Increased T regulatory cells and decreased Th1 pro-inflammatory cytokines correlate with culture-positive infection in febrile neutropenia childhood oncology patients.

BACKGROUND: Paediatric oncology patients with febrile neutropenia are usually hospitalised and treated with empirical broad-spectrum antibiotic therapy to counter the risk of infection. However, there is currently no method available to rapidly identify bacteremia in these patients. T-helper-type-1 (Th1) cytokines are required for effective immune response to many pathogenic organisms and T regulatory cells are known suppressors of Th1 cells. We hypothesized that characterization of reduced intracellular Th1 cytokines and increased T regulatory cells (Tregs) may prove useful in identifying infection in childhood oncology patients with febrile neutropenia. METHODS: Intracellular Th 1 cytokines and Tregs were enumerated in peripheral blood from a group of childhood oncology patients with febrile neutropenia using multiparameter flow cytometry. RESULTS: There was a significant increase in the percentage of CD25(+) CD127(-) CD8(-) CD3(+) Tregs and a significant decrease in Th1 intracellular cytokines IFNγ, IL-2 and TNFα in the blood of culture positive patients compared with culture negative patients. CONCLUSIONS: Enumeration of Tregs and intracellular Th1 cytokines may provide a sensitive, specific test for determining infection in childhood oncology patients before blood culture results become available.

Toll-like receptors.

Toll-like receptors are a family of transmembrane receptors responsible for recognition and initiation of a response to invading microbes by the immune system. As part of the innate immune system, Toll-like receptors recognise pathogen-associated molecular patterns, highly conserved components that are essential to microbial function. Some of ten toll-like receptors identified in humans are able to recognise several pathogen-associated molecular patterns.

CD 271 (P75 neurotrophin receptor).

P75NTR (or CD271) is a member of the Tumor Necrosis Factor receptor (TNFR) super family of transmembrane proteins that share significant homology in their extracellular domains. Subsets of TNF receptors, including CD271, have a cytoplasmic death domain, although CD271 has unique intracellular structure and downstream signaling partners. CD271 is also differentiated from other members of the TNFR receptor family in that it binds pro and mature neurotrophins and affects the growth, differentiation and death of the nervous system. The ligands for CD271 are neurotrophins, which are Nerve Growth Factor (NGF), Brain-Derived Growth factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4/5 (NT4/5). Recent studies have provided evidence that CD271 also serves as a receptor for the pro-forms of these neurotrophins.

Assessing the potential of immunohistochemistry for systematic gene expression profiling.

Immunohistochemistry (IHC) is a powerful technique for identifying sites of protein expression in tissues at the cellular and sub-cellular level. Here we have investigated the potential of using IHC for genome-wide expression screening by measuring the success rate and specificity of a panel of 35 monoclonal antibodies recognizing 5 well characterised CD antigens. Antibodies were pre-screened on acetone fixed frozen sections of spleen, tonsil and colon tissues. 19/35 antibodies gave staining with a success rate of 0/7 for JAM-2, 1/4 for CD99, 3/6 for CD138, 5/8 for CD45 and 10/10 for MHC-class II. 16/19 of these antibodies also gave staining on formalin fixed paraffin embedded tissue sections of tonsil and colon. All antibodies that had given staining were then profiled on tissues presented in human tissue microarrays. In the frozen microarrays 216 cores from 29 normal tissue types were present and in the formalin fixed paraffin array 344 cores from 35 normal and 4 cancers were represented. Where multiple antibodies were positive, there was evidence of consistent staining of the same tissues with several antibodies. In some cases differences in staining were observed potentially due to differential splice variants, polymorphisms or protein modification. With some antibodies there was evidence of cross-reactivity to inappropriate cells or structures. In addition the staining intensity with formalin fixation was changed quantitatively for some antibodies and in a few cases qualitatively, representing differential sensitivity of specific and non-specific epitopes to fixation. Accordingly, whilst IHC has potential for describing protein expression of unknown genes, these results emphasise a need to systematically address issues of specificity and sensitivity if appropriate profiles are to be described.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

In silico evaluation of two mass spectrometry-based approaches for the identification of novel human leukocyte cell-surface proteins.

The identification and quantitation of cell-surface proteins expressed by leukocytes currently use the wide availability of monoclonal antibodies (mAb) in immunohistochemical and flow cytometric assays. Presently, approximately 400 such proteins have been characterized; however, analysis of the completed human genome sequence indicates that it may contain several thousand as-yet unidentified molecules, which may be expressed on the leukocyte cell surface. Recent advances in protein isolation and analysis using mass spectrometry illustrate that it is now feasible to identify the protein composition of a complex sample such as a plasma membrane extract. Such an approach may be useful for the identification of the cell-surface proteins that have not been identified using mAb techniques. Here, we detail the results of an in silico evaluation of the peptides isolated using two methods used to label plasma membrane proteins to determine whether these methods are suitable for the identification of known leukocyte cell-surface proteins by mass spectrometry. The labeling of cell-surface proteins before isolation and characterization is a valuable means of differentiating between plasma membrane and internal membrane proteins The results indicate that although the majority of cell-surface proteins can be identified using either of the approaches, others known to be important diagnostically and/or therapeutically would not be identified using either approach. The implication of this for the use of these techniques in the discovery of new leukocyte cell-surface proteins is discussed.

Detecting antibodies with similar reactivity patterns in the HLDA8 blind panel of flow cytometry data.

The blind panel collected for the 8th Human Leucocyte Differentiation Antigens Workshop (HLDA8; ) included 49 antibodies of known CD specificities and 76 antibodies of unknown specificity. We have identified groups of antibodies showing similar patterns of reactivity that need to be investigated by biochemical methods to evaluate whether the antibodies within these groups are reacting with the same molecule. Our approach to data analysis was based on the work of Salganik et al. (in press) [Salganik, M.P., Milford E.L., Hardie D.L., Shaw, S., Wand, M.P., in press. Classifying antibodies using flow cytometry data: class prediction and class discovery. Biometrical Journal].

Coexpression of IL-2 receptor p55 and p75 by circulating blood lymphocytes.

By using high-sensitivity fluorescence and flow cytometry, it is possible to show that 30-40% of lymphocytes from PBL express the p55 chain of the IL-2 receptor, whereas the p75 chain is expressed at low concentrations on most lymphocytes without in vitro activation. The availability of a second fluorochrome capable of high sensitivity allows simultaneous analysis of p55 and p75, albeit with some sacrifice in sensitivity. Two-color analysis shows that a small proportion of cells (1-6%) coexpress measurable concentrations of both chains of the IL-2 receptor, and three-color studies show that these cells are predominantly CD4-positive T cells and express the CD45R0 isoform of the leucocyte-common antigen, i.e., have the phenotype of activated helper T cells. These cells may be a useful indicator of immune activation.

CD antigens 2001.

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

Rapid detection of immunoglobulin gene somatic hypermutation using heteroduplex formation.

In order to identify somatic hypermutation of rearranged human immunoglobulin genes, we have examined heteroduplex formation between cloned VH6 genes. In test systems, the presence of five or more point mutations could be detected by examining the formation of heteroduplexes between a known germline VH6 gene and other sequenced genes using polyacrylamide gel electrophoresis. If a mutated sequence was used, then the presence of two or more mutations could be detected. The method was used for rapid screening of VH6-D-JH rearrangements for the presence of point mutations before sequencing, and to distinguish between different highly mutated sequences, allowing clones containing the same rearrangement to be identified indirectly.

Delineation of serologically distinct monomorphic determinants of human MHC class II antigens: evidence of heterogeneity in their topographical distribution.

Three monoclonal antibodies (mAbs), FMC4, FMC14 and FMC15, which react with invariant sites of major histocompatibility complex (MHC) class II (Ia-like) molecules were studied in various serological assays. Sequential immunodepletion experiments show that all three epitopes are present on the same class II molecules. However, a minor subset may exist which does not express epitope 15. In competitive binding assays, using several different B-lymphoblastoid cell lines (B-LCLs), e.g. BRISTOL-8 8392, B-85, RAJI, 8866, CESS-B and LD-B, FMC4 did not block the binding of FMC14, or FMC15, and vice versa. In contrast, mutual inhibition was observed between FMC14 and FMC15. Furthermore, pairwise combinations of saturating amounts of FMC4 + FMC14 and FMC4 + FMC15 gave additive binding whilst FMC14 + FMC15 did not. These results demonstrate that epitopes 4 and 14/15 are spatially distinct; 14 and 15 on the other hand appear to be spatially related. However, contrary to this partial and reciprocal inhibition was consistently observed between FMC4 and FMC14 on two other LCLs, namely DAUDI and BALM-2. Furthermore, on certain cells, FMC14 and FMC15 show markedly disparate binding. Taken together, these observations indicate that the juxtaposition of certain epitopes on class II antigens can vary according to the cell type. This demonstrates a hitherto unreported heterogeneity of antigenic determinants and of their topographical distribution on the class II molecule.

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

Immunoabsorbents prepared from cell membrane antigens: efficiency of incorporation and shedding of protein during use.

Immunoabsorbents for the removal of antibodies against cell membrane antigens have been prepared by a variety of techniques in common use. Cells were surface-labelled by lactoperoxidase-catalysed radioiodination, and the different immunoabsorbent preparations were compared in terms of efficiency of incorporation and shedding of membrane material during use. Whole cells, either unfixed or fixed with glutaraldehyde, shed substantial amounts of material during absorption. Detergent extracts of surface-labelled cells were coupled covalently to agarose beads by cyanogen bromide activation, periodate oxidation, or epoxy activation of the gel. Although these immunoadsorbents were much more stable than whole cells, shedding could not be eliminated entirely. Minimal shedding was achieved using freshly washed epoxy-based immunoadsorbents. The possible consequences of shed protein in immunoabsorbed sera are emphasised.

Fractionation of human lymphocytes using rosette formation with papain-treated mouse erythrocytes.

Human B lymphocytes from rosettes with mouse erythrocytes. If the erythrocytes are pretreated with papain the rosettes are sufficiently stable to allow separation of T and B lymphocytes. The technique is complementary to the sheep erythrocyte rosette method, and gives cell populations with a similar degree of purity.

A simple technique for evaluation of methods of cell separation.

A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.

Removal of erythroid cells from umbilical cord blood mononuclear cell preparations using magnetic beads and a monoclonal antibody against glycophorin A.

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.

Preparation of human-mouse heterohybridomas against an immunising antigen.

The production of murine monoclonal antibodies against specific antigens by hybridomas is a well utilised technique. The production of hybridomas secreting specific human antibodies would have many advantages in therapeutic applications of monoclonal antibodies. The immortalised human lymphocytes themselves would also provide valuable tools in research on lymphocyte development. Preparation of human-human hybridomas has been limited by a lack of suitable fusion partners. This protocol paper describes the production of human-mouse heterohybridomas by two independent laboratories. The purpose of this protocol is to provide a basis for the development of heterohybridoma technology in laboratories with limited hybridoma experience.

The purification of mouse monoclonal antibodies from ascitic fluid.

A method is described for the purification of monoclonal antibody from mouse ascitic fluid. The fluid is clarified and the lipid removed using silicon dioxide powder, before the immunoglobulin is precipitated using polyethylene glycol. The method provides IgM antibody in high yield and good purity. In the case of IgG antibodies the purity is 30-40% after PEG precipitation and the yield is high. The enriched IgG is adequate for many purposes and is suitable for further purification on an ion exchange column.

Analysis of chemical and biochemical properties of membrane molecules in situ by analytical flow cytometry with monoclonal antibodies.

Methods are described for analysing chemical and biochemical properties of membrane antigens against which monoclonal antibodies are available. The methods are based on gentle manipulation of whole viable cells and quantitative flow cytometric fluorescence analysis of the effect of such treatment on monoclonal antibody binding. Proteolytic enzymes, glycosidases, inhibitors of biosynthesis and various mild chemical treatments have been used to derive information on the chemical nature of individual membrane antigens, their insertion in the membrane, and their turnover. The nature of the particular epitope detected by a monoclonal antibody may be probed in a similar way.

Purification of cord blood lymphocytes.

When cord blood is separated using standard methods based on Ficoll-Hypaque, the mononuclear fraction is contaminated with erythrocytes and also with nucleated cells that do not express the leucocyte marker CD45. The contamination with CD45-negative cells can exceed 50%, and will interfere with phenotypic, mRNA or functional analysis. A large proportion of these cells are erythrocyte precursors. The contaminating cells may be removed by lysis with hypotonic ammonium chloride; when the cells are required for studies which are adversely affected by ammonium chloride (such as antigen processing), high purity can be attained by two rounds of density separation.