Structure of an anti-DNA fab complexed with a non-DNA ligand provides insights into cross-reactivity and molecular mimicry.

Antibodies that recognize DNA (anti-DNA) are part of the autoimmune response underlying systemic lupus erythematosus. To better understand molecular recognition by anti-DNA antibodies, crystallographic studies have been performed using an anti-ssDNA antigen-binding fragment (Fab) known as DNA-1. The previously determined structure of a DNA-1/dT5 complex revealed that thymine bases insert into a narrow groove, and that ligand recognition primarily involves the bases of DNA. We now report the 1.75-A resolution structure of DNA-1 complexed with the biological buffer HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid). All three light chain complementarity-determining regions (CDRs) and HCDR3 contribute to binding. The HEPES sulfonate hydrogen bonds to His L91, Asn L50, and to the backbone of Tyr H100 and Tyr H100A. The Tyr side-chains of L32, L92, H100, and H100A form nonpolar contacts with the HEPES ethylene and piperazine groups. Comparison to the DNA-1/dT5 structure reveals that the dual recognition of dT5 and HEPES requires a 13-A movement of HCDR3. This dramatic structural change converts the combining site from a narrow groove, appropriate for the edge-on insertion of thymine bases, to one sufficiently wide to accommodate the HEPES sulfonate and piperazine. Isothermal titration calorimetry verified the association of HEPES with DNA-1 under conditions similar those used for crystallization (2 M ammonium sulfate). Interestingly, the presence of 2 M ammonium sulfate increases the affinities of DNA-1 for both HEPES and dT5, suggesting that non-polar Fab-ligand interactions are important for molecular recognition in highly ionic solvent conditions. The structural and thermodynamic data suggest a molecular mimicry mechanism based on structural plasticity and hydrophobic interactions.

Isolation of anti-mesothelin antibodies from a phage display library.

Phage display has been used to select single-chain Fvs (scFvs) against mesothelin, a surface antigen present on mesothelial cells as well as mesotheliomas and non-mucinous ovarian cancers. Several attempts to produce anti-mesothelin hybridomas from spleen cells of mice immunized with recombinant mesothelin were unsuccessful. This report describes the isolation of anti-mesothelin scFvs from a phage display library made from the mRNA of the same spleens. Panning on recombinant antigen produced in E. coli or on antigen positive cells was employed. Several scFvs which bind specifically to mesothelin were isolated. Panning on recombinant antigen yielded five different scFvs. Panning on cells selected two different scFvs which also differ from the scFvs selected by recombinant antigen. The heavy chains of the scFvs selected on recombinant antigen are derived from four different heavy chain gene families and the scFvs selected on cells are derived from two of these families. In contrast, the light chains of all of these scFvs are derived from family XI. Moreover, the light chains of the two scFvs selected on cells are very similar to the light chains of two of the scFvs selected by panning on recombinant mesothelin except for a few point mutations. One of these scFvs which have been studied in detail has been shown to bind specifically to mesothelin positive transfected cells.

Enzymes transducing extracellular signals in acute myeloid human leukemias.

The ectoenzymes gamma-glutamyltransferase (gamma-GT) and the PIP2 phospholipid phospholipase-C (PLC), a second messenger generating enzyme active on the cytoplasmic membrane bilayer, were biochemically investigated in leukemic cells isolated from 67 patients with acute leukemia. Six groups were distinguished on the basis of morphology, cytochemistry and immunophenotyping according to the FAB classification: M0, M1-M2, M3, M4, M5 and CML blast crisis (CML-BC). The activity of PLC ranged from 0.6 to 14.5 nmol/min/mg without a significant difference among groups, whereas the gamma-GT activity varied significantly from 0 to 31.6 nmol/min/mg. The highest mean activity was measured in monoblastic leukemia (M5), followed by groups M4, CML-BC and M0 (undifferentiated) while the lowest activity was found in M1-M2 and M3 groups. Within each group, activity distribution profiles of both enzymes never correlated with each other, suggesting that in leukemic cells functional-structural constituents of both membrane leaflets were independently affected by the neoplastic process.

Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup.

Staphylococcal protein A (SPA) is a bacterial membrane protein that possesses, in addition to its Fc gamma-binding activity, a distinct specificity for the Fab region of some IgM, IgA, IgG, and IgE. The Fab site that binds to SPA has been localized to the V region of the Ig H chain. In a previous study of human monoclonal and polyclonal IgM, we demonstrated that binding to SPA was highly restricted to molecules of the VHIII subgroup, and that nearly all VHIII IgM were able to bind SPA. The present study examines the VH composition of SPA-binding and SPA-nonbinding fractions of purified human polyclonal IgA, and IgG F(ab')2 fragments. We found that 22% of the IgA and 15% of the IgG F(ab')2 bound to SPA-agarose. Analysis with VH subgroup-specific antisera indicated that the SPA-binding fraction of IgA was dominated by the VHIII subgroup, and the SPA-binding fraction of IgG F(ab')2 contained only VHIII molecules. Furthermore, substantial portions of the total VHIII protein in IgA and in IgG F(ab')2 bound to SPA. We conclude that Fab binding to SPA is both restricted to and highly prevalent among human VHIII molecules, regardless of Ig class. These results suggest that protein A is an Ig superantigen.

Update on the prognostic implication of morphology, histology, and karyotype in primary myelodysplastic syndromes.

The prognostic value of the FAB classification, bone marrow histology, Bournemouth score, and chromosome findings was studied in 88 patients with primary myelodysplastic syndromes. The median survival for the whole group of patients was 22 months (RA 61.7 months, RARS 31.6 months, CMML 15.7 months, RAEB 10.3 months, and RAEBt 8.2 months). Chromosomal abnormalities were found in 37 of the 70 patients investigated (52%). Only the differences in survival between patients with complex versus normal karyotype were statistically significant (p = 0.02). The presence of small blastic cells, located away from the endosteal surface (abnormal localization of immature blasts or ALIP) appears to be a major prognostic factor in predicting the duration of survival and progression to ANLL, especially in the FAB subgroups RA and RARS. Median survival for the 22 ALIP- cases with RA/RARS was 65 months, compared with 31 months for the ALIP+ cases (p = 0.0006). Nine ALIP+ patients (53%) developed ANLL in contrast to 3 (13%) of the ALIP- cases (p = 0.008). By redefining ALIP and evaluating the number and characteristics of the accompanying cells, histological subtypes were distinguished correlating largely with the FAB subgroups. Our findings demonstrate the prognostic importance of bone marrow biopsy and quantifying the complexity of bone marrow chromosome changes. It should be helpful in evaluating current attempts to find effective treatment for patients with MDS.

IgG anti-IgE in circulating immune complexes in the hyper-IgE syndrome.

We fractionated, by gel chromatography, sera with high IgE content from atopic subjects and five cases with the hyper-IgE syndrome, and measured the presence of IgE in high molecular weight (HMW) fractions. Two out of four asthmatics and four out of five hyper-IgE had HMW IgE. The same serum fractions gave positive results for conglutinin binding IgG (all six) and IgA (three cases) as well as C1q binding complexes (five cases). IgG auto-antibodies to IgE were also detected together with IgE in HMW fractions. Anti-F(ab)'2 activity was present in five cases (one of them negative for IgG anti-IgE). Our data indicate that complexes made of IgE and IgG anti-IgE are present mainly in patients with chronic allergic symptoms and most frequent in cases of hyper-IgE syndrome.

Both Fc alpha domains of human IgA are involved in in vitro interaction between secretory component and dimeric IgA.

The sites of interaction between 125I labelled human secretory component (SC) and dimeric IgA were located by studying the inhibitory effect of various antibodies to IgA. Several Fab' fragments were isolated from three sera of hyperimmunized rabbits. The specificity of these different antibody preparations, as determined by a RIA inhibition test or by ELISA, showed that two were directed against both domains of Fc alpha, two against C alpha 2, two against C alpha 3 and one against Fd alpha. A monoclonal antibody against C alpha 3 was also used. The results indicate that both the C alpha 2 and C alpha 3 domains are equally and independently involved in the interaction between SC and dimeric IgA.

Cell surface markers in acute lymphoblastic leukemia.

During the last nine years, two important methodologies have been used to characterize the cell surfaces of normal lymphocytes and malignant lymphoblasts. Normal mature T-cells have a receptor for sheep erythrocyte (E+) while mature B-cells bear membrane-bound immunoglobulin molecules (sIg+). These two findings can be used to divide acute lymphoblastic leukemia of childhood into three major groups; B-cell leukemia (sIg+ E-), which is rare (approximately 2 percent) and has the poorest prognosis, T-cell leukemia (sIg-, E+) which is more common (10 percent) but also has a poor prognosis and null cell leukemia (sIg-, E-) which is the most common (85 percent) and has the best prognosis. By the use of additional immunological methods, subgroups within T-cell leukemia and null cell leukemia have also been proposed. One of the most valuable of these additional methods is the detection of surface antigens. Three of the more commonly detected antigens currently being evaluated are (1) common leukemia antigen (cALL), (2) a normal B Lymphocyte antigen the Ia antigen (Ia) which is not generally expressed on most T lymphocytes and (3) a normal T lymphocyte antigen (T) not expressed on B lymphocytes. Within null cell leukemia, the most commonly identified and probably the largest subgroup if Ia+, cALL+, T-, E-, sIg-. In another but smaller subgroup within null cell leukemia, the lymphoblasts contain cytoplasmic immunoglobulin but do not express surface immunoglobulins or E receptors. This subgroup is designated pre B-cell leukemia. Subgroups with T-cell leukemia have also been suggested. These include T+ E-, T+ E+, in which the rosettes are thermolabile and T+ E+, in which the rosettes are thermostable. Whether or not there are any prognostic differences in these three subgroups remains to be determined.