Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

Potent depressant effects of adenosine analogs on hippocampal slow-wave activity in the unanesthetized rat

Summary Adenosine and its analogs have previously been shown to exert a depressant effect on several measures of hippocampal excitability in the hippocampal slice and intact anesthetized preparation. In the present report, we examined the effects of intraventricular injections of adenosine analogs on hippocampal slow-wave activity in the freely moving rat. Each of three adenosine analogs— 5-N-ethylcarboxamidoadenosine (NECA) and N⁶-(phenylisopropyl) adenosine (L- and D-PIA) — were found to strongly suppress hippocampal electroencephalographic (EEG) activity. For instance, low doses of NECA (0.5 g) produced an 80–90% decrease in the amplitude of the hippocampal EEG. NECA was approximately 20-fold more potent than L-PIA, and L-PIA was twice the potency of D-PIA. In separate experiments in the anesthetized rat, NECA and L-PIA were found to block completely the activation of the hippocampal theta rhythm elicited with brainstem stimulation. The effects of adenosine analogs on both the hippocampal EEG and theta rhythm were very effectively reversed with methylxanthine, 8-para-sulphophenyl-theophylline (8-PSPT). The present findings demonstrate that adenosine analogs exert a powerful depressant effect on the hippocampal EEG in the natural unanesthetized state, and suggest that changes in the levels of endogenous adenosine may play a significant role in modulating the normal activity and function of the hippocampus.This work was supported in part by National Science Foundation grant BNS-8403544 to RPV     

Immunochemical characterization of mouse brain-associated theta (Thy-1) antigen.

The Thy-1 antigens or rat brain and thymus have been isolated and chemically characterized, but those of mice have not been identified. Moreover, it is uncertain whether the antigens are glycolipids or glycoproteins. This study with highly purified preparations of gangliosides GM₁, ¹GD_1a, GD_1b and GT_1b from bovine brain and several ganglioside fractions from mouse brain showed that Thy-1 activity does not reside in gangliosides, but rather in the chloroform-methanol-insoluble residue of brain remaining after extraction of gangliosides. The antigen could be solubilized from this residue with a non-ionic detergent. The antigenic activity of the solubilized preparation was heat-labile but resistant to periodate. The chemical properties of the Thy-1 antigen of mouse brain are discussed.     

Activity of human erythrocyte gangliosides as a receptor to HVJ

Liposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained gangliosides isolated from human erythrocytes. Sialosylparagloboside, which has a terminal sequence of NeuAcα2?3Ga1β1?4GlcNac, has a much higher receptor activity to the virus than GD_1a, GD_1b, GT_1b, and GT_1a, all of which contain the terminal sequence of NeuAcα2?3Galβ1?3GalNAc or NeuAcα2?8NeuAcα2?3Galβ1?3GalNAc. The activity of sialosylparagloboside is comparable to that of glycophorin, a major sialoglycoprotein of human erythrocytes, when compared on the basis of the required amount (as sialic acid) of compounds. The high affinity of sialosylparagloboside to the viral HANA protein is also suggested by the finding that it showed high inhibitory activity against HVJ-mediated binding of glycophorin liposomes to erythrocytes. Sialosylparagloboside was also highly susceptible to the viral sialidase, the other biological function of HANA protein.     

Strain-Dependent Migration of CD4 and CD8 Lymphocyte Subsets to Lymph Nodes in NOD (Nonobese Diabetic) and Control Mice

Subpopulations of lymphoid cells were compared with respect to their ability to migrate into peripheral lymphoid organs of nonobese diabetic (NOD) mice and various strains of control mice. In short-term, in vivo homing studies, no major differences in the pattern of homing of B and T cells were observed among all mouse strains studied. On the other hand, CD4 cells localized consistently more efficiently than CD8 cells in both PP and LN of adult NOD and BALB/c mice, whereas both populations migrated roughly equivalently in LN of adult DBA/2, CBA, and C57BL/6 mice. No age-dependent differences in the homing of CD4 and CD8 cells were observed in BALB/c mice. On the contrary, in 2-week-old NOD mice, CD4 and CD8 cells migrated equally well. The preferential entry of CD4 cells in adult NOD and BALB/c did not result from increased blood transit time of CD8 cells. On the other hand, the preferential migration of CD8 cells was observed in the liver, whereas the two T-cell subsets migrated equally well in the lungs. The differences in the homing characteristics of CD4 and CD8 cells among NOD, BALB/c, and C57BL/6 mice were not related to modifications in the level of expression of adhesion molecules such as MEL-14, LFA-1, and Pgp-1.     

Two dinucleotide repeat polymorphisms at the D8S1442 and D8S1443 loci

Two polymorphic dinucleotide (CA) repeat clones were isolated from cosmids, cCI8-1121 and cCI8-1199, mapped to chromosome 8p11.2-p12.     

Analysis of 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation

In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20-60 Gy of gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA.     

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.     

In vivo and in vitro correlates of food allergy.

Sera of 86 patients clinically sensitive to foods were tested by passive sensitization of human and/or monkey lung (127 tests) and the radioallergosorbent test (RAST) (72 tests), using whole-food antigens; the results were compared with skin (prick) testing. Results of the prick test correlated with history in 76% of cases; lung sensitization correlated with history in 37% and with prick test in 57%; and RAST correlated with history in 54% and prick test in 72%. It is concluded that a very large percentage of adverse reactions to foods are IgE-mediated. The prick test is of use in diagnosis, particularly when combined with RAST; the lung sensitization test is technically impractical and not a reliable indicator. The best diagnostic method is careful history with food challenge and withdrawal and rechallenge; the latter is safe except in patients with a history of violent reaction.