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Comparison between the conventional tube technique and the gel technique in direct antiglobulin tests.

OBJECTIVES: The direct antiglobulin test (DAT) is a diagnostic procedure demonstrating in-vivo antibody or complement coating on red cells. The gel technique (GT) for this test is sensitive and easier to do than the conventional tube technique (CTT). METHODS: We tested 52 newborns with hyperbilirubinemia and 6 children and 17 adults with autoimmune hemolytic anemia (AIHA) using the (DAT) in the form of the conventional tube technique (CTT) and the gel technique (GT) simultaneously. The gradings of the agglutination reactions of both techniques were scored as 12, 10, 8, 5, 3, 0 for 4+, 3+, 2+, 1+ and w+ and negative, respectively. RESULTS: The GT yielded higher scores than the CTT (p < 0.01). The overall sensitivity and specificity of the GT were 93.5 and 88.6%, respectively. The specificity of the DAT-positive results in newborns was determined by IgG only, while in AIHA, it was determined by IgG and/or C3d and, in only one sample, by IgM. CONCLUSION: The GT is equal to or better than the CTT. Since the GT is simple, the exposure of blood bank personnel to the blood sample is low. We highly recommend the GT, especially in areas where HIV infection is prevalent.

Adolescent↗

gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4).

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.

Antibodies, Monoclonal↗

Unexpected limitations in the use of commercial antiglobulin reagents.

The majority of antiglobulin sera used in blood banks in the USA are commercially prepared immune rabbit sera, designed to be reactive only with human red blood cells sensitized with immunoglobulins or complement components. Current manufacturing methods result in a product that gives reliable specific reactions provided that the test red blood cells are normal, particularly with respect to sialic acid levels. This report describes our findings of false positive antiglobulin tests caused by incompletely absorbed antiglobulin reagents when cells with low sialic acid levels were tested. An evaluation of nine commercially prepared antiglobulin reagents revealed, in many of them, the presence of anti-species antibody, anti-T, and anti-Tn. Blood bank personnel must be aware of these characteristics especially when testing either enzyme premodified or polyagglutinable cells. The use of incompletely absorbed reagents might account for positive direct antiglobulin tests that are encountered occasionally in apparently normal healthy individuals. Furthermore, recent protocols advocating the use of trypsinized cells for the evaluation of anti-C3d activity of antiglobulin sera are invalid if the reagent is inadequately absorbed of anti-species agglutinins.

Agglutinins↗

Autoantibodies against gp140, the Epstein-Barr virus and C3d receptor in sera from rheumatoid arthritis patients.

gp140 is the Epstein-Barr virus receptor and the C3d receptor (EBVR/C3dR) of human B lymphocytes. Recently, we have shown that cross-linking of EBVR/C3dR on cell surface by polyclonal anti-gp140 induced B cell activation, in presence of T cell factors. Immunoregulatory abnormalities of EBV-induced B cell activation have been demonstrated in rheumatoid arthritis (RA) patients. These data prompted us to analyze the putative presence of anti-EBVR/C3dR autoantibodies in human sera. The IgG fractions from eleven RA and 10 normal sera were tested for their ability to: (a) bind to Raji cell surface; (b) inhibit the binding to cell surface of 3 anti-EBVR/C3dR monoclonal antibodies (mAb), which recognized different epitopes on gp140; (c) inhibit the binding of particle-bound C3d and (d) react with 1% Nonidet-P40-solubilized gp140 from Raji cell membranes, in immunoblotting assays. Three RA sera carry anti-EBVR/C3dR autoantibodies which react with gp140 expressed on Raji cell surface or its solubilized form. The purification of monomeric IgG fraction of selected RA sera ruled out involvement of immune complexes carrying C3 molecules, which could interfere in these assays. One of these 3 RA sera was able to inhibit the binding to cell surface of anti-EBVR/C3dR mAb and particle-bound C3d. However, the 2 other RA sera, found positive by immunoblotting, did not inhibit particle-bound C3d and presented differences in their inhibitory effect on anti-EBVR/C3dR mAb binding to Raji cell surface. These data allow us to demonstrate differences which exist in the properties of anti-EBVR/C3dR autoantibodies. These autoantibodies were not detected in all the normal and other RA sera. Anti-EBVR/C3dR autoantibodies could play a role "in vivo" in B lymphocyte activation of RA patients.

Arthritis, Rheumatoid↗

Release of platelet-activating factor from HL-60 human leukemic cells following macrophage-like differentiation.

Platelet-activating factors (PAF), a phospholipid mediator of anaphylaxis, is also known to be released in vitro from both phagocytic polymorphonuclear neutrophils (PMN) and monocytes in response to a variety of stimuli. The fact that human myeloid cells of the HL-60 line can be made to differentiate in vitro into macrophage-like cells by 12-O-tetradodecanoylphorbol-13-acetate (TPA) prompted us to investigate the generation and release of PAF during this transformation. Both passive release of PAF at pH 9.5, and active release, following phagocytosis of C3b- and C3d-opsonized yeast spores, and stimulation with C5a anaphylatoxin from untreated and TPA-treated HL-60 cells, PMN, and plastic-adherent normal human monocytes were studied. It was found that after 3 days of TPA treatment, HL-60 cells released PAF following phagocytosis of C3b- and C3d-opsonized yeast spores. Inhibition of PAF release by a selective inhibitor of phospholipase A2 and labeling of PAF with sodium 14C-acetate indicated that PAF generation is a two-step process: (1) release of PAF precursor from cell membranes and (2) its acetylation. A model for the in vivo study of mechanisms and metabolic events involved in PAF generation and release could perhaps be built on these findings.U

Cell Line↗

Relationship between different cell surface markers in chronic lymphocytic leukemia.

Peripheral blood lymphocytes from 15 patients with chronic lymphocytic leukemia (CLL) and 13 normal controls were examined for the presence of surface markers: CLL antigen, surface immunoglobulins, receptors for sheep erythrocytes, receptors for the IgG Fc, C3b, C3d and receptor(s) for mouse erythrocytes. Leukemic lymphocytes were characterized by the presence of CLL antigen and a significant increase in the capacity to bind mouse erythrocytes. CLL antigen was correlated with surface immunoglobulins and receptor of IgG Fc, however, the receptor for mouse red cells was not correlated with other surface markers. Neuraminidase treatment of CLL lymphocytes significantly increased proportion of mouse erythrocyte rosettes. The routine use of mouse erythrocyte rosettes in the immunodiagnostics of B lymphoproliferative disorders is postulated.

Animals↗

In situ identification of mononuclear cells in cutaneous infiltrates in discoid lupus erythematosus, sarcoidosis and secondary syphilis.

The inflammatory mononuclear cell infiltrates observed in lesional skin from patients with discoid lupus erythematosus (DLE), sarcoidosis, and secondary syphilis have been characterized in situ. Immunological markers (human T lymphocyte antigens and receptors for sheep erythrocytes, C3b, C3d and Fc gamma) were studied by using immunofluorescence tests with IgG F (ab')2 preparation of anti-T lymphocyte serum, hemadsorption with tissue sections, and tests with soluble immune complexes of peroxidase. In DLE, T lymphocytes were the dominant cell type. In sarcoidosis, the epithelioid cells, including giant cells, had markers similar to macrophages. The lymphohistiocytic cells consisted mainly of macrophages, some T lymphocytes and a few B lymphocytes. In secondary syphilis (condylomata lata), macrophages and T lymphocytes were the dominant cell types, and relatively few B lymphocytes were detected.

Antigens↗

In situ characterization of cell infiltrates in the dermis of human scabies.

Cell infiltrates in the dermis of biopsy specimens from skin lesions of seven patients with scabies were characterized by receptors for sheep erythrocytes (T-lymphocytes), Fc C3b (macrophages and B-lymphocytes) and C3d receptors (B-lymphocytes) using hemadsorption to cryostat sections in closed chamber. T-lymphocytes were also identified using an indirect immunofluorescence technique with rabbit anti-T-lymphocyte serum. All sections showed perivascular mononuclear cell infiltrates in the dermis as judged by sections stained by hematoxylin and eosin. T-lymphocytes were the predominant cells in the infiltrates. Fewer macrophages and very few B-lymphocytes were detected. Cell-mediated immune response may be of importance in the pathogenesis of scabies.

Animals↗

Epstein-Barr virus receptors on human pharyngeal epithelia.

The apparently strict tropism of Epstein-Barr virus (EBV) for B lymphocytes has been attributed to the existence of a B-lineage-specific surface molecule, the C3d receptor, which also functions as a receptor for EBV. Two monoclonal antibodies against different determinants on the EBV/C3d receptor of B cells were shown to react with pharyngeal epithelia in a cell differentiation-dependent manner. These findings, which raise the possibility of direct virus entry into a naturally exposed epithelium, strengthen the evidence in favour of an epithelial reservoir of EBV infection in vivo and identify a means whereby the virus/epithelium interactions leading to nasopharyngeal carcinoma might be initiated.

Antibodies, Monoclonal↗

Biotinylation of human C3.

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.

Avidin↗

Diagnostic value of C4d in renal biopsies.

PURPOSE OF REVIEW: Capillary C4d is now an established marker of antibody-mediated rejection in graft biopsies. The technique is widely used to further define the clinical relevance of humoral alloreactivity in various patient subgroups. These include highly sensitized patients, recipients with late graft failure and also some with 'stable' graft function. RECENT FINDINGS: The C4d technique compares favourably with other techniques that are explored, for example detection of C3d. Capillary C4d can be associated with any graft pathology, including transplant glomerulopathy. C4d is related to circulating alloantibodies but not autoantibodies, and is probably not derived from local sources. Presensitization and de-novo sensitization are important scenarios of humoral alloreactivity that require refined analysis and treatment. SUMMARY: Detection of C4d in graft biopsies has emerged as an important tool that could substantiate the clinical significance of antibody-mediated rejections. The comprehensive analysis of humoral alloreactivity in the posttransplantation period is still ongoing and will hopefully result in improved patient care and better long-term graft survival.

Autoantibodies↗

Follicular dendritic cells contain a unique gene repertoire demonstrated by single-cell polymerase chain reaction.

Follicular dendritic cells (FDCs) form a dense network between B cells within the germinal center and are thought to be an important component of this B-cell microenvironment. Previous immunophenotypic studies have been inconclusive in determining the cellular origin of FDCs. Gene coexpression within individual and highly enriched FDCs was determined using polymerase chain reaction. FDCs contain a very restricted mRNA pattern with high levels of message for the C3d receptor (CR2, Epstein Barr-virus/EBV receptor, CD21) and lack of mRNA for CD20, CD45, CD4, fibronectin, and platelet-derived growth factor receptor alpha and beta. These observations are consistent with the hypothesis that FDCs may not be of classical hematopoietic or fibroblastic origin. The absence of interferon-gamma, tumor necrosis factor-alpha, interleukin-3, and interleukin-6 mRNA provides preliminary evidence that these cells might produce only a very restricted set of cytokines limited to the germinal center.

Antigens, CD↗

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. II. C3 receptor switching during human B cell differentiation.

Our experiments showed that two C3 receptors (C3-R) appeared on membrane surfaces in a distinct sequence during the differentiation of human B cells. The experimental results supporting this conclusion were (1) the lymphoblastoid cells established from immature B cells expressed more EACm-R (C3bi and C3d receptors) than EACh-R (C3b receptor), (2) the lymphoblastoid cells originated from mature B cells showed more EACh-R than EACm-R, (3) the above characteristic expression of C3-R was demonstrated on every clone isolated in soft agar from cells in B cell lineage at various differentiation stages, indicating that a cell line consists of homogeneous population in terms of C3-R expression. From these results, it was concluded that C3-R switched from EACm-R to EACh-R during B cell differentiation.

B-Lymphocytes↗

Association of T cell dysfunction with the presence of IgG autoantibodies on CD4+ lymphocytes in haemophilia patients; results of a 10-year study.

HIV induces progressive dysfunction followed by numerical depletion of CD4+ lymphocytes. IgG autoantibodies and gp 120-containing immune complexes have been implicated in the pathogenesis of AIDS. We carried out a longitudinal study in 19 HIV- and 72 HIV+ haemophilia patients over a 10-year period in order to investigate a possible relationship between the occurrence of autoantibodies and CD4+ lymphocyte changes. IgM, IgG, C3d and gp120 on the surface of CD4+ lymphocytes were determined in heparinized whole blood with flow cytometry and double-fluorescence. The in vitro response of autoantibody-coated cells was tested in cell cultures with concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PWM) anti-CD3 MoAb or pooled allogeneic stimulator cells (MLC). After a 10-year follow up, 12 of 71 HIV+ and 16 of 19 HIV- haemophilia patients showed no evidence of immunoglobulins on circulating CD4+ lymphocytes. HIV- haemophilia patients without autoantibodies had CD4+ and CD8+ cell counts in the normal range (957+/-642/microliters and 636+/-405/microliters) and normal T cell responses in vitro (mean relative response (RR) > or = 0.7). In contrast, HIV+ haemophilia patients showed immunological abnormalities which were associated with the autoantibody and immune complex load of CD4+ blood lymphocytes. HIV+ patients without autoantibodies had a mean CD4+ lymphocyte count of 372+/-274/microliter, a mean CD8+ lymphocyte count of 737+/-435 microliter, and normal T lymphocyte stimulation in vitro (mean RR > or = 0.7). HIV+ patients with complement-fixing IgM on CD4+ lymphocytes had somewhat lower CD4+ (255+/-246/microliters, P = NS) and CD8+ (706 +/- 468/microliters, P = NS) lymphocyte numbers, and also normal T lymphocyte stimulation (mean RR > or = 0.7) in vitro. However, patients with complement-fixing IgG autoantibodies showed a strong decrease of CD4+ (150 +/- 146/microliters, P< 0.02) and CD8+ (360 +/- 300 microliters, (P<0.02) lymphocytes and impaired CD4+ lymphocyte stimulation in vitro with a mean RR of 0.5+/-0.5 for Con A (P = NS), 0.7 +/- 0.8 for PHA (P<0.03), 0.4 +/- 0.4 for PWM (P = NS), 0.8 +/- 1.2 for anti-CD3 MoAb (P<0.04) and 0.7 +/- 1.0 for pooled allogeneic stimulator cells (P=0.05). Patients with gp120-containing immune complexes on CD4+ blood lymphocytes demonstrated strongly decreased CD4+ (25+/-35/microliters, P<0.0001) and CD8+ (213+/-212/microliters, P<0.006) lymphocyte counts as well as strongly impaired T lymphocyte responses in vitro upon stimulation with PHA (RR 0.2+/-0.1, P<0.02), PWM (RR 0.2+/_0.2, P=0.05), anti-CD3 MoAb(RR 0.1+/-0.1, P<0.04), and allogeneic stimulator cells (RR 0.2+/-0.1, P<0.02). These data led us to speculate that autoantibody formation against CD4+ lymphocytes is an important mechanism in the pathogenesis of AIDS. We hypothesize that autoantibodies against circulating CD4+ lymphocytes inhibit CD4+ cell function, especially the release of cytokines, and induce CD4+ cell depletion. The reduction and dysfunction of CD4+ lymphocytes may be responsible for the CD8+ cell depletion observed in HIV+ patients.

Antigen-Antibody Complex↗

Identification of a C3b/iC3 binding protein of rabbit platelets and leukocytes. A CR1-like candidate for the immune adherence receptor.

Immune adherence is the attachment of C-bearing immune complexes via the major activation fragment of the third component of C(C3b) to C3b binding membrane proteins. On primate E, the C3b-R, termed CR1, mediates immune adherence. In nonprimates, immune adherence involves platelets instead of E. However, these functional data have not been corroborated by the identification of the binding protein. In this work, we have identified a C3b/iC3 binding protein of rabbit platelets and characterized it as a single chain structure with a Mr of 150 kDa (nonreducing) or 175 kDa (reducing). This protein binds to rabbit iC3 or C3b but not C3d. This specificity of binding and the ability to rebind to a second column of iC3- or C3b-thiol-Sepharose are comparable to human CR1. Also, a molecule with the identical Mr as well as other structural and binding characteristics is present on rabbit PBMC. No such protein was isolated from rabbit E. Our data strongly suggest that the C3b/iC3 binding protein of rabbit platelets is the homologue of human CR1. If so, this represents an interesting evolutionary switch in the tissue specific expression of the immune adherence R from platelets in the nonprimate to E in the primate.

Animals↗

Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. I. Incidence and class specificity.

Gambian children with past or present Plasmodium falciparum malaria were investigated for the incidence of Coombs positivity using monospecific antisera. Approximately 50% were positive and the most frequent form of erythrocyte sensitization was with C3d. Other specificities, EIgG, EIgGC3d and EIgGC4bC3d were less common. Erthyrocytes were never found sensitized with IgA or IgM. There was no correlation between a positive test and age, tribal status or level of parasitaemia at presentation, although a positive test was often found in association with anaemia. Sensitized erythrocytes were present in the circulation for a period of up to 6 weeks following initial observation. The mechanism of erythrocyte sensitization is not known, but the results suggest a Type III complex-mediated hypersensitivity involving parasite antigen-antibody complexes. It is likely that these reactions contribute to the pathogenesis of the anaemia in falciparum malaria.

Anemia↗

Genetically determined low C4: a predisposing factor to autoimmune chronic active hepatitis.

Of 26 patients with autoimmune chronic active hepatitis (CAH) starting in childhood 18 (69%) had low C4 and 5 (19%) had low C3 serum levels. Impaired hepatic synthesis and immune-consumption were unlikely since transferrin levels were normal in all patients, albumin levels were persistently low in only 3, and only 3 had raised levels of activation fragment C3d. C4d was normal in all patients studied. In the families of 12 probands with low C4, 7 parents had low C4 and 2 had levels which were at the lower limit of normal. 5 of 10 siblings from 5 families had low C4. These results suggest that low C4 levels in CAH are genetically determined. C4 phenotyping in 20 patients and in 26 parents showed that 90% and 81%, respectively, had null allotypes at either the C4A or C4B locus compared with 59% in controls, indicating that defective expression of structural genes may contribute to the observed C4 deficiency.

Adolescent↗

Phenotype characteristics of human B cells studied by Epstein-Barr virus infection. III. In vitro modulation of C3 receptor expression on human lymphoid cells.

An addition of dimethyl sulfoxide (DMSO) in culture medium enhanced the expression of receptor (R) for EACh (C3b-R) of the lymphoblastoid cells derived from immature B cells which were expressing little or no EACh-R. EACm-R (C3bi- and C3d-R) on the lymphoblastoid cells of bone marrow cells decreased remarkably (25 to 5%) during short term cultivation, though other markers remained unchanged. In addition, C3-R expression of the lymphoblastoid cell lines from bone marrow cells, which were established by EBV infection before and after 4-day cultivation, was quite similar to that of the cell of origin. These results, in accordance with our previous paper (Kudo, T. and Tachibana, T. (1984) Immunol. Lett. 8,343-348), support the idea that the sequential switching of C3 receptors, EACm-R----EACh-R, occurs during B cell differentiation.

B-Lymphocytes↗