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The aim of the present study was to isolate and characterize the immune complexes detected by the Raji cell assay in metasatic breast cancer. However, the Raji cell binding material could not be separated from monomeric IgG by Sephacryl S-3(H) gel chromatography in any of the 16 sera investigated. The parallel elution profile of monomoric IgG and the Raji cell binding activity suggested that anti-Raji cell antibodies, rather than immune complexes of low molecular weight, were present in these sera. This was further substantiated by pepsin digestion of the Raji cell binding IgG fractions. The binding of F(ab')2 fragments was quantitatively comparable to the binding of undigested IgG, and the bound F(ab') 2 fragments could be visualized by immunofluorescence at the cell membrane. The presence of antibodies against Raji cells was further confirmed by the complement-dependent cytotoxicity assay. These antibodies did not react with untransformed lymphocytes and there was no correlation with anti-Epstein-Barr virus antibodies. The incidence of cytotoxic anti-Raji cell antibodies in breast cancer was 12% compared to 20% in malignant lymphoma, to 0% in normals and patients with degenerative cardiovascular diseases, and to 5% in patients with auto-immune diseases. [ABSTRACT FROM AUTHOR]

The article discusses the use of an electro-transfection of dendritic cells (DC) with human reverse transcriptase (hTERT) mRNA that enables a human leukocyte antigen (HLA) independent whole antigen approach potentially targeting a wide range of hTERT epitopes. It details how the DCs of a healthy individual were transfected with green fluorescent protein (GFP) mRNA.

Quantifying urease activity is an important task for Microbial Induced Calcite Precipitation research. A new urease activity microplate assay using a fluorescent pH indicator is presented. The method is also suitable for automated measurements during microbioreactor experiments. The assay reagent consists of the green fluorescent pH-indicator fluorescein, urea and a phosphate buffer. After sample addition, the microbial urease hydrolyses urea, which results in a pH and hence fluorescence increase. The fluorescence signal can be measured with a microplate reader or with the microbioreactor system BioLector, allowing for automated urease activity measurements during cultivation experiments. In both measurement systems, the fluorescence signal slope highly correlates with the urease activity measured offline with standard methods. Automated measurement is possible, as no sample preparation such as centrifugation or adjusting of the optical density is required. The assay was developed so that the culture samples turbidity, salinity or buffer concentration does not have a negative impact on the fluorescence signal. The assay allows for straightforward, non-hazardous, parallelized, cheap and reliable measurements, making research on ureolytic bacteria for Microbial Induced Calcite Precipitation more efficient. The assay could be adapted to other enzymes, which have a strong impact on the pH value., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lapierre, Bolz, Büchs and Huber.)

Complex formation of the enolizable chromophor 1-n-butyl-5-(4-nitrophenyl)barbituric acid 1 with multiple binding sites for supramolecular assemblies and its corresponding adducts produced with the Proton Sponge (1,8-bis(dimethylamino)naphthalene), PS) and the adenine-mimetic 2,6-diacetamidopyridine (DAC) have been studied by means of solid-state proton NMR spectroscopy under fast magic-angle spinning, X-ray analysis, and UV/vis spectroscopy. Both NMR data and X-ray results reveal that the enolic chromophor undergoes self-aggregation to hydrogen-bonded dimers which are involved in stacked arrangements. Depending on the nature of the added base, this dimeric assembly is preserved in the formed enolate anion but can be broken in the presence of complementary hydrogen-bonding pattern leading to supramolecular complexes. Molecular recognition of these structural different bases significantly influences the chromophoric pi-system of 1.

The use of hydrogen-bonding patterns in the same way as is known from DNA building blocks is a challenge for the construction of novel types of suitable chromophoric probes. This feature has been utilised for the construction of a novel type of UV/Vis probe for detection of supramolecular AAD or DAD sequences (A=hydrogen bond acceptor, D=hydrogen bond donor). Here we report on the structure of the enolisable chromophore 1-n-butyl-5-(4-nitrophenyl)barbituric acid (1), which has an adjustable hydrogen-bonding pattern. The position of the keto-enol equilibrium of this dye is strongly influenced both by the solvent polarity and by the chemical environment. Furthermore, the recognition properties of the barbiturate were examined by the use of seven artificial receptors: the pyridine bases 2,6-diaminopyridine (DAP), 2,6-diacetamidopyridine (DAC) and 2,6-bis(trifluoroacetamido)pyridine (TFA), as well as the nucleic acid bases 9-ethyladenine (EtAd), 9-ethylguanine (EtGu), 1-n-butylcytosine (BuCy) and 1-n-butylthymine (BuTy). It was found that 1 can interact with these bases either through acid-base interaction or by hydrogen-bonding complexation. The balance between the interactions is dependent both on the basicity strength and on the presence of a suitable recognition sequence in the base. The induced formation of the enol form of 1 thus causes a significant UV/Vis shift as function of the nature of the base.

The human t(14;18) chromosomal translocation is assumed to result from illegitimate rearrangement between BCL-2 and D(H)/J(H) gene segments during V(D)J recombination in early B cells. De novo nucleotides are found inserted in most breakpoints and have been thus far interpreted as nontemplated N region additions. In this report, we have analyzed both direct (BCL-2/J(H)) and reciprocal (D(H)/BCL-2) breakpoints derived from 40 patients with follicular lymphoma with t(14;18). Surprisingly, we found that more than 30% of the breakpoint junctions contain a novel type of templated nucleotide insertions, consisting of short copies of the surrounding BCL-2, D(H), and J(H) sequences. The features of these templated nucleotides, including multiplicity of copies for 1 template and the occurrence of mismatches in the copies, suggest the presence of a short-patch DNA synthesis, templated and error-prone. In addition, our analysis clearly shows that t(14;18) occurs during a very restricted window of B-cell differentiation and involves 2 distinct mechanisms: V(D)J recombination, mediating the breaks on chromosome 14 during an attempted secondary D(H) to J(H) rearrangement, and an additional unidentified mechanism creating the initial breaks on chromosome 18. Altogether, these data suggest that the t(14;18) translocation is a more complex process than previously thought, involving the interaction and/or subversion of V(D)J recombination with multiple enzymatic machineries.

The development of rapid PCR protocols for amplification of rearranged IgH gene sequences has greatly facilitated the identification of clonal IGH rearrangements in non-Hodgkin's lymphomas (NHL) and leukemias. However, the 15-35% incidence of false negative results with this approach has been a constant and unresolved problem. To assess the reliability of a previously published framework region 3 (FR3A) IgH-CDR3-PCR for detection of monoclonal IgH gene rearrangements we compared the PCR and Southern results in a series of 44 NHL and leukemias of B cell lineage showing a JH-rearrangement in Southern analysis with genomic DNA and hybridization with a IgH joining region (JH) probe. IgH-CDR3 regions were amplified using DNA extracted from clinical specimens by PCR using fluorescent dye-labeled consensus primers homologous to conserved regions within the variable (VH) and the joining (JH) gene segments. The PCR products were size separated on a high resolution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an automated DNA sequencer. With commonly used DNA polymerases monoclonal IgH-CDR3 junctions were identified in 36/44 samples (82%). However, in the remaining eight cases (18%) with pathohistologically clearly demonstrated B cell malignancies which were also monoclonal on JH-Southern analysis, monoclonality could be demonstrated by FR3A-IgH-CDR3-PCR only with the proofreading UITma DNA polymerase. In four of these monoclonal VH--N--DH--N--JH junctions sequence analysis was performed which showed a point mutation in one and a single nucleotide deletion at the 3' terminus of the primer target site in the other case. In the remaining two cases no primer mismatches could be identified. Thus we conclude that the marked improvement of the PCR-detection rate of monoclonal IgH-CDR3 junctions was achieved at least in part due to the ability of UITma DNA polymerase to remove mismatched bases at the 3' terminus of the primers with respect to the target during the first amplification cycles. Our results suggest, that UITma is the DNA polymerase of choice for amplification of IgH-CDR3 junctions with consensus FR3A-VH- and JH-primers.

Lymph node biopsies from 140 cases of Hodgkin's disease (HD) and from 30 non-malignant lesions were screened for the presence of t(14;18) translocations involving the major breakpoint region (mbr) of the bcl-2 gene and the joining region (JH) of the immunoglobulin heavy chain gene, using a polymerase chain reaction (PCR) assay with subsequent nucleotide sequencing of amplified bcl-2/JH junctional regions. Expression of the bcl-2 protein within the Hodgkin and Reed-Sternberg (HRS) cells was investigated in 86 cases of HD by immunohistochemistry on cryostat or paraffin sections. Although bcl-2 expression could be found in a proportion of neoplastic cells in up to one-third of HD cases, the frequency of t(14;18) gene fusions detected by PCR was low. We identified such gene fusions in only 3 out of 140 (2 per cent) HD cases, one biopsy of which presented with four clonally distinct bcl-2/JH sequences. No t(14;18) was found in any of 30 reactive lymph node lesions. All fusion gene sequences were unique regarding the localization of the chromosome 14 and 18 breakpoints and the extranucleotide N-insertions. None of these gene fusions conformed to t(14;18) breakpoint sequences previously characterized in our laboratories. Our findings point to a mere coincidence in some cases of HD lesions and cells carrying a t(14;18) in the same biopsy and argue against a significant role of bcl-2 in the pathogenesis of HD.

We have examined 165 unselected cases of non-Hodgkin's lymphomas for rearrangements involving the t(14;18) major breakpoint region using a polymerase chain reaction (PCR) and direct sequencing of amplified major breakpoint region bcl-2/JH junctional regions. The lymphomas, diagnosed according to the updated Kiel classification, consisted of 33 centroblastic-centrocytic, 37 centroblastic, 27 immunocytic, 10 immunoblastic, 10 centrocytic, 2 lymphoblastic, 2 Ki-1-positive anaplastic large cell, 14 peripheral T-cell, and 4 unclassified lymphomas. In addition 18 chronic lymphocytic leukemias, 2 hairy cell leukemias, and 6 plasmacytomas were studied. In 17 cases a bcl-2/JH gene fusion sequence was amplified by PCR. A bcl-2/JH gene fusion was detected only in three lymphoma subgroups: 13 of 33 centroblastic-centrocytic (39%), 2 of 37 centroblastic (6%), and 2 of 27 immunocytic (8%) were positive. In two cases, major breakpoint region bcl-2 rearrangements verified by genomic Southern analysis were not detected by PCR. Direct sequencing of all 17 PCR-amplified, previously uncharacterized t(14;18) junctional regions provided corroborating evidence for the specificity of the assay. The procedure gave sequencing results even from limited amounts of lymphoma cells as obtained by fine needle aspiration of lymph nodes or from clinically uninvolved sites. Clone-specific sequences were identified due to the involvement of different JH segments, the variations among the exact JH and bcl-2 breakpoint positions, and the extensive incorporation of junctional region (D-) N-nucleotides. These clone-specific sequences allow accurate identification of clinically occult lymphoma cells and reduce the threat of false positive results. The finding of exceptionally long intervening sequences in some of the junctions and the partial homology with published DH segments in three cases support the view that some of the putative N-regions harbor DH regions.

The fluid phase C1q binding, the solid phase C1q binding and the Raji cell assay are the most widely employed methods for the detection of complement fixing immune complexes in human disease. However, their binding characteristics for complexes formed in native serum have not been compared and studied in detail. For this purpose, Tetanustoxoid (TT): anti-TT complexes were formed closely to in vivo conditions by incubating sera of immunized people with TT. Then the molecular characteristics of those complexes, which could be detected by the 3 immune complex assays, were defined. The methods used are precipitation curve, gel chromatography and measuring the complexed antibody with a sensitive Elisa method. All 3 assays detected only complexes near the equivalence zone; the Raji cell assay being the most sensitive detecting 1.5 micrograms complexed antibody/ml serum followed by the solid phase (6 micrograms/ml) and the fluid phase C1q binding assay (50 micrograms/ml). As estimated by gel chromatography, both the C1q binding assays measured most sensitively complexes with a molecular weight distinctly over 2,000,000 daltons, whereas the Raji cell assay detected preferentially complexes in the range of 2,000,000 daltons. Smaller TT: anti-TT complexes with a molecular weight less than 600,000 daltons were not detectable by the 3 assays. In conclusion, the 3 assays, while differing in sensitivity, showed similar binding patterns with preference for high molecular complexes near equivalence. Subsequently, they might be insensitive to small complexes persisting in the circulation.

During recent years cloning of the genes encoding the immunoglobulin (Ig) and T-cell (TCR) antigen receptor has made it possible to analyze clonal expansions of B- or T-cells at the molecular level. We here describe the usefulness of the Ig- and TCR-gene rearrangements in selected cases of malignant lymphoma. In contrast to all other cases investigated, one non-Hodgkin's lymphoma (NHL) exhibited the infrequent phenomenon of oligoclonality, i.e. three distinct B-cell clones were discerned by Southern blot analysis. Detection of clonal TCR- and Ig-gene rearrangements unequivocally documented bone marrow involvement in four of eight NHL-patients, where conventional histologic and cytologic examination remained inconclusive. As expected, analysis of bone marrow DNA revealed clonal Ig-gene rearrangements in three cases with clear histologic evidence of bone marrow infiltrations by the NHL. In one case of a patient with acute mixed lymphoid-myeloid lineage leukemia a previously identified clonal Ig-gene rearrangement vanished after successful chemotherapy. Analysis of Ig- and TCR-gene rearrangements promises to be a very useful diagnostic tool in selected cases of lymphoid neoplasia.