Characterization of porcine platelet glycoproteins recognized by human natural "anti-gal" antibodies.

Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.

Simultaneous presence, in one serum, of four monoclonal antibodies that might correspond to different steps in a clonal evolution from polyreactive to monoreactive antibodies.

Three monoclonal IgG of different subclasses (IgG1, IgG2, and IgG4) and one IgA1 were isolated from the serum of patient Per suffering from an immunocytic sarcoma. All four monoclonal Ig shared the same N-terminal sequence of their H chains (VH3). Furthermore, their kappa-chains exhibited identical isoelectric charges and N-terminal sequences (VK2) and expressed the same private idiotope. A strong antitubulin activity was found in IgA1Per and in two of the three monoclonal IgGPer. The specificity was restricted to tubulin for IgA1Per and IgG4Per, whereas IgG1Per also displayed significant polyreactive bindings and IgG2Per failed to react with any of the Ag tested. The monoreactive IgG4Per, as well as the polyreactive IgG1Per, bound a large peptide in the central part of both alpha and beta subunits of tubulin (amino acid position 100 to 300). In contrast, the monoreactive IgA1Per bound to a rarely detected epitope close to residue 310 of these subunits. The tubulin epitope recognized by polyreactive IgG1Per was similar to that of germ-line-encoded polyreactive antibodies. It is hypothesized that IgG4Per- and IgA1Per-producing cells derive from the IgG1Per polyreactive clone after somatic events leading to the production of monoreactive antibodies.

Autoantibody activity of immunoglobulins isolated from B-cell follicular lymphomas.

Previous work with monoclonal Igs (MIgs) has demonstrated that a high proportion of paraproteins bind to self-antigens such as the Fc fragment of IgG, Ii blood group antigens, cytoskeleton proteins, DNA, and myelin-associated glycoprotein (MAG). Recent work in CLL indicates that CD5+ B lymphocytes are frequently committed to production of autoantibodies. We have examined the antibody specificity of MIgs derived from the tumor cells of 31 different patients with CD5- B-cell lymphomas. Our results indicate that the tumor cells from 8 of these 31 patients (25.8%) express Igs with autoantibody activity. In two cases antibody activity was multispecific. In four cases, antibody activity was exclusively directed against the Fc fragment of IgG, whereas the two other cases bound to both Fc fragment of IgG and nuclear antigens. Most non-Hodgkin's lymphomas (NHL) are derived from CD5- B cells. These results indicate that like CLL, NHL also express Igs that frequently have autoantibody activity.

B Cell Malignancies Frequently Target the Autoreactive B Cell Repertoire.

Autoreactive B cells constitute a substantial part of B cell repertoire. They frequently secrete polyspecific natural autoantibodies, which are the expression of germinal genes. During recent years considerable evidence has accumulated indicating that this autoreactive B cell repertoire frequently undergoes malignant transformation. Studies on antibody activity of paraproteins and some B-cell malignancies have clearly demonstrated an impressive high frequency of autoantibody activity in these disorders. About 30% of IgM paraproteins and at least 10% of IgG and IgA monoclonal Ig display rheumatoid factor, cold agglutinin, anti-myelin associated glycoprotein or polyreactive activity. Similar high frequencies of autoantibody activity have been observed when CD5*** CLL B lymphocytes and CD5-follicular lymphoma B lymphocytes are induced to secrete Ig. The activation of this autoreactive B-cell repertoire through continuous challenge by self antigens could create suitable conditions for the occurrence of mutations and chromosomal translocations.

Monoclonal and/or Oligoclonal Immunoglobulins in Sera of Patients with Non-Hodgkin's Lymphomas, Determined by Iso-Electric Focusing and Immunoblotting.

Sera from 60 patients with non-Hodgkin's lymphomas (NHL) were tested by isoelectric focusing (IEF) and immunoblotting (IB) techniques for the presence of monoclonal or oligoclonal immunoglobulins (Ig). Of the 60 NHL patients, 18 were newly diagnosed and had not received any treatment while the rest had received some chemotherapy in the past. Two serum samples were obtained from 27 patients over the treatment period. Oligoclonal Ig patterns were detected in 34 of the 60 (56.6%) sera, and were further identified by IB as IgG of mixed kappa (K) and lambda (λ) types. Seven of the 18 (38.8%) newly diagnosed NHL patients had these oligoclonal bands, compared with 27 of the 42 (69%) who had received chemotherapy in the past. In 11 of these 27 (40.7%) patients, follow-up studies revealed the appearance of new Ig bands (9 cases) with the disappearance of preexisting ones (2 cases). After treatment of sera with 2-mercaptoethanol, monoclonal IgM was detected in 39 of the 60 (65%) NHL patients. These monoclonal Ig spikes occurred together with oligoclonal IgG (20/60 cases) or appeared in the absence of oligoclonal IgG (19/60 cases), while oligoclonal IgG could also appear as the sole serum finding (14/60 cases). There was a higher prevalence of monoclonal IgM among patients with stage IV disease and of oligoclonal IgG among patients with high grade of malignancy NHL. Our results demonstrate that among patients with NHL there is a high prevalence of monoclonal or oligoclonal Ig bands detected by highly sensitive methods such as IEF and IB.

Evidence that chronic lymphocytic leukemia B lymphocytes are frequently committed to production of natural autoantibodies.

CD5+ B lymphocytes have been postulated to be primarily involved in autoantibody production. To investigate whether CD5 B lymphocytes from chronic lymphocytic leukemia (CLL) patients display the same function, we fused B lymphocytes from 23 CD5+ B-CLL with nonsecreting murine myeloma X-63 cells. Hybrids were derived from 18 of the 23 patients, but only hybrids from 13 patients were found to secrete immunoglobulin (Ig) (IgM kappa 4, IgM lambda 8, and only kappa light chain 1). Supernatants of these hybrids were tested against an extensive panel of antigens by enzyme-linked immunosorbent assay, indirect immunofluorescence, and hemagglutination. At the same time, peripheral blood mononuclear cells (PBMC) from 15 of these patients, including most patients from whom secreting hybrids had not been obtained, were stimulated with phorbol myristate acetate (PMA). Our results indicated that secreting heterohybrids from CLL B lymphocytes are frequently obtained; however, this is highly dependent on the light chain phenotype, since 8 of 9 lambda-expressing CLL produced hybrids secreting complete Igs, as compared with 4 of 12 kappa-expressing CLL. In addition, B lymphocytes of most patients from whom we failed to derive secreting hybrids also failed to secrete Ig on PMA stimulation. Secondly, CD5+ B-CLL lymphocytes were frequently committed to the production of natural autoantibodies, since in 8 of 15 cases (including hybrids and PMA stimulation) anti-Fc activity was found; three of these also displayed a multispecific binding pattern.

CD5 and immunoglobulin V gene expression in B-cell lymphomas and chronic lymphocytic leukemia.

We studied the expression of CD5 and immunoglobulin variable gene families in a panel of monoclonal Epstein-Barr virus (EBV) transformed lines, chronic lymphocytic leukemias (CLLs) and CD5+ and CD5- B-cell lymphomas. The CD5 gene expression was in all cases identical to that of T-cell malignancies. The utilization of the various VH and VK gene families was roughly proportional to the estimated gene family size in EBV lines obtained from adult healthy subjects. In contrast we found a statistically significant biased usage of VH6 in CLL and VH5 in CD5+ lymphomas as compared with EBV lines, and of VKIII in both CLL and CD5+ lymphomas as compared with EBV lines. Some differences in the variable gene usage were also noted when comparing CD5+ and CD5- lymphomas. These findings are analyzed in the context of possible mechanisms involved in the malignant transformation of CD5+ B cells.

Evidence that the B lymphocyte proliferating in B-CLL and in other B-cell malignancies is frequently committed to production of natural autoantibodies.

Autoreactive B-cells constitute a substantial part of B-cell repertoire. They frequently secrete polyspecific natural autoantibodies which are the expression of germinal genes. During recent years, considerable evidence has accumulated indicating that autoreactive B-cells frequently undergo malignant transformation. This evidence arose from the study of monoclonal immunoglobulins (MIgs) and from recent studies of chronic lymphocytic leukemia (CLL) and non Hodgkin lymphoma (NHL) B lymphocytes. The present review, includes two sections. The first one reviews evidence supporting the idea that autoreactive B-cells constitute a substantial part of autoreactive B-cell repertoire. The second one is devoted to review evidence favouring the view that this autoreactive B-cell repertoire is frequently involved in B-cell malignancies.

Autoantibodies against nuclear envelope-associated proteins in primary biliary cirrhosis.

An antinuclear immunofluorescence pattern displaying a thin ring confined to the nuclear envelope was assessed in sera from 38 patients with primary biliary cirrhosis and in sera from a control group of 277 patients with other antinuclear antibody-positive diseases. This rim-like antinuclear reactivity was present in sera from 20 primary biliary cirrhosis patients (52.6%) but in only two patients from the control group (0.7%) (p less than 0.001). Furthermore, this autoantibody was present in three of four primary biliary cirrhosis patients without antimitochondrial antibodies. Presence of this rim-like pattern in primary biliary cirrhosis did not correlate with the presence of associated autoimmune diseases nor with other clinical, biochemical, nor histological features of the disease. The antigenic specificity of sera displaying this antinuclear immunofluorescence pattern was characterized by Western blot analysis using an antigenic extract containing nuclear envelope proteins purified from rat liver. Sixteen of the 20 positive sera showed a common pattern of reactivity with a set of nuclear envelope-associated proteins approximately 200 kD in size. In conclusion, sera from primary biliary cirrhosis patients showing a rim-like fluorescent nuclear pattern have antinuclear autoantibodies that react specifically with components of the nuclear envelope. The high specificity of these new autoantibodies in primary biliary cirrhosis indicate that they might be a serological marker of the disease, particularly useful in patients without antimitochondrial antibodies.

CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes.

In this study data are presented indicating that the molecule identified by the recently described CD43 cluster of monoclonal antibodies (mAb; Oxford, 1986) is the human analogue to the one originally described in rat as leukocyte sialoglycoprotein (LSGP). This conclusion is based on several criteria. Both molecular mass (105 kDa) and cellular distribution are similar to the antigens previously described in the rat with the mAb W3/13 and subsequently in humans with L10 mAb. Sialidase treatment of the molecule immunoprecipitated by 84-3C1 mAb (CD43) resulted in a decreased electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. L10 mAb inhibited the binding of 84-3C1 mAb to the cell membrane suggesting that both mAb recognized the same molecule. Moreover, the presence in the CD3-4-8- thymic populations of the sialidase-sensitive epitope recognized by mAb 84-3C1 suggests that there is no simple correlation between the thymic maturation and the degree of sialylation.

A second signal for T cell mitogenesis provided by monoclonal antibodies CD45 (T200).

The induction of T cell mitogenesis through CD3 is a complex process that requires at least two signals. The first one can be provided by Sepharose-bound CD3. The second one is normally provided by monocytes. The signal provided by Sepharose-bound CD3 is unable by itself to induce mitogenesis in monocyte highly depleted cells (MHDC). We describe here that the monoclonal antibody (mAb) 72-5D3 belonging to CD45 (T200), which was not mitogenic by itself, could replace monocytes when MHDC were activated by Sepharose-bound CD3. That is to say, in the absence of monocytes, mAb 72-5D3 gave a second signal necessary for T cell proliferation. Using eleven anti-CD45 mAb from other investigators we show that this effect is not a peculiar characteristic of 72-5D3 mAb. The effect of the mAb 72-5D3 was only effective in CD4-positive cells and was not observed when MHDC were activated with either soluble CD3 or concanavalin A. As both phorbol myristate acetate and mAb 72-5D3 can replace monocytes, a comparative study of their effects was undertaken. Phorbol myristate acetate but not mAb 72-5D3 induced proliferation of MHDC when recombinant interleukin 2 (rIL2) was added. On the other hand mAb 72-5D3 induced IL2 production in MHDC activated by Sepharose-bound CD3 and increased the IL2 production in Sepharose-bound CD3-activated peripheral blood mononuclear cells. In conclusion, data presented in this report indicate that the T200 molecule could be involved in T cell proliferation by giving a signal that induces the production of IL2 and bypasses the necessity of monocytes.

An antiplatelet monoclonal antibody that inhibits ADP and epinephrine-induced aggregation.

A monoclonal antibody (Mab) named EDU-3, was produced by fusing splenocytes from one Balb/c mouse, immunized with a mixture of platelets and non-T cells from heparinized human peripheral blood, with the HAT-sensitive myeloma line P3-NS1/1.Ag4.1. By indirect immunofluorescence (IF) it was seen that this Mab reacted with all normal human platelets and bone marrow megakaryocytes, but did not react with lymphoid cells from normal donors, or platelets from Glanzmann's thrombasthenia (GT) patients. Immunoprecipitation and SDS-PAGE experiments demonstrated that this Mab recognized an epitope on the IIb-IIIa glycoprotein complex (GPC). EDU-3 inhibited platelet aggregation and release of ATP induced by ADP and epinephrine. Aggregation induced by arachidonic acid, ristocetin and bovine factor VIII were not inhibited by EDU-3. The difference between EDU-3 and other Mab directed against the IIb-IIIa GPC is discussed.

Further evidence against preeclampsia as an immune complex disease.

Sera from 25 preeclamptic women were examined for the presence of circulating immune complexes (CICs) by the radioisotopic C1q binding assay (C1q-BA), the polyethylene glycol precipitation-complement consumption (PEG-CC) test, and the microcomplement consumption test (MCT). Five abnormal values were found by the MCT; 4 became normal after delivery, as did the preeclamptic clinical signs. Normal values were obtained by the C1q-BA and the PEG-CC test in all preeclamptic patients. In addition, maternal HLA antibodies and complement levels were investigated. There was no correlation between lymphocytotoxic activity and CICs in sera from patients with preeclampsia. Complement levels were in the normal range in all cases. It is concluded that CICs do not play a significant role in the pathogenesis of preeclampsia.