Heddy Zola

Isolation of whole mononuclear cells from peripheral blood and cord blood.

Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll‐Hypaque density gradient centrifugation—a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L‐leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues. Curr. Protoc. Immunol. 85:7.1.1‐7.1.8.

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.

Surface marker analysis in acute myeloid leukaemia and correlation with FAB classification.

Summary. The immunological phenotype of blast cells in 102 patients with acute myeloid leukaemia (AML) was analysed with a panel of 20 monoclonal antibodies and the enzyme terminal transferase, and correlated with the FAB classification. Although a partial correlation between these two approaches could be observed, almost every morphological group contained patients from more than one immunological phenotype. The M1 and M5a leukaemias showed the most undifferentiated phenotype, often lacking in specific myelomo‐nocytic antigens. The M3 formed a uniform group defined as My7+, Ia‐, FMC8+, a phenotype which was also observed in two cases of the microgranu‐lar variant. The granulocytic (CDwl5) and monocytic (CDwl4) antibodies crossreacted with some M5b and M2 leukaemias, respectively. Compared with M5a, the M5b leukaemias showed a large increase in the expression of CDw l4 antigen, confirming the validity of the morphological differentiation. Giyco‐phorin‐A was present in four out of five M6 leukaemias. TdT activity was demonstrated in 10% of AML cases, with a higher incidence among the monocytic variants: M4 and M5‐. Eleven AML were considered as unclassifi‐able according to the FAB criteria and in seven of them a megakaryoblastic cell population (GP IIb/IIIa+, GPIb+) was demonstrated; this confirms the need to include the subgroup of megakaryoblastic leukaemias within the AML. Finally, a possible immunological classification for AML is proposed.

The Preparation and Properties of Monoclonal Antibodies against Human Granulocyte Membrane Antigens

Summary. A series of four monoclonal antibodies has been prepared by hybridization of mouse myeloma cells with spleen cells from mice immunized with human blood granulocytes. The four antibodies all react specifically with granulocytes, failing to stain lymphocytes and other blood cells. Lymphocytic leukaemia cells are not stained, whereas myeloid leukaemia cells give a varied reaction with the antibodies. Studies on marrow, leukaemic cells and cell lines suggest that the four antibodies react with distinct differentiation antigens which are absent from myeloblasts, appear at the promyelocyte, myelocyte or metamyelocyte stage (depending on the antibody in question) and are expressed on all mature granulocytes.

Transforming Growth Factor-β Levels in Maternal Milk and Expression in Postnatal Rat Duodenum and Ileum

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-β1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-β. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-β1, and in situ hybridization for TGF-β1 mRNA was also performed on the duodenum. TGF-β levels in milk were assayed by ELISA. TGF-β2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-β1 was not detected. In contrast, the cell number and intensity of staining of TGF-β1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-β1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-β2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-β production in the small intestine increases during the weaning period.

Genome-Wide Identification of Human FOXP3 Target Genes in Natural Regulatory T Cells

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3+ Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25+FOXP3+ Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

Immune Activation in Irritable Bowel Syndrome: Can Neuroimmune Interactions Explain Symptoms?

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.

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.

Membrane antigens of human cells of the monocyte/macrophage lineage studied with monoclonal antibodies

Summary Three monoclonal antibodies, FMC17, FMC32, and FMC33 directed against human cells of the monocyte‐macrophage lineage are described. The antibodies react strongly with blood monocytes and weakly, if at all, with granulocytes. Lymphoid cells are not stained. In tissue sections macrophages and interdigitating reticulum cells are stained. Lymphoid leukemia cells generally do not react with the antibodies, while myeloid leukemia cells give a variable pattern, with relatively differentiated cells more likely to react than undifferentiated cells. Differences between the 3 antibodies in their reactivity with leukemic cells and tissue macrophages indicate that they are directed against distinct antigens, which may serve as differentiation markers in the monocyte/macrophage lineage.

Isolation of antigen-specific B cells

Cell separation techniques are important in immunology. Major cell populations can be separated successfully with high purity. However, isolation of cells which are specific for particular antigens is more challenging because of the relatively small numbers of antigen‐specific cells, and the lack of independent markers available to determine the purity of the isolated population. In this review, the literature describing three principal techniques used to separate antigen‐specific cells has been reviewed. Particular emphasis has been placed on yield and purity; the two most important parameters of any purification method. The most promising isolation methods have used immunomagnetic sorting and multiparametric flow cytometric analysis.