Papain digestion fragments of human IgM globulins.

Papain digestion of two Waldenström IgM globulins produced a high amount of small peptides and resulted in the formation of two end products, the Fabmicro and Fcmicro fragments. The Fcmicro fragment is characterized by a fast electrophoretic mobility, a high content in carbohydrate, and a high molecular weight. It was demonstrated that this fragment is made of heavy chain pieces belonging to several disulfide-linked monomeric subunits, presumably representing the carboxy-terminal end of the micro-chains. Fc fragments from the two macroglobulins could not be distinguished immunologically. An appreciable proportion of IgM molecules apparently underwent degradation without the formation of a stable Fc fragment. An Fc-like fragment, analogous to the reduced Fc fragment, was obtained at early stages of papain digestion of the IgM subunits. The Fabmicro fragment, with slow and individually distinct electrophoretic mobility, bears many physicochemical and immunological similarities to the Fabgamma fragment. It consists of one light chain and one Fd piece, both of which were isolated. The interaction of these two constituents was demonstrated by gel diffusion studies. Fab fragments of both IgM globulins were resolved into two subpopulations with different electric charges. In addition to these fragments, intermediary split products were observed at early stages of the degradation process, together with a high yield of small peptides mainly derived from the papain-sensitive region of the heavy chains. Immunologic data strongly suggested that this segment of micro-chains is situated between the Fd piece and the portion included in the Fc fragment. Several experiments indicated the importance of conformational antigenic specificity in both Fab and Fc regions of the IgM globulins.

Ultrastructure of papain and pepsin digestion fragments of human IgM globulins.

The ultrastructure of papain and pepsin-digested products of human IgM globulins has been analyzed. Papain digestion was performed both in the presence and absence of cysteine. The Fcmicro fragment was found to represent the central ring structure in the intact IgM molecule, plus a minor part of the appendages extending from the ring. The Fcmicro ring structure was occasionally seen to be composed of dimers of short rods, probably identical with the endpieces of two micro-chains. Such dimeric structures, released from the intact Fcmicro rings, had a tendency to aggregate sidewise, producing complexes of varying size. The dimensions of the Fcmicro fragments were: outer diameter approximately 85 A, inner diameter about 40 A. The length of the protrusions varied from 20-30 A. The Fabmicro preparations contained long strands of sidewise aggregated, short rod-shaped fragments. No aggregates were seen in the F(ab'')(2)micro preparations. The two Fab''micro units in the dimeric F(ab'')(2)micro fragments were usually parallel to each other. The dimensions of the Fabmicro and F(ab'')(2)micro fragments were 50-80 A x 30 A and 75-80 A x 55 A, respectively. These findings provide morphological evidence that the C-terminal ends of the micro-chains (the Fcmicro fragment) make up the central ring structure in the IgM molecule. They further indicate that the F(ab'')(2)micro fragments constitute about (3/4) of the appendages extending from this ring structure.

Antigenic determinants common to human immunoglobulins G and M: importance of conformational antigens.

Antiserums produced against certain isolated human myeloma IgGglobulins and absorbed in order to show only individual antigenic specificity crossreact with certain Waldenstrom IgM-globulins. Some of the common antigenic determinants revealed by these cross-reactions depend on the tertiary and quaternary structure of the IgG and IgM molecules and are independent of their K and L light-chain antigenic types.

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.

Antibodies to native and denatured deoxyribonucleic acid in systemic lupus erythematosus.

The relative reactivities with native and denatured DNA of 35 lupus sera were investigated by quantitative complement fixation and precipitin studies and showed great variations. The use of purified native DNA demonstrated that, in at least 22 of these 35 sera, the anti-DNA antibodies reacted with the native form, independently of denatured contaminants. Systemic lupus sera were shown to contain three main types of DNA antibodies: those reacting only with denatured DNA, those reacting to the same extent with both forms of DNA, and those reacting preferentially with native DNA. In some instances, the latter antibodies fix complement and precipitate only with native DNA but are inhibited by the denatured form. This finding points to the importance of conformation in the antigenic structure of DNA. The simultaneous occurrence of different varieties of DNA antibodies was demonstrated in several sera. Evidence was obtained that some of these human antibodies to DNA can belong to the IgM class. Thus, DNA antibodies from systemic lupus patients differ in many respects from most of the experimentally produced antibodies capable of reacting with DNA.

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.

Functional and physicochemical studies of hemoglobin St. Louis beta 28 (B10) Leu replaced by Gln: a variant with ferric beta heme iron.

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.

Imbalances of gamma globulin subgroups and gene defects in patients with primary hypogammaglobulinemia.

Analysis of immunoglobulin classes, gammaG subgroups, and Gm genetic markers from 59 patients with various types of immune deficiencies was undertaken to assess the function of the several cistrons concerned with synthesis of gamma globulins. 13 patients including two sibling pairs were found to have gammaG subgroup imbalances. All of these patients had non sex-linked disease. 11 of the 13 had preponderance of the gammaG3 subgroup. In most instances of gammaG3 preponderance it was the Gm(b) type of gammaG3 that was selectively retained; the Gm(g) type, controlled by the allelic gene was markedly depressed but not absent in the cases where it could be studied. Other imbalances, either seen concomitantly with gammaG3 preponderance or independently, included predominance of the gammaG2 subgroup and selective absence of single gammaG subgroups.One family was encountered with probable structural gene abnormalities in the autosomal Gm loci. Both parents had different abnormal gene complexes detectable by absence of specific Gm markers and the propositus received both types from the parents. Similar gene complexes have been seen previously in rare instances through population screening but only in the heterozygous state and were not associated with clinically evident hypogammaglobulinemia. Of several other families of patients with subgroup imbalance, two were informative in that structural gene defects could be excluded. Studies on 22 first degree relatives of patients with subgroup imbalances indicated that the most common abnormality detected was in gammaA which was absent in 3 and markedly decreased in 2 others; other abnormalities included decreased levels of specific genetic types of gammaG globulin. It is concluded that gammaG subgroup imbalances are frequently found in non sex-linked immunoglobulin deficiency disorders and in some instances may be associated with family abnormalities suggesting either regulator or structural gene defects.

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.

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.

Association of the human type C retrovirus with a subset of adult T-cell cancers.

To determine whether the human T-cell lymphoma-leukemia virus (HTLV) is associated with particular cancers, patient sera were surveyed for HTLV-specific antibodies. An association was seen with aggressive cancers of mature T-cells, specifically Japanese adult T-cell leukemia (ATL) and T-cell lymphosarcoma cell leukemia (TLCL), a similar cancer of Caribbean blacks. Ninety to 100% of these patients possessed HTLV-specific antibody. Forty-seven and 20% of relatives of ATL and TLCL patients, respectively, and 12 and 4% of healthy donors from ATL and TLCL endemic areas were also antibody positive. Visceral organ involvement, hypercalcemia, and skin manifestation, features of ATL and TLCL, were often seen in other antibody-positive patients. Childhood cancers, most cutaneous T-cell and all non-T-cell leukemias and lymphomas, myeloid leukemias, Hodgkin's disease, and solid tumors were not associated with HTLV. Healthy United States donors and European patients with non-malignant diseases were antibody negative. HTLV is thus associated with a subtype of adult T-cell leukemia-lymphoma, clustered in viral endemic areas, with apparent racial and geographic predilection.

T cell-derived blast crisis in chronic myelocytic leukemia.

We describe herein the occurrence of a T cell-derived blast crisis of chronic myelocytic leukemia which presented as a typical T cell leukemia on clinical (thymic mass and high white blood cells) and immunological grounds (T6+ T4+ T8+), with rearranged T cell receptor genes and germ line immunoglobulin genes. A Philadelphia chromosome was detected in all blast cell mitoses. The significance of T cell involvement during the chronic and acute phases of chronic myelocytic leukemia is discussed.

Immunochemical, clical, and pathological features of alpha-chain disease.

Making the diagnosis of alpha-chain disease in the laboratory is difficult. Immunologic and structural characteristics of the abnormal protein were shown in results of cellular studies. There are clinicopathological features associated with the protein abnormality. The peculiar geographic origin of patients affected with alpha-chain disease and the possible reversibility of the hyperplastic process at its early stage lead to some considerations of cause.

mu-chain disease. Report of two new cases.

We report two cases of mu-heavy-chain disease. Both patients were affected with a lymphoproliferative disease that shared several suggestive features with the previously reported cases of mu-chain disease: the presence of vacuolated plasma cells in bone marrow, a small amount of alpha 2 moving abnormal mu-chain protein, and urinary kappa Bence Jones protein in one case.

Impaired T-lymphocyte-dependent immune responses to microbial antigens in patients with HIV-1-associated persistent generalized lymphadenopathy.

T-cell mediated and humoral responses directed to microbial antigens were investigated, at the time of the initial visit, in a group of 139 patients with HIV-1-related persistent generalized lymphadenopathy (PGL) enrolled in a longitudinal study. In vivo and in vitro cell-mediated responses to tuberculin were lower in patients than in controls. Differences were not significant for candidin and streptococcal antigen in vitro, whereas higher responses were observed in the patient group for cytomegalovirus antigen. Following immunization, a subgroup of patients did not have a significantly raised serum antitetanus antibody level, whereas in vitro lymphocyte proliferative responses to tetanus toxoid were lower than in controls. No association was found between these abnormalities and other immunological parameters, including the blood level of CD4+ lymphocytes. Lower responses to most microbial antigens were observed in patients with HIV-1-related symptoms in addition to lymphadenopathy, or the patients who progressed to AIDS in the 2 years following the study. Moreover, intravenous drug users showed higher responses than homosexual patients, possibly because of the influence of previous infections on immunological responses to microbial antigens.

Gamma heavy chain "disease": heterogeneity of the clinicopathologic features. Report of 16 cases and review of the literature.

This review underscores the diversity of the clinical manifestations and hematopathological features of gamma heavy chain disease based on the detailed report of 16 patients evaluated in our chemical department, the analysis of 12 cases diagnosed in our laboratory, and the study of 81 cases previously reported. This condition is defined by the presence in the serum of immunoglobulin molecules composed of deleted gamma heavy chains devoid of light chains. The production by the monoclonal B cells of these peculiar proteins appears to result from multiple defects (deletions, insertions, and mutations) in both heavy and light chain genes leading to abnormal mRNA splicing. Gamma heavy chain disease is currently underdiagnosed. The diagnosis established by immunoelectrophoresis using specific antisera combined, in some instances, with the immunoselection procedure, can easily be missed on serum electrophoretic patterns: a narrow abnormal band suggestive of a monoclonal component was found in only 10 of our 28 cases. The amount of heavy chain disease protein in urine ranges from trace to 20 g/day and is usually moderate. Gamma heavy chain disease most often presents as a lymphoproliferative disorder featured by lymphadenopathies, splenomegaly, and constitutional symptoms. Extra-hematopoietic tumor localizations, such as cutaneous or subcutaneous involvement or thyroid tumor, may occur. Autoimmune disorders, notably rheumatoid arthritis and autoimmune hemolytic anemia or thrombocytopenic purpura, are frequent (26% of cases). There is no specific histological pattern. The most frequent is a pleomorphic malignant lymphoplasmacytic proliferation mainly seen in bone marrow and lymph nodes. Some cases present with a predominantly plasmacytic proliferation or chronic lymphocytic leukemia. Other patients are affected with non-Hodgkin lymphoma of various morphologic types. Immunocytologic studies showed that a gamma heavy chain disease protein may occur in the context of a double monoclonal lymphoproliferative process or in various B or T cell malignancies that are not directly involved in the production of the abnormal immunoglobulin. In some patients, the histologic appearance of the enlarged lymphoid organs showed only a moderate lymphoplasmacytic infiltration of uncertain malignancy. More important, some patients showed no evidence of an underlying lymphoproliferative disorder after several years of follow-up. The clinical course of gamma heavy chain disease varies from an asymptomatic state to a rapidly progressive malignancy. The choice of therapy should entirely rely on the underlying clinicopathologic features, without taking into account the presence of the abnormal protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Clinical and pathologic features of Waldenström's macroglobulinemia in seven patients with serum monoclonal IgG or IgA.

The clinical, hematologic and pathologic findings in seven patients were similar to those of Waldenström's macroglobulinemia, but unexpectedly the serum monoclonal immunoglobulin belonged to the IgG class in five patients and to the IgA class in two. The bone marrow and lymph node lymphoid proliferation was pleomorphic, with the simultaneous presence of small lymphocytes, normal mature plasma cells and transitional lymphoplasmacytic cells. Immunofluorescence studies showed that a monoclonal immunoglobulin similar to that found in the serum was detectable on the membrane or in the cytoplasm of all the proliferating cells, which thus belonged to the same B cell clone. The study of these patients is in accordance with the concept that lymphoid disorders featured by a pleomorphic monoclonal B cell proliferation constitute a distinct clinicopathologic entity, which is not restricted to IgM-producing clones.

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.

Cytogenetic studies in four cases of alpha chain disease.

Cytogenetic studies were performed in four cases of alpha chain disease. Chromosomal abnormalities were found in the lymphoid cells of the mesenteric lymph nodes of three patients, two of whom had not reached the stage of overt malignant lymphoma. In two instances, a rearrangement of 14q32, resulting from a t(9;14)(p11;q32) and a t(2;14)(p12;q32) was observed. One case showed complex rearrangements including t(5;9). No abnormalities were found in the intestinal tumor of the fourth case with immunoblastic lymphoma. It is concluded that alpha chain disease is a clonal proliferation with frequent alteration of chromosome #14 at band q32 resulting from translocations that differ from those observed in the vast majority of other non-Hodgkin lymphomas.