Alpha-chain disease: a new immunoglobulin abnormality.

A new type of pathological immunoglobulin was found in the serum, urine, and saliva of a young Arab patient with abdominal lymphoma and diffuse lymphoplasmacytic infiltration of the small intestine. This protein is devoid of light chains and is closely related to the alpha polypeptide chains of the gamma(A1) (Le) subclass of immunoglobulin A. It is characterized by electrophoretic heterogeneity, tendency toward polymerization, and a high carbohydrate content. No intracellular synthesis of light chain was detected.

Immunoglobulins on the surface of lymphoid cells in Waldenström's macroglobulinemia.

Immunofluorescence study of viable and fixed cells was performed on marrow and blood samples from 25 patients with Waldenström's macroglobulinemia. Whereas intracytoplasmic staining for IgM was restricted to the plasma cells and to a limited number of lymphocytic cells, the vast majority of the pleomorphic "lymphoid" proliferating cells in the marrow were shown to bear the monoclonal IgM on their surface. All IgM-secreting plasma cells displayed membrane positivity. Most lymphocytic proliferating cells carried the monoclonal IgM on their surface in the absence of detectable amounts of intracytoplasmic IgM. Despite the usual absence of increase in blood lymphocyte counts, a large number of circulating lymphocytes were shown to bear membrane-bound monoclonal IgM in patients with active disease. The number of fluorescent spots, their size and brightness varied greatly from cell to cell in marrow and blood samples of a given patient, and this finding is in contrast to the homogeneous fluorescent pattern observed in chronic lymphocytic leukemia. The data suggest that macroglobulinemia represents the proliferation of a clone of B cells which continues to mature and differentiate.

Structure of a monoclonal kappa chain of the V kappa IV subgroup in the kidney and plasma cells in light chain deposition disease.

That structural abnormalities may be responsible for nonamyloid immunoglobulin (Ig) light chain deposition disease (LCDD) is suggested by previous results of Ig biosynthesis studies, but this hypothesis was not documented at the molecular level. We report on the first complete primary sequence deduced from cDNA analysis of a kappa light chain responsible for LCDD associated with an apparently nonsecretory myeloma. Bone marrow myeloma cells contained intracellular kappa chains and no heavy chains by immunofluorescence. Kidney biopsy showed typical nonamyloid PAS-positive kappa chain deposits. SDS-PAGE analysis of material extracted from a kidney biopsy specimen and of Ig produced by the myeloma cells revealed kappa chains of abnormally high apparent molecular mass (30,000). Comparison of the NH2-terminal aminoacid sequence of the kappa chain deposited in the kidney and of the complete sequence of several identical kappa cDNA clones from bone marrow cells showed the identity of the tissue deposited and plasma cell kappa chain. The kappa mRNA had an overall normal structure and corresponded to the V kappa IV gene rearranged to J kappa 1 and followed by a normal constant exon of the Km(3) allotype. The variable sequence differed from the V kappa IV germline gene by nine point mutations, including an Asp----Asn substitution at position +70 resulting in a potential N-glycosylation site. In vitro biosynthesis experiments and treatment with N-glycosidase provided evidence for the intracellular glycosylation of the monoclonal kappa chain. The peculiar sequence and the glycosylation of a kappa chain of the rare V kappa IV subgroup might be responsible for structural abnormalities leading to tissue deposition.

Autoantibodies to B lymphocytes in a patient with hypoimmunoglobulinemia. Characterization and pathogenic role.

In a young woman with ulcerative colitis, hypoimmunoglobulinemia, and humoral immunodeficiency, lymphocyte counts vary between 600 and 1,000 per mm(3) with 0.5-1.5% bone marrow-derived (B) cells and 98-99% thymus-derived (T) cells. Anti-lymphocyte antibodies were detected by immunofluorescence and by microlymphocytotoxicity with increased reactivity at +4 degrees C. They belonged to the IgM class and were polyclonal. Studies performed with various normal lymphocyte subpopulations, several lymphoblastoid cell lines and lymphocytes from immunodeficiency patients showed that these antibodies reacted with B cells. The corresponding antigen(s) is distinct from membrane-bound immunoglobulins, is not an alloantigen, and is probably unrelated to the la-like molecules. Pokeweed mitogen stimulated B cells appear to lose this antigen. Cells from various lymphoproliferative disorders were tested. T-derived and "non T-non-B" leukemic cells did not react with the antibody. Malignant cells from B-derived lymphomas and prolymphocytic leukemias were reactive. The incidence of positivity of the leukemic cells among patients with common B chronic lymphocytic leukemia was surprisingly low (one-third of the patients). The autoantibody nature of the anti-B-cell antibodies and their pathogenic role in the genesis of the patient's hypoimmunoglobulinemia was demonstrated by the effect of removal of antibodies by massive plasmaphereses which were followed by a dramatic and transitory increase of B-cell figures. Whereas most primary immunodeficiency syndromes appear to result from an arrest in the differentiation capabilities of immunologically competent cells, autoantibodies to circulating B lymphocytes may be incriminated in the pathogenesis of some cases of hypogammaglobulinemia.

Immunochemical studies in four cases of alpha chain disease.

Studies of a number of properties of the pathological gammaA-proteins in the first four cases of the recently recognized alpha-chain disease demonstrate that, as in gamma-heavy-chain disease, the abnormal protein is devoid of light chains and represents a portion of the alpha-heavy chain related to the Fc-fragment. In two patients, serum electrophoresis showed a broad abnormal band, whereas in the two others the pathological protein was not noticeable on the electrophoretic pattern. The diagnosis of alpha-chain disease can be established without purification of the protein by immuno-electrophoresis and gel diffusion experiments using selected antisera to gammaA and a reference alpha-chain disease protein. All four proteins belonged to the alpha1-subclass, displayed electrophoretic heterogeneity, and showed a strong tendency to polymerize. The polymers occurred in vivo and were held together both by disulfide bonds and by strong noncovalent forces. Two of the three purified proteins had a very high carbohydrate content. The abnormal protein was always found in concentrated urines in variable but generally low amounts. It was not detected in parotid saliva but was present in significant amounts in jejunal fluid of all four patients. The alpha-chain disease protein was shown to be associated with the secretory piece in external secretions of two patients. The clinicopathological features were strikingly similar in the four patients. All patients were affected with a neoplastic and mostly plasmacytic proliferation involving primarily the whole length of the small intestine and the mesenteric nodes and all exhibited a severe malabsorption syndrome. While Israeli authors have emphasized the frequency of this type of abdominal lymphoma in young Arabs and non-Ashkenazi Jews, two of our patients were Kabyles, one a Syrian Arab, and one an Eurasian. Cellular studies showed that the pathological protein was synthesized by the proliferating cells in the lymphoid tissue of the digestive tract and in the mesenteric nodes, and that there was no detectable light-chain synthesis at the intracellular level.

Acute lymphoblastic leukemia with Burkitt's lymphoma cells: membrane markers and serum immunoglobulin.

Blast cells from 24 patients with acute lymphoblastic leukemia of Burkitt's cell type were studied for lymphocyte surface markers. The leukemia cells were of B-cell origin in 23 cases and showed a non-B, non-T phenotype in 1 case. Surface immunoglobulins on blast cells were monoclonal, with a striking predominance of cases with light chains of lambda type. They consisted most often of high-density IgM usually without associated IgD. However, 3 patients had cells with surface IgG and 1 had surface IgA. The blast cells lacked detectable IgG Fc receptors in more than half the patients. Serum immunoglobulins were studied in 15 cases: A monoclonal IgM was found in 5 patients (whose blast cells had surface IgM) and a Bence Jones protein was found in 2 others, both of whom had blasts with surface IgG lambda.

Membrane markers in "histiocytic" lymphomas (reticulum cell sarcomas).

Neoplastic cells from 9 patients affected with a "histiocytic" lymphoma were studied with 5 membrane markers of B or T lymphocytes. In 2 patients a monoclonal B-cell proliferation was found; they had been affected previously with well documented B-cell proliferations: chronic lymphocytic leukemia or Waldenström's macroglobulinemia. The blast cells of 2 other patients had T-cell features; in a fifth case, the abnormal cells carried only a strong receptor for the Fc fragment of IgG, which suggested their truly monocytic origin. In 4 patients, the cells had no detectable surface markers. These findings demonstrated that this group of lymphomas is heterogenous, that the term "histiocytic" appears to be wrong in most instances, and that the cellular origin of the malignant cells frequently remains unidentified and thus prevents a satisfactory new classification.

Characterization of jacalin, the human IgA and IgD binding lectin from jackfruit.

The lectin jacalin from the jackfruit Artocarpus heterophyllus reacts by precipitation and western blotting with human IgA1 and IgD but not with IgA2 (nor IgG and IgM). However, it weakly binds IgA2 as shown by affinity chromatography and competitive ELISA. Predominantly reactive carbohydrates are D-galactose and N-acetyl D-galactosamine. Jacalin has an apparent Mr of about 54,000 and is probably made up of three non-glycosylated and one glycosylated non-covalently linked subunits. Its electrophoretic properties and amino acid and carbohydrate composition are indicated.

Structural and functional similarities of breadfruit seed lectin and jacalin.

Aquous extracts from seeds of Artocarpus altilis (breadfruit) and Artocarpus heterophyllus (jackfruit) were compared by polyacrylamide gel electrophoresis. Two bands of the same size (12 and 15 kD) as the jacalin subunits were the major components in breadfruit seed extract. They strongly reacted with anti-jacalin antibodies by western blotting. The breadfruit lectin displayed the same IgAl and IgD precipitation specificity as jacalin in gel double diffusion experiments. It also stimulated in vitro proliferation of human peripheral blood mononuclear cells. These results suggest that lectins from both species of Artocarpus are very similar.

Structural and functional properties of membrane and secreted IgD.

More than 35 years ago, study of an unknown immunoglobulin (Ig) in the serum from a myeloma patient led to the discovery of IgD. Subsequently, the finding that it also exists as a membrane-bound Ig stimulated a large number of studies during the 70s. Then, the interest on IgD shrank, largely because of the lack of known function of secretory IgD (secIgD) and of a stagnating knowledge of the functions of surface IgD. In the recent years, very significant advances followed the tremendous accumulation of data on the physiology of the B cell receptor, of which IgD is the major component, on the role of secIgD in normal and diseased individuals. This review, which is focused on human IgD but integrates data in the mouse and other species when needed, summarizes present data on the structure, synthesis and functions of both membrane and secIgD, IgD receptors and the involvement of IgD in various diseases, especially the hyperIgD syndrome.

Production of an abnormal mu chain with a shortened VHIV subgroup variable region in a Burkitt's lymphoma cell line.

The Burkitt's lymphoma cell line Ly66 produces a short mu chain which lacks 4 kDa in apparent molecular mass. Study of the corresponding messenger RNA showed it to be 0.3 kb shorter than normal mu transcripts. The cDNA sequence of the mu transcripts began by a short VH region consisting of the first one-third of a VHIV subgroup gene segment. It was followed by a normal mu constant region. This VH region coded for 38 amino acids, thus differing from two truncated VHIV regions previously reported in other Burkitt's lymphoma cell lines, which were interrupted at codon +26 by an alternate splicing event. In addition, the Ly66 variable sequence bore several point mutations and contained two potential N-glycosylation sites.

Stimulation of peripheral blood lymphocytes of HIV-infected patients by jacalin, a lectin mitogenic for human CD4+ lymphocytes.

The proliferative response of peripheral blood mononuclear cells stimulated by jacalin was investigated in 157 samples from 104 HIV-infected patients at various clinical stages of disease. Jacalin proliferative response correlated with the absolute numbers and percentages of CD4+ lymphocytes and with the percentages of Leu 8 lymphocytes. It correlated negatively with the percentages of CD8+ and CD38+ lymphocytes. It was depressed during acute HIV infection and at advanced stages. The response in Centers for Disease Control groups II and III was heterogeneous and similar; in both, some patients showed a very low response. Further follow-up of the present patients should indicate whether jacalin responsiveness has a prognostic value.

Measurement of anti-Haemophilus influenzae type b capsular polysaccharide antibodies by ELISA.

Measurement of antibodies to the capsular polysaccharide polyribosylribitolphosphate (PRP) of Haemophilus influenzae type b by ELISA is made difficult by the poor binding of this antigen to the solid phase. Six coating conditions were compared using immune and non-immune human sera. Direct coating with PRP was inefficient. Precoating with protamine or poly-L-lysine (PLL) yielded irreproducible results and high background levels. Assays with PRP conjugated with PLL as coat were not sensitive enough. In addition, anti-PRP antibodies, especially those belonging to the IgM class, crossreacted with PLL. Coating with avidin or streptavidin followed by incubation with biotin-coupled PRP was not satisfactory either, due to binding of certain sera in the absence of PRP. Coating with PRP coupled to tyramine resulted in low backgrounds and acceptable specific binding levels. However, the finding that the binding of a few sera was only partially inhibited by soluble PRP led us to include an inhibition step in every experiment. Only optical densities inhibited by the antigen were taken into account. In view of the lack of parallelism of dilution curves from different sera, no attempt was made to express the results in weight units. They were expressed in arbitrary units calculated by comparison with internal standards. Under such conditions, the assay permitted a reproducible (interassay coefficients of variation around 10%) determination of PRP-Ab belonging to the various immunoglobulin classes and IgG subclasses and showed a good correlation with results obtained using the Farr assay.

Affinity immunoblotting detection of serum monoclonal immunoglobulins reactive with glycosphingolipids.

The characterization of serum monoclonal IgM reactive with glycosphingolipid present in the nervous system is of major importance in patients with certain neuropathies. A rapid, sensitive and specific one-step method which permits such a characterization is described. Serum immunoglobulins separated by high resolution agarose electrophoresis are transferred to affinity filters (nitrocellulose sheets coated with glycolipids), and revealed with enzyme-tagged monospecific anti-immunoglobulin antibodies.

Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes.

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

Measurement of serum IgG4 levels by a competitive immunoenzymatic assay with monoclonal antibodies.

A competitive indirect ELISA is described for the measurement of IgG4 levels. It uses a monoclonal anti-subclass and antibody and purified monoclonal IgG4 as standards. This method is sensitive and reproducible and more accurate than hemagglutination inhibition and radial immunodiffusion. Serum IgG4 levels in 173 normal adults were less than 0.01-2.1 mg/ml (mean 0.30 mg/ml) in women and less than 0.01-1.87 mg/ml (mean 0.465 mg/ml) in men.

Evaluation of monoclonal antibodies with specificity for human IgA, IgA subclasses and allotypes and secretory component. Results of an IUIS/WHO collaborative study.

51 monoclonal antibodies (McAb) with putative specificity for human IgA, the IgA subclasses, Am allotypes or secretory component (SC) were evaluated for immunoreactivity and specificity by nine laboratories employing immunodiffusion, agglutination, immunohistological assays, immunoblotting and direct binding and competitive inhibition enzyme immunoassays. McAbs specific for IgA PAN (n = 24), IgA1 (n = 7), IgA2 (n = 3), IgA2m(2) (n = 2), non-IgA2m(2) (n = 4) and SC or secretory IgA (n = 5) were identified that were immunoreactive and specific in the assays employed. The McAbs identified as IgA- or SC-reactive were shown to be non-reactive to human IgG, IgM, IgD, IgE, kappa and lambda by direct binding and competitive inhibition immunoassays. Interestingly, no McAbs with restricted specificity for IgA2m(1) were identified. Some McAbs displayed higher affinity and/or better performance in one or several of the assay groups. The IgA- and SC-specific McAbs identified in this international collaborative study have potential as immunochemical reference reagents to identify and quantitate monomeric and polymeric IgA in human serum and secretions.

Synthesis of abnormal immunoglobulins in lymphoplasmacytic disorders with visceral light chain deposition.

Three patients presented with renal or more diffuse tissue deposits of a nonamyloid material reactive with anti-kappa antibody by immunofluorescence. All patients had progressive renal failure with the nephrotic syndrome and extensive tubular basement membrane deposits. Glomerular lesions were conspicuous but heterogeneous. One patient also had hepatic deposits with peliosis at histopathologic examination. An underlying lymphoplasmacytic disorder was found in all patients: multiple myeloma in one, pleomorphic lymphoplasmacytic malignancy analogous to Waldenström's macroglobulinemia in one and bone marrow monoclonal plasmacytosis without overt myeloma in one. Biosynthesis experiments in two cases showed production of abnormal kappa chains which were not detected in appreciable amounts in serum and urine. These light chains had an aberrant size (abnormally short or large), their apparent molecular weight was larger in secretion than in cytoplasmic extracts (suggesting their glycosylation) and they were secreted as polymers. These results suggest a causal relationship between production of abnormal light chains and tissue deposition.

Reactivity of a human monoclonal anti-GM1 and anti-GD1b IgM antibody with human neurons in cultures.

A serum containing a monoclonal IgM lambda with anti-GM1 and anti-GD1b activity was obtained from a patient with upper motor neuron syndrome. By indirect immunocytochemical techniques with double staining, the patient's IgM strongly stained membranes of neurons in primary cultures of fetal central and peripheral nervous system. It was cytotoxic for neurons in two human neuroblastoma established cell lines in a complement-dependent chromium release assay. These results are in keeping with the hypothesis of a direct pathogenetic role of such monoclonal anti-GM1 and GD1b IgM antibodies.

Effect of protein A immunoadsorption on panel lymphocyte reactivity in hyperimmunized patients awaiting a kidney graft.

Six hyperimmunized patients awaiting a kidney graft underwent immunoadsorption of separated plasma through a protein-A column to remove HLA antibodies. This procedure was partially limited by constant and rapid antibody resynthesis in spite of strong immunosuppression with cyclophosphamide and prednisolone. Only two patients could be grafted with previously positive--currently negative crossmatches. The first died of infection on day 40, having developed early chronic vascular rejection and the other returned to hemodialysis on day 285 after the development of transplant glomerulopathy. However, immunoadsorption seems to be effective in removing HLA antibodies having a titer below 1/2. Such extremely hyperimmunized patients should probably be excluded from the immunoadsorption program.