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.

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.

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.

Cell type-specific expression of endogenous cardiac Troponin I antisense RNA in the neonatal rat heart.

Since the number of detected natural antisense RNA is growing, investigations upon the expression pattern of the antisense RNA become more important. As we focused our work on natural occurring antisense transcripts in human and rat heart tissues, we were interested in the question, whether the expression pattern of antisense and sense RNA can vary in different cell types of the same tissue. In our previous analysis of total neonatal rat heart tissue, we demonstrated the co-expression of both cTnI RNA species in this tissue. Now we investigated the expression of antisense and sense RNA quantitatively in neonatal cardiomyocytes (NCMs) and neonatal cardiac fibroblasts (NCFs). Performing northern blot as well as RT-PCR, we could detect natural antisense and sense RNA transcripts of cTnI in NCM and NCF implying that these transcripts are co-expressed in both cell types. The absolute amounts of the RNA transcripts were higher in NCM. Both RNA species showed identical sizes in the northern blot. Quantification by real-time PCR revealed a higher relative level of natural antisense RNA in NCF compared to NCM which points out to a cell type-specific expression of sense and antisense RNA. Our observations suggest that antisense RNA transcription may contribute to a cell type-specific regulation of the cTnI gene.

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 that facilitates the comparison of antibody specificities by reusing the same immunoblot at least three times in order to detect an abundantly expressed autoantigen in total cellular extracts.

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 that 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.

Use of non-radioactive detection method for north- and southwestern 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 northwestern and southwestern blot protocol for the evaluation of the DNA and RNA binding abilities of a novel putative methyl transferase HSPC133 (METTL5).

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.

Regulation of the human atrial myosin light chain 1 promoter by Ca2+-calmodulin-dependent signaling pathways.

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor substances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to approximately 50%. This suggests that the Ca2+-calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the nucleus upon vasopressin stimulation. Inhibition of protein kinase C (PKC) isoforms by bisindolylmaleimide had no significant influence on human ALC-1 promoter activity. Thus, our results demonstrate a dominant role of Ca2+-calmodulin-dependent signaling pathways in the regulation of human ALC-1 expression.

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.

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model.

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17 +/- 4 microg hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8 +/- 2.3 to 45.1 +/- 3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7 +/- 89.8 to 2,181 +/- 135.4 mmHg/s in the contraction rate, and 713 +/- 60.2 to 1,364 +/- 137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.

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.

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.

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).

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.