missing, ALK protein expression cannot by regarded as the method of choice for identification of patients eligible for treatment with ALK inhibitors.

Native polyacrylamide gels.

Usually proteins are separated by polyacrylamide gel electrophoresis (PAGE) in the presence of a detergent and under (heat-) denaturing and (non- or) reducing conditions. The most commonly used detergent is sodium dodecyl sulfate (SDS). The major function of SDS is to shield the respective charge of the proteins present in the mixture to be analyzed and to provide all proteins with a negative charge. As a consequence, the proteins will be separated according to their molecular weight. Electrophoresis of proteins can also be performed in the absence of SDS. Using such "native" conditions, the charge of each of the proteins, which will depend on the primary amino acid sequence of the protein (isoelectric point) and the pH during electrophoresis, will mainly influence the mobility of the respective protein during electrophoresis. Here we describe a starting protocol for "native" PAGE.

Gel drying methods.

For some instances, protein gels need to be dried after SDS-PAGE, for example, if autoradiography should be performed from radioactive-labeled proteins after their separation on SDS-polyacrylamide gels. Another reason may be to simply store the gel in the laboratory book. Aside from expensive commercial solutions, especially for storage of the dried gel in the lab book, the simple and cheap drying protocol here presented may be sufficient.

Coomassie-Brilliant Blue staining of polyacrylamide gels.

Over the past, a series of staining procedures for proteins were published. The most commonly used staining dye for proteins is still Coomassie-Brilliant Blue. The major reason is Coomassie-Brilliant Blue staining is simple, fast, and sensitive. As Coomassie-Brilliant Blue is almost insoluble in water, a series of procedures including colloidal aqueous procedures were described.

Silver staining techniques of polyacrylamide gels.

Although the main application for polyacrylamide gels is the separation and subsequent blotting of proteins for immunodetection, there are tasks that need staining of proteins in the polyacrylamide gel. Several different staining techniques exist for protein staining in SDS gels that differ in their sensitivity, their expenditure of time, and other aspects. Still, silver staining is the most sensitive and reliable staining technique. Because this technique was developed in the 1970s, a huge number of variations exist. Therefore, we will provide herein three methods, which are robust and easy to perform.

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses.

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target cells.

Environmental isocyanate-induced asthma: morphologic and pathogenetic aspects of an increasing occupational disease.

Occupational diseases affect more and more people every year. According to the International Labour Organization (ILO), in 2000 an estimated amount of at least 160 million people became ill as a result of occupational-related hazards or injuries. Globally, occupational deaths, diseases and injuries account for an estimated loss of 4% of the Gross Domestic Product. Important substances that are related to occupational diseases are isocyanates and their products. These substances, which are used in a lot of different industrial processes, are not only toxic and irritant, but also allergenic. Although the exposure to higher concentrations could be monitored and restricted by technical means, very low concentrations are difficult to monitor and may, over time, lead to allergic reactions in some workers, ending in an occupational disease. In order to prevent the people from sickening, the mechanisms underlying the disease, by patho-physiological and genetical means, have to be known and understood so that high risk groups and early signs in the development of an allergic reaction could be detected before the exposure to isocyanates leads to an occupational disease. Therefore, this paper reviews the so far known facts concerning the patho-physiologic appearance and mechanisms of isocyanate-associated toxic reactions and possible genetic involvement that might trigger the allergic reactions.

Unexpected recombinations in single chain bispecific anti-CD3-anti-CD33 antibodies can be avoided by a novel linker module.

CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). In a first attempt for immunotargeting of AML blasts we constructed two bispecific antibodies in the single chain bispecific diabody (scBsDb) format by fusing the variable domains of monoclonal antibodies directed against CD3 and CD33. Unfortunately, protein expression of both scBsDbs resulted in varying mixtures of fragmented and full length proteins. As the non-functional fragments competed with the functional full length antibodies we tried to understand the reason for the fragmentation. We found that the anti-CD3 and anti-CD33 antibody genes show striking sequence homologies: during B cell development the same V(h) J558 heavy and V(l) kk4 light chain genes were selected. Moreover, the closely related D genes DSP2 (9 and 11) were combined with the same JH4 gene. And finally, during VJ recombination of the light chain the same JK5 element was selected. These homologies between the two monoclonal antibodies were the reason for recombinations in the cell lines generated for expression of the scBsDbs. Finally, we solved this problem by (i) rearranging the order of the heavy and light chains of the anti-CD3 and anti-CD33 domains, and (ii) a replacement of one of the commonly used glycine serine linkers with a novel linker domain. The resulting bispecific antibody in a single chain bispecific tandem format (scBsTaFv) was stable and capable of redirecting T cells to CD33-positive tumor cells including AML blasts of patients.

Retargeting of T cells to prostate stem cell antigen expressing tumor cells: comparison of different antibody formats.

BACKGROUND: Prostate cancer (PCa) is the most common malignant disease in men. Novel treatment options are needed for patients after development of metastatic, hormone-refractory disease or for those who have failed a local treatment. The prostate stem cell antigen (PSCA) is expressed in >80% of primary PCa samples and bone metastases. Its expression is increased both in androgen-dependent and independent prostate tumors, particularly in carcinomas of high stages and Gleason scores. Therefore, PSCA is an attractive target for immunotherapy of PCa by retargeting of T cells to tumor cells. METHODS: A series of different bispecific antibody formats for retargeting of T cells to tumor cells were described but, only very limited data obtained by side by side comparison of the different antibody formats are available. We established two novel bispecific antibodies in different formats. The functionality of both constructs was analyzed by FACS and chromium release assays. In parallel, the release of pro-inflammatory cytokines was determined by ELISA. RESULTS AND CONCLUSIONS: Irrespective of the underlying antibody format, both novel bispecific antibodies cause an efficient killing of PSCA-positive tumor cells by pre- and non-pre-activated T cells. Killing and release of pro-inflammatory cytokines requires an antigen specific cross-linkage of the T cells with the target cells.

A novel modular antigen delivery system for immuno targeting of human 6-sulfo LacNAc-positive blood dendritic cells (SlanDCs).

BACKGROUND: Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767-777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. METHODOLOGY/PRINCIPAL FINDINGS: Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. CONCLUSIONS/SIGNIFICANCE: In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines.

Analysis of KRAS Mutations of Exon 2 Codons 12 and 13 by SNaPshot Analysis in Comparison to Common DNA Sequencing.

Due to the call for fast KRAS mutation status analysis for treatment of patients with monoclonal antibodies for metastatic colorectal cancer, sensitive, economic, and easily feasible methods are required. Under this aspect, the sensitivity and specificity of the SNaPshot analysis in comparison to the commonly used DNA sequencing was checked. We examined KRAS mutations in exon 2 codons 12 and 13 with DNA sequencing and SNaPshot analysis in 100 formalin-fixed paraffin-embedded tumor tissue samples of pancreatic carcinoma, colorectal carcinoma, and nonsmall cell lung cancer specimens of the primary tumor or metastases. 40% of these samples demonstrated mutated KRAS genes using sequencing and SNaPshot-analysis; additional five samples (45/100) were identified only with the SNaPshot. KRAS mutation detection is feasible with the reliable SNaPshot analysis method. The more frequent mutation detection by the SNaPshot analysis shows that this method has a high probability of accuracy in the detection of KRAS mutations compared to sequencing.

Search for and identification of novel tumor-associated autoantigens.

During the development of tumors, autoantibodies against aberrant or overexpressed autoantigens can be induced. Several hundreds of tumor-associated autoantibodies (TAAB) with more or less specificity for tumors have been found until now by molecular cloning and proteomics technologies. Many TAAB are detectable in preclinical stages of the disease and may be indicators of tumor development. The screening for autoantibody responses in tumor patients may lead to new diagnostic tumor markers and may be a simple and effective way to identify concomitantly cytotoxic T-lymphocyte (CTL) reactivity. However, most of the TAAB lack sufficient sensitivity and specificity for use as biomarkers in the clinical practice. For further use TAAB should be selected for their specificity regarding malignancies and for their potential clinical application. If selected for high specificity, for the screening of risk groups the sensitivities of most TAAB are too low. A combined determination of two or more tumor-specific autoantibodies may overcome this problem. Therefore, a further evaluation of the relevance of known autoantibody specificities as well as the search for novel diagnostically relevant TAAB by different methodologies is necessary. An optimal combination of highly specific TAAB in multiparametric assays as well as the standardization of the autoantibody analysis is necessary to exhaust the potential of TAAB in the early (presymptomatic) diagnosis and monitoring of malignancies.

A miniaturized blotting system for simultaneous detecting of different autoantibodies.

Sera of tumor patients frequently contain autoantibodies to tumor-associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.