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BackgroundTumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC).Methods/ResultsTiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23+, CD80+) and produced immunoglobulin (Ig; IgG secretion in 55% of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigen-experienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV.ConclusionIn summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specific antigens.

Ligands specifically binding to leukemia cells may be used for drug targeting, resulting in more effective treatment with less side effects. Little is known about receptors specifically expressed on acute myeloid leukemia (AML) cells or ligands thereof. We selected random phage display peptide libraries on Kasumi-1 AML cells. A peptide with the sequence CPLDIDFYC was enriched. Phage displaying this peptide strongly bound to Kasumi-1 and SKNO-1 cells and binding could be inhibited by the cognate peptide. Both, Kasumi-1 and SKNO-1 cells carry the chromosomal translocation t(8;21), leading to aberrant expression of the fusion protein AML1/ETO. CPLDIDFYC also strongly and specifically bound primary AML1/ETO-positive AML blasts as well as U-937 cells with forced AML1/ETO expression, suggesting that the CPLDIDFYC receptor may be upregulated upon AML1/ETO expression. Gene expression profiling comparing a panel of CPLDIDFYC-binding and CPLDIDFYC-nonbinding cell lines identified a set of potential receptors for the CPLDIDFYC peptide. Further analysis suggested that a4b1 integrin (VLA-4) is the CPLDIDFYC receptor. Finally, we showed that the CPLDIDFYC-phage is internalized upon receptor binding, suggesting that the CPLDIDFYC-receptor-ligand interaction may be exploitable for targeting drugs or gene therapy vectors to leukemia cells carrying the suitable receptor.

Patient-specific sequential epitopes were identified by peptide chip analysis using 15mer peptides immobilized on glass slides that covered the topoisomerase IIa protein with a frameshift of five amino acids. Binding specificities of serum antibodies against sequential epitopes were confirmed as being mono-specific by peptide chip re-analysis of epitope-affinity-purified antibody pools. These results demonstrate that serum samples from colon carcinoma patients contain antibodies against sequential epitopes from the topoisomerase IIa antigen. Interactions of patients' antibodies with sequential epitopes displayed by peptides on glass surfaces may thus mirror disease-specific immune situations. Consequently, these data suggest epitope-antibody reactivities on peptide chips as potential diagnostic readouts of individual immune response characteristics, especially because monospecific antibodies can be interrogated. Subsequently, the clonality of the antibodies present in the mono-specific antibody pools was characterized by 2D gel electrophoresis. This analysis suggested that the affinity-purified antibodies were oligoclonal. Similarly to large-scale screening approaches for specific antigen-antibody interactions in order to improve disease diagnostic, we suggest that "protein-wide" screening for specific epitope-paratope interactions may help to develop novel assays for monitoring of personalized therapies, since individual properties of antigen-antibody interactions remain distinguishable.

Biomaterials such as applied in microcapsules may have harmful effects on encapsulated cells. Up to now, there are no adequate assays available for testing the function and viability of cells in capsules. Therefore, we investigated whether the combination of MTS proliferation assay and live-dead viability assay is suitable for testing microencapsulated L929 fibroblasts in long-time culture. Proliferation of L929 cells was shown by a significant increase of formazan absorbance within the first 3 weeks (Day 0: 0.132 +/- 0.047; Day 7: 0.404 +/- 0.101; Day 14: 0.728 +/- 0.239; Day 21: 0.877 +/- 0.224) followed by stagnation and decrease thereafter. This was confirmed by an increasing proportion of dead cells measured by the live-dead assay. Thus, proliferation of encapsulated L929 can be reliably investigated by the MTS assay. In combination with life-dead assays, the proliferation can be correlated to the survival rate of the encapsulated cells.

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ObjectiveTo determine the spectrum of anti-neutrophil cytoplasmic antibody (ANCA) antigen-specificities in eosinophilic granulomatosis with polyangiitis (EGPA), an ANCA-associated vasculitis (AAV) entity. MethodsWe conducted a retrospective analysis of 73 EGPA patients from three German tertiary referral centres for vasculitis. In addition to in-house ANCA testing, pentraxin 3 (PTX3)-and olfactomedin 4 (OLM4)-ANCA were determined using a prototype cell-based assay for research (EUROIMMUN, Lübeck, Germany). Patient characteristics and clinical manifestations were evaluated and compared based on ANCA status. Results Myeloperoxidase (MPO)-ANCA positive patients (n=8; 11%) significantly more frequently displayed peripheral nervous system (PNS) and pulmonary involvement and less frequently heart involvement compared to MPO-ANCA negative patients. PTX3-ANCA positive patients (n=5; 6.8%) had a significantly higher prevalence of ear, nose and throat, pulmonary, gastrointestinal and PNS involvement, and a lower prevalence of renal and central nervous system involvement compared to PTX3-ANCA negative patients. Proteinase 3 (PR3)-ANCA and OLM4-ANCA were detected in 2 patients (2.7%) each with multiorgan involvement. One PR3-ANCA positive patient was also positive for bactericidal permeability increasing protein (BPI)-ANCA. ConclusionIn addition to MPO, the spectrum of ANCA antigen specificities includes various other target antigens such as PR3, BPI, PTX3, and OLM4, potentially segregating further EGPA subgroups. A lower prevalence of MPO-ANCA was detected in this study compared with other studies. OLM4 is reported as novel ANCA antigen-specificity in EGPA, and thus AAV.