Complement regulators in human disease: lessons from modern genetics.

First identified in human serum in the late 19th century as a 'complement' to antibodies in mediating bacterial lysis, the complement system emerged more than a billion years ago probably as the first humoral immune system. The contemporary complement system consists of nearly 60 proteins in three activation pathways (classical, alternative and lectin) and a terminal cytolytic pathway common to all. Modern molecular biology and genetics have not only led to further elucidation of the structure of complement system components, but have also revealed function-altering rare variants and common polymorphisms, particularly in regulators of the alternative pathway, that predispose to human disease by creating 'hyperinflammatory complement phenotypes'. To treat these 'complementopathies', a monoclonal antibody against the initiator of the membrane attack complex, C5, has received approval for use. Additional therapeutic reagents are on the horizon.

Hyperphosphorylation of autoantigenic targets of paraproteins is due to inactivation of PP2A.

Paratarg-7, a frequent autoantigenic target, and all other autoantigenic targets of human paraproteins molecularly defined to date are hyperphosphorylated in the respective patients compared with healthy controls, suggesting that hyperphosphorylation of autoantigenic paraprotein targets is a general mechanism underlying the pathogenesis of these paraproteins. We now show that hyperphosphorylation of paratarg-7 occurs because of an additional phosphorylation of Ser17, which is located within the paraprotein-binding epitope. Coimmunoprecipitation identified phosphokinase C ζ (PKCζ) as the kinase responsible for the phosphorylation of most, and phosphatase 2A (PP2A) as the phosphatase responsible for the dephosphorylation of all hyperphosphorylated autoantigenic targets of paraproteins. Single-nucleotide polymorphisms (SNPs) or mutations of PKCζ and PP2A were excluded. However, PP2A was inactivated by phosphorylation of its catalytic subunit at Y307. Stimulation of T cells from healthy carriers of wild-type paratarg-7 induced a partial and transient hyperphosphorylation between days 4 and 18, which was maintained by incubation with inhibitors of PP2A, again indicating that an inactivation of PP2A is responsible for the hyperphosphorylation of autoantigenic paraprotein targets. We conclude that the genetic defect underlying the dominantly inherited hyperphosphorylation of autoantigenic paraprotein targets is not in the PP2A itself, but in genes or proteins controlling PP2A activity by phosphorylation of its catalytic subunit.

Complement fixation by rheumatoid factor.

The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum.

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.

Fixation of the first component of complement (C'la) by human antibodies.

The fixation of the first component of complement (C'1a) by human antibodies and human cells has been studied by the use of the C'1a fixation and transfer test (C'1a FT test) of Borsos and Rapp.Cold agglutinin antibodies appear to require no more than one antibody molecule to fix one molecule of C'1a. Most warm agglutinin antibodies are IgG in immunoglobulin type and require at least two molecules of antibody to fix a molecule of C'1a. Donath-Landsteiner antibody has the same requirements for C'1a fixation. A single example of a warm agglutinin antibody which appears to require one molecule of antibody for the fixation of C'1a was found. Antibodies of the Rh system do not fix significant amounts of C'1a in the absence of anti-antibody when antiserum of a single Rh specificity was used. However, when three antisera at different specificity are present, C'1a may be fixed. Under these conditions cells from a patient with paroxysmal nocturnal hemoglobinuria may be lysed when fresh serum is added to provide the other components of complement. The presence of IgG antibodies could be detected by the use of anti-IgG(Hu) antiserum and a one-to-one relationship between the concentration of antiserum in the reaction and the amount of C'1a fixed could be established. The effect of temperature, ionic strength, papainization of the red cells, and repeated washing of the red cell-antibody aggregates on the amount of C'1a fixed was investigated. Conditions of maximal C'1a fixation were established for each class of antibodies. Globulins present in normal isologous or autologous serum are absorbed in small amounts to normal red cells in a manner analogous to warm agglutinin antibody. Their presence is detectable by the C'1a fixation and transfer test only with antiglobulin antiserum. Within certain limits, the C'1a fixation and transfer test provides a quantitative measure of the reaction of human red cells and antibodies to antigens on their surface.

The structure of human high density lipoprotein and the levels of apolipoprotein A-I in plasma as determined by radioimmunoassay.

The major apoprotein of high density lipoprotein is apolipoprotein A-I (ApoA-I). In addition to being a structural component of this class of lipoproteins, ApoA-I also has a physiologic role as an activator of lecithin-cholesterol acyl transferase, an enzyme important in the metabolism of all lipoproteins. To measure ApoA-I content in human plasma, to assess its immunologic activity in hyperlipoproteinemia, and to carry out certain structural studies of high density lipoproteins, we have developed a double antibody radioimmunoassay. ApoA-I, isolated by gel filtration, was used to produce monospecific antisera. ApoA-I was iodinated by chloramine-T and the resulting [(125)I]-ApoA-I was purified by gel filtration. > 85% of [(125)I]-ApoA-I was precipitated by antibody, and 90% of bound [(125)I]ApoA-I was displaced by "cold" ApoA-I. Other lipoproteins and apoproteins did not react. Plasma and high density lipoprotein from normals and subjects with hyperlipoproteinemia displaced counts in parallel with ApoA-I, suggesting that the same antigenic determinants were reacting with antibody on lipid-free and lipid-associated ApoA-I. However, less than 5% of ApoA-I of high density lipoprotein reacted in the assay. Removal of the lipid by extraction increased the reactivity of ApoA-I in high density lipoprotein 15-20-fold; thus more than 95% of the ApoA-I molecules in "intact" high density lipoprotein are unreactive with antibody. Normal and hyperlipoproteinemic plasma and high density lipoproteins isolated from the same subjects continued to display parallelism with ApoA-I standard after lipid extraction, suggesting that ApoA-I of normal and hyperliproteinemic subjects are immunologically identical. About 90% of ApoA-I was in the d 1.063-1.21 fractions of normal plasma, trace quantities were found in the lipoproteins of d < 1.063, and the rest (about 10%) was in the d > 1.21 fraction. Normal plasma levels, assessed in extracted plasmas with a precision of 8%, were 100+/-35 mg/dl. Levels were normal in small groups of subjects with types II and IV hyperlipoproteinemia and high in pregnancy. However, larger population studies need to be performed to determine the distribution of ApoA-I levels in the various hyperlipoproteinemias.

Idiopathic (asymptomatic) monoclonal gammopathies.

Asymptomatic forms of monoclonal gammopathies (MG) are recognized with increasing frequency; their recognition and differentiation from the symptomatic forms of MG appear imperative, since the therapeutic approaches are different. Available clinical and laboratory indexes lack specificity required for useful and practical discrimination; presently, we must still rely on the timecourse monitoring of such laboratory values as hemoglobin levels, M-protein concentrations, and presence of Bence Jones proteins. Elucidation of histocompatibility A and W antigenic profiles, as well as the functions and kinetics of B-lymphocytes from such patients, appear most promising. Evidence of the causative role of extrinsic and intrinsic antigenic stimulation in MG production is increasing; segregation into two distinct concentration ranges of M-proteins in the asymptomatic and symptomatic groups suggests two control levels of the expression of immune response (Ir) genes, due to partial or complete derepression of the latent Ir gene function, reflecting "partial" (asymptomatic, benign MG) and "complete" (symptomatic, malignant MG) monoclonal immune responders.

Monoclonal gammopathy in lymphoma.

Serum protein electrophoresis was performed in 68% of 1,682 consecutive patients with lymphoma. Of 400 patients with chronic lymphocytic leukemia and lymphocytic lymphoma, 2.3% had an IgG peak, a frequency significantly higher than that found in normal individuals of comparable age. IgM peaks occurred in 4.5% of patients with lymphomas characterized by diffuse histologic infiltration of lymph nodes. This frequency was about 100 times greater than predicted and indicated that most IgM peaks detected by electrophoresis are produced by lymphomas. Clinical responses to therapy were associated with reductions of tumor mass usually ranging from 10% to 50% of the pretreatment level. A much greater magnitude of tumor reduction is needed to improve remission duration for patients with chronic lymphocytic leukemia and lymphocytic lymphoma.

Blast cell leukemia with IgM monoclonal gammopathy and intracytoplasmic vacuoles and Auer-body-like inclusions.

A patient with acute leukemia and an IgM, kappa (IgMkappa) monoclonal gammopathy, Bence-Jones proteinuria, and blasts containing intracytoplasmic vacuoles with peroxidase-positive inclusions is discussed. Special stains, immunofluorescence, and electron microscopy suggested that the vacuoles were autophagosomes containing Auer-body-like inclusions, and that the blast cells did not synthesize the paraprotein. Chemotherapy with cyclophosphamide, vincristine, and prednisone resulted in transient improvement of the leukemia, but the level of the paraprotein was unchanged. Other case reports involving monoclonal gammopathy in association with acute leukemia are reviewed and contrasted with this case.

Flow cytometric measurement of antiplatelet antibodies.

A flow cytometric technic was developed to detect platelet surface-bound immunoglobulin in patients with thrombocytopenia. Elevated platelet surface IgG and/or IgM was detected in 90.9% of patients with immune thrombocytopenia purpura (ITP). False positive results occurred in 9.3% of patients with nonimmune thrombocytopenia usually associated with sepsis. False negatives occurred most frequently in adults with chronic ITP. Measurement of platelet surface immunoglobulin with this flow cytometric technic helps differentiate immune from nonimmune thrombocytopenia.

Cytoplasmic cytokines in lymphoproliferative disorders: multiple cytokine production in angioimmunoblastic lymphadenopathy with dysproteinemia.

Cytokines play an important role in the pathogenesis of lymphomas via autocrine or paracrine mechanisms, or both. Here we determined the proportion of CD3-positive T lymphocytes containing various types of cytokines in enlarged lymph nodes. Lymph nodes were obtained from 16 patients with various lymphoproliferative disorders, including 3 cases with angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), 3 cases with adult T cell leukemia/lymphoma (ATLL), 2 cases with T-cell nonspecific malignant lymphoma (T-ML), 3 cases with B-cell diffuse large malignant lymphoma (BDL), 3 cases with histiocytic necrotizing lymphadenitis (HNL), and 2 cases with non-specific lymphadenitis (NSL). The percentages of T lymphocytes positive for cytoplasmic cytokines IL-2, IL-4, IL-5, IL-6, IL-13, TNF-alpha, and INF-gamma were determined. The percentage of INF-gamma positive T lymphocytes was high in reactive lymphadenopathy of NSL and HNL. AILD showed a high proportion of TNF-alpha positive T-lymphocytes, and in addition, the percentages of IL-2, IL-4, IL-5, IL-6, IL-13 and INF-gamma positive T-lymphocytes were relatively higher than in other diseases. Our results supported the state of multiple hypercytokinemia typically seen in AILD and suggested that the source of the cytokines is the lymph nodes. Our results also suggested that multiple cytokine networks play an important role in the clinical and histopathological features of AILD. Modulation of the cytokine network may be the logical objective in future therapeutic strategies designed for AILD.

[Circulating immunocomplexes in patients with various morbid conditions]. / Immunocomplessi circolanti in pazienti con varie condizioni morbose.

The polyethylenglycol (PEG) precipitation technique has been employed for the measurement of immune complexes in the circulation of 100 normal subjects and in 14 patients suffering from a variety of diseases (systemic lupus erythematodes, nephrosic syndrome, cryoglobulinaemia, Buckley's syndrome). Values higher than 0.80 UA on the absorption scale were considered pathological, namely 2 standard deviations above the mean (0.32 UA) in the subjects examined; in the patients, values between a minimum of 0.98 UA and a maximum of 2.38 UA were observed. Longitudinal study of these cases also pointed to the disappearance of immune complexes during therapy. The results suggest that the PEG precipitation technique can play an important part as a screening test in situations in which circulating IC pathology is suspected; it is also a sensitive means of monitoring treatment.

[The significance of cryoglobulins and cryohemagglutinins. Immunological and clinical study of 39 cases]. / Sul significato delle crioglobuline e delle crioemoagglutinine. Studio immunologico e clinico di 39 casi.

In about 5 years of clinical and immunological research, 39 cases of cryoplasmopathies (5 with cryoglobulinaemia and 34 with cryohaemoagglutinaemia) were identified on the basis of immunological typing studies (immunoelectrophoresis, immunodiffusion, identification and isolation of cryoantibodies, cryocrit, titration, identificcation of antibody specificity). Discussion and physiopathological interpretation of possibly associated cases and diseases are conducted on the basis of personal immunological data and the most recent literature on matters of clinical immunohaematology.

[Sjögren's syndrome, mixed cryoglobulinemia and Waldenström's purpura (author's transl)]. / Síndrome de Sjögren, crioglobulinemia mixta y púrpura hipergammaglobulinémica de Waldenström.

A 66-year-old patient was hospitalized after the appearance of a deep ulcer in the posterior face of the right calf. She had purpuric spots on the lower limbs only, as well as parotiditis, especially on the left side, and was affected with dryness of the mouth but not of the eyes. The patient also presented, in contact with the cold, Raynaud's phenomenon. Physical examination revealed as pathologic signs: purpura of the lower limbs, a deep ulceration on the right calf, parotiditis, and a reduction in the pallaesthesia of the lower limbs. Biologically an increased sedimentation rate, elevation of the total proteins (particularly the gammaglobulins), positive antinuclear factor, reduction of the total complement and of the C3 fraction, circulating immunocomplexes, positive latex test, positive Sia test, and the presence of mixed cryoglobulin IgG-IgM type kappa, stood out. The patient was diagnosed as having Sjögren's syndrome, mixed cryoglobulinemia and Waldenström's purpura. Probably the two latter conditions characterized by the presence or circulating immunocomplexes are part of the same process. According to recently studies described in the literature the relationships between these three entities are discussed, as well as the possibility of developing lymphoproliferative syndromes under certain immune stimuli.