Ivan Lefkovits

Limiting Dilution Analysis

Publisher Summary This chapter discusses the limiting dilution analysis. The objective of the microculture technique is to analyze the frequency of rare cells involved in the immune response. The precursors of antibody-forming cells (B cells), helper T cells, and suppressor T cells are considered rare cells. The microculture assay further allows the assessment of the average clone size of the progeny of precursors, the class, the allotype, and other characteristics of the products of B cells. Many small aliquots of lymphoid cells are cultured in the presence of antigen. The number of cells in each aliquot is chosen so that a considerable fraction of cultures will not contain any precursor cell. The analysis of the fraction of nonresponding cultures is performed, as a rule, on the culture fluid. The samples of culture fluid are removed by an automated replicator and deposited on an assay plate. Complement-dependent zones of lysis mark the cultures that have produced antibody.

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria‐containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP‐1 and β‐hexosaminidase. Combining subcellular fractionation with two‐dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.

Clonal proteomics: One gene – family of proteins

The work presented here attempts to consolidate our knowledge on cellular transcriptome and proteome. It takes into account that a typical activated cell (lymphocyte) contains 40 000 mRNA molecules at any time, and it represents about 5000 different molecular species of transcripts. Such a cell has about 1 000 000 000 protein molecules, some of them being present at 10 000 000 copies while others at a very low copy number (say 1 to 10 copies per cell). By studying cell free expression of individual cDNA clones (or pools of known complexity) we address to those rare molecular components that will remain undetected by the current analytical means. For our analysis we use cell free translation systems (wheat germ or rabbit reticulocyte origin) and we study polypeptide products originating from intact, or restriction endonuclease‐treated cDNA clones. We conclude that in most instances expressed genes yield transcript(s) that translate into several, and often very numerous families of polypeptide species. In our ISODALT two‐dimensional gel system we characterize the proteomic profile of the clonal polypeptide families in terms of their molecular mass, charge, multiple products, and appearance.

Integrated Epigenetics of Human Breast Cancer: Synoptic Investigation of Targeted Genes, MicroRNAs and Proteins upon Demethylation Treatment

Background The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2′-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. Methods and Findings Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. Conclusions In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.

Limiting dilution analysis: from frequencies to cellular interactions

Abstract Molecular biology techniques have provided the means to pursue the precise definition of molecular components in a single cell type and its clonal progeny. However, to study the workings of the individual components of the immune system as well as the system itself, additional tools and supplementary approaches are required.

Global analysis of gene expression in cells of the immune system I. Analytical limitations in obtaining sequence information on polypeptides in two-dimensional gel spots

We have outlined various aspects and limitations of the collective analysis of protein species of a cell (lymphocyte). We have indicated research directions that, in to our opinion, deserve more attention. We have evaluated mainly the approach used in our laboratory and we recognize that a bulk of important research on the interface of proteomics and genomics remains to be dealt with. It is of great value that we can proceed in our quest by trial and error. But as much as the human genome initiative was not implemented by trial and error, but by formulating new technological approaches, we hope that our approach can be incorporated in the mainstream of proteomics. We need several integrating research directions, some of which are outlined in this communication, namely the use of ordered cDNA libraries, cell‐free expression systems, high density filter hybridization, identification of two‐dimensional (2‐D) gel spots in terms of their amino acid composition through biosynthetic labeling and identification of restriction sites in the corresponding coding sequences. In the accompanying paper the cDNA ordered library approach will be described in some detail.

Light chain heterogeneity in the amphibian Xenopus

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

Internal standard-based analysis of microarray data. Part 1: analysis of differential gene expressions

Genome-scale microarray experiments for comparative analysis of gene expressions produce massive amounts of information. Traditional statistical approaches fail to achieve the required accuracy in sensitivity and specificity of the analysis. Since the problem can be resolved neither by increasing the number of replicates nor by manipulating thresholds, one needs a novel approach to the analysis. This article describes methods to improve the power of microarray analyses by defining internal standards to characterize features of the biological system being studied and the technological processes underlying the microarray experiments. Applying these methods, internal standards are identified and then the obtained parameters are used to define (i) genes that are distinct in their expression from background; (ii) genes that are differentially expressed; and finally (iii) genes that have similar dynamical behavior.

Simultaneous isolation of DNA, RNA, and proteins for genetic, epigenetic, transcriptomic, and proteomic analysis.

Analysis of DNA, RNA, and proteins for downstream genetic, epigenetic, transcriptomic, and proteomic analysis holds an important place in the field of medical care and life science. This is often hampered by the limited availability of sample material. For this reason, there exists an increasing interest for simultaneous isolation of DNA, RNA and proteins from a single sample aliquot. Several kit-systems allowing such a procedure have been introduced to the market. We present an approach using the AllPrep method for simultaneous isolation of DNA, RNA and proteins from several human specimens, such as whole blood, buffy coat, serum, plasma and tissue samples. The quantification and qualification of the isolated molecular species were assessed by different downstream methods: NanoDrop for measuring concentration and purity of all molecular species; DNA and RNA LabChip for fractionation analysis of nucleic acids; quantitative PCR for quantification analysis of DNA and RNA; thymidine-specific cleavage mass array on MALDI-TOF silico-chip for epigenetic analysis; Protein LabChip and two-dimensional (2D) gel electrophoresis for proteomic analysis. With our modified method, we can simultaneously isolate DNA, RNA and/or proteins from one single sample aliquot. We could overcome to some method limitations like low quality or DNA fragmentation using reamplification strategy for performing high-throughput downstream assays. Fast and easy performance of the procedure makes this method interesting for all fields of downstream analysis, especially when using limited sample resources. The cost-effectiveness of the procedure when material is abundantly available has not been addressed. This methodological improvement enables to execute such experiments that were not performable with standard procedure, and ensures reproducible outcome.

Increased protein synthesis after T cell activation in presence of cyclosporin A.

BACKGROUND The immunosuppressive drug, cyclosporin A (CsA), blocks immune responses by inhibiting the calcineurin-dependent dephosphorylation of the nuclear factor of activated T cells (NFAT). We have previously reported that T cells activated in presence of CsA exhibit particular properties. In our study, we have tested the hypothesis that T cells activated in presence of CsA display a differential pattern of gene expression. METHODS T lymphocytes were activated in vitro by Concanavalin A with or without CsA. The cells were: (1) pulsed with 35S-methionine to label the newly synthesized proteins that in turn were revealed by 2D-gel electrophoresis; (2) analyzed by flow cytometry for activation markers expression; and (3) examined by gel electrophoresis for early tyrosine phosphorylation events. RESULTS The proteomic patterns of T lymphocytes activated by Concanavalin A, with or without CsA, were compared. In keeping with the well-known effect of the immunosuppressor, many polypeptides were not found in its presence. Remarkably, several newly synthesized polypeptides were detected only when activation was carried out in presence of CsA. In addition, immunologically relevant proteins, such as CD44 and CD69, escape CsA-inhibitory action. Furthermore, CsA did not modify the early protein tyrosine phosphorylation events resulting from T cell triggering. CONCLUSIONS The present data show that the effect of CsA on protein synthesis is more complex than anticipated. Signaling provided by T cell activation and the blockade of the calcineurin-dependent pathway by CsA results in an altered program of gene expression.