Interaction between actomyosin complexes and Fc receptors on human peripheral mononuclear blood cells

The soluble FcRI shed from HPMBC following 4–37°C temperature shift interacts with AM, A-HMM and A-S1, respectively, but do not bind to A, M, HMM or S1. In contrast to the monovalent soluble FcRI the multivalent A-HMM-FcRI and A-S1-FcRI complexes agglutinate EA cells. Due to the interaction of soluble FcR with muscle proteins an alteration in the binding properties of the receptor was observed. FcRI in soluble form inhibits only the EA rosette formation of FcRI⁺ HPMBC in contrast to FcRI-A-HMM and FcRI-A-S1 complexes inhibiting the rosette formation of FcRI⁺ and FcRII⁺ cells as well. Based on these observations one can suppose that depending on their anchoring to cytoskeletal structures the FcRs possess one or two binding sites.     

Beta-2-microglobulin-specific autoantibodies cause platelet aggregation and interfere with ADP-induced aggregation.

The anti-platelet activity of beta-2-microglobulin (beta 2m) specific autoantibodies isolated from sera of patients with autoimmune diseases was tested in direct and ADP-induced aggregation assays. It was established that human anti-beta 2m autoantibodies and heterologous rabbit anti-beta 2m antibodies evoke a dose-dependent aggregation of human platelets. Anti-beta 2m autoantibodies also impaired ADP-induced platelet aggregation. Antibodies with anti-beta 2m activity could be desorbed from the platelets and lymphocytes of a patients with systemic lupus erythematosus who was not thrombocytopenic. The possibility that such autoantibodies may alter platelet function is considered.     

Mitogenic Properties of Fab and F(ab'')2 Fragments of Rabbit Anti-Human β2-Microglobulin for Human Lymphocytes

Bivalent F(ab')2 fragments and monovalent Fab fragments of rabbit anti-human beta2-microglobulin (anti-beta2m) stimulated DNA synthesis in human lymphocytes. Mitogenicity of anti-beta2m antibodies can therefore be ascribed to the antigen-binding site and not to the Fc portion of the molecule. The mitogenic response to F(ab')2, and sometimes Fab, fragments of anti-beta2m IgG was comparable to that obtained with original IgG antibodies when tested at the same protein concentration. Since Fab monomers of anti-beta2m can cause lymphocyte activation, 'cross-linking' of hypothetical beta2-microblobulin-containing lymphocyte receptors does not seem necessary for activation. F(ab')2, as well as Fab, fragments of anti-beta2m blocked the cytotoxic effect of anti-beta2m IgG, showing that the fragments did indeed react with beta2-microblobulin on the cell surface. F(ab')2 dimers, but not Fab monomers, of anti-beta2m were capable of inhibiting the cytotoxic effect of an anti-HLA-A2 antiserum. The mitogenic activity of both anti-beta2m IgG and Fab monomers of such antibodies disappeared after absorption with highly purified beta2-microblobulin. The mitogenic effect of anti-beta2m IgG was inhibited to a minor extent by exposure of cells to high concentrations of pooled multispecific anti-HLA antibodies. This effect was probably nonspecific.     

Study of lymphocyte membrane antigens and receptors with antibodies to beta2-microglobulin (author's transl)]

Anti-beta 2 microglobulin sera (beta2m AS) rendered specific after extensive absorptions were obtained following immunization of rabbits with highly purified beta2m prepared from urine of tubular proteinuria. beta2m AS were cytotoxic upon addition of selected rabbit complement for human T and B lymphocytes. Partial inhibition of sheep red blood cells receptor recognition on T lymphocytes (E rosettes) and C3 component recognition on B lymphocytes (EAC rosettes) was obtained only with high concentrations of Fab'2 anti-beta2m, eliminating a direct association of beta2m and those receptors. Fab'2 anti-beta2 induced very little inhibition of Fc portion receptor recognition (EA rosettes), but they had no effect on lysis of targets covered with IgG anti-targets (ADCC), a function mediated through that receptor. Anti-beta2m antibodies in excess inhibited antigen-induced proliferation (PPD) and the mixed lymphocyte reaction (MLR) performed in AB serum and fetal calf serum containing media ; whereas a potentiation of the response occurred in the presence of an antigen excess (beta2m) brought by the culture medium (AB serum) suggesting involvement of immune complexes. Pretreatment of responding cells with beta2m AS did block unilateral MLR ; conversely, treatement of stimulating cells had no effect. Independent migration of T cell membrane antigens (HTLA) and beta2m upon addition of suitable ligands, as well as the lack of inhibition by Fab'2 anti-beta2m of complement dependent lysis with IgG anti-HTLA, excluded possible association of HTLA and beta2m.     

Inhibitory Effects of Scutellaria Barbata D. Don. and Euonymus Alatus Sieb. on Aromatase Activity of Human Leiomyomal Cells

It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase, we have examined the aromatase‐inhibiting potency of the Scutellaria barbata D. Don. (SB) and Euonymus alatus Sieb. (EA) in myometrial and leiomyomal cells which contain aromatase. We have also used human placental tissues. Although SB and EA are approximately equipotent in a cell‐free aromatase system (human placental microsomes), EA is consistently 10–30 times more potent than SB in inhibiting intracellular aromatase in myometrial and leiomyomal cells. To provide insights into the effect of SB and EA on aromatase activity in leiomyomal cells, we examined the cell lines, which is induced to differentiate toward the more transformed cell phenotype by 12‐tetradecanoylphorbal‐13‐acetate (TPA) as a protein kinase C activator and transforming growth factor‐β1 (TGF‐β1). Enzyme activity was inhibited in a time‐and dose‐dependent fashion by SB and EA and by either 1–50 nM TPA or 0.01–0.5 ng/ml TGF‐β1, with maximal responses after 2–3 h exposure.     

Alpha-1-antitrypsin-induced inhibition of complement-dependent phagocytosis

In a previous investigation, inhibition of complement-dependent rosette formation by alpha₁-antitrypsin (α₁-AT) was observed, and it was demonstrated that α₁-AT interacts through its carbohydrate portion with C3 and its fragments. In the present study, the effect of α₁-AT on the complement-receptor-mediated phagocytosis by human peripheral blood monocytes was examined.Purified α₁-AT inhibited in a dose-dependent manner phagocytosis of C3-carrying yeast particles. Inhibition was selective, concerned only C3-receptor-mediated phagocytosis, neither Fc-receptor-mediated phagocytosis nor uptake of untreated yeast particles was blocked by α₁-AT. It was demonstrated that α₁-AT exerted its inhibitory effect through binding to C3-carrying particles. The activity of α₁-AT towards C3 and fragments of C3 was not mediated by its antiprotease effect, but by its carbohydrate moiety. This finding suggests that α₁-AT may have an impact on various immune functions involving complement receptors.     

Mouse spleen cells and sensitized sheep red blood cells: An Fc-rosette-forming system allowing the detection of “activated” Fc structures

From a variety of Fc receptor-bearing cell/sensitized red blood cell combinations, mouse spleen cells, and sensitized SRBC were selected as an Fc-specific EA rosette assay system because only this mixture combined a high percentage (about 50%) of rosette-forming cells with complete absence of spontaneous rosettes and showed no influence of complement on the rosette formation. From studies on the minimal structural requirement of IgG both for mediation and inhibition of EA rosettes using IgG and several well-defined fragments, it appeared that both the C_H2 and the C_H3 domain of Fe are needed for optimal interaction with the lymphocyte Fc receptor. Finally, it was demonstrated that the assay system is able to detect “activated” Fc structures (here: heat-aggregated IgG) and to differentiate between varying amounts of such structures.     

Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.     

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of beta2-GPI

Autoantibodies targeting beta2-glycoprotein l (beta2-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-beta2-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-beta2-GPI and only 1.6% for IgG anti-beta2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human beta2-GPI and full-length human beta2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-beta2-GPI ELISA assays. Domain-deleted mutants D--345 and D--45 inhibited the binding in the IgA anti-beta2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the beta2-GPI molecule. These results clearly show that IgA anti-beta2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.     

Autoantibodies specific to beta-2-microglobulin inhibit the Fc receptor-dependent phagocytosis of human monocytes

Effect of anti-beta2-microglobulin (beta2m) antibodies was investigated on the in vitro Fc and C3 receptor dependent phagocytic capacity of human peripheral monocytes. Both rabbit and human anti-beta2m antibodies inhibit the Fc receptor-mediated phagocytosis. Anti-beta2m antibodies do not influence the spontaneous or C3b receptor-mediated ingestion. The inhibitory activity by human autoantibodies but not by rabbit anti-human beta2m antibodies was diminished when incubation was performed at 37 degrees C instead of 20 degrees C.     

Activation of Human Lymphocyte Subpopulations by Rabbit Anti-Human β2-Microglobulin and by Lipopolysaccharide

Rabbit anti-human beta2-microglobulin (anti-beta2m) was found to increase DNA synthesis in peripheral blood lymphocytes (PBLs) and in cells from abdominal lymph nodes, spleen, tonsil, adenoid, appendix, and bone marrow. The response to anti-beta2m was highest in cells originating from abdominal lymph node, appendix, and spleen. These organs were shown to contain a high proportion of surface-Ig-positive cells. No response to anti-beta2m was seen in thymus cells or in B-cell-depleted lymphocyte populations. Lipopolysaccharide (LPS) increased DNA synthesis in spleen cells, bone marrow cells, tonsil cells, and, sometimes, in cells from abdominal lymph nodes but weakly or not at all in PBLs. To study whether anti-beta2m and LPS activated the same subpopulation of lymphocytes, cultures were exposed to both mitogens in various concentrations. The effect on DNA synthesis in spleen cells was almost additive. This may indicate that these two polyclonal B-cell activators (PBAs) stimulate mainly distinct subsets of B cells in spleen. On the other hand, these two mitogens have a synergistic effect on DNA synthesis in PBLs. Since anti-beta2m is the first described selective B-cell mitogen activating human PBLs, it might be of clinical importance in the functional characterization of lymphocyte subpopulations.     

Relationships Between β2-Microglobulin and Alloantigens Coded for by the Major Histocompatibility Complexes of the Rabbit and the Guinea Pig

Treatment of rabbit and guinea pig lymphocytes with Fab' fragments of anti-beta2-microglobulin completely inhibited the cytotoxic effects of alloantisera to RLA or GPLA antigens, respectively. Aggregation of beta2-microglobulin on the lymphocyte surface by successive incubations with goat anti-beta2-microglobulin on the lymphocyte surface by successive incubations with goat anti-beta2-microglobulin and F(ab')2 fragments of rabbit anti-goat IgG also made rabbit lymphocytes resistant to lysis by anti-RLA, and guinea pig lymphocytes resistant to lysis by anti-RLA, and guinea pig lymphocytes resistant to lysis by anti-GPLA. The two kinds of pretreatment of guinea pig lymphocytes did not affect the cytotoxicity of antisera directed against guinea pig Ia antigens. These results in conjunction with previous findings in the mouse and in man suggest that beta2-microglobulin on the lymphocyte surface in mammals is generally associated with major serologically defined histocompatibility antigens but not with I-region-associated antigens.     

Epitope studies with anti-beta 2-glycoprotein I antibodies from autoantibody and immunized sources

This paper examines the methodology of anti-beta(2)-glycoprotein I (beta(2)-GPI) epitope determination and provides further epitope studies using human sera containing anti-beta(2)-GPI autoantibodies. Studies in this field may be misleading as the antigen coating density using mutant forms of beta(2)-GPI may be below the threshold required for monogamous divalent binding by low affinity anti-beta(2)-GPI autoantibodies, while being easily detected by high affinity anti-beta(2)-GPI from immunized animals. The antigen density threshold effect is found in anti-beta(2)-GPI autoantibodies from humans and from monoclonal anti-beta(2)-GPI derived from mice with models of autoimmune disease. Anti-beta(2)-GPI from an autoimmune mouse and from 18/21 human sera did not bind above background levels to a domain-I-deleted mutant. In addition, single point mutations in domain I result in dramatic changes in the binding of many human sera containing anti-beta(2)-GPI. These findings support a conclusion that domain I of beta(2)-GPI contains significant epitopes for the anti-beta(2)-GPI antibodies found in the antiphospholipid syndrome.     

Serum beta 2-microglobulin (beta 2m) and anti-beta 2m antibody in chronic hepatitis

Serum beta 2-microglobulin (beta 2m) concentration is increased in pathological processes associated with lymphocyte activation. beta 2m and anti-beta 2m autoantibody (anti-beta 2m) determinations were made in the sera of 41 patients with chronic hepatitis and cirrhosis, respectively, in 19 with systemic lupus erythematosus (SLE) as well as in 27 healthy controls. A pathologically high beta 2m value was found in one-third of inactive persistent hepatitis, and in more than two-thirds of active hepatitis and postnecrotic cirrhosis cases. In SLE it occurred in 16 out of 19 cases. The beta 2m level was elevated mainly in HBV-negative and circulating immune complex-positive hepatic patients. Anti-beta 2m antibody occurred in one-third of chronic hepatitis, but only in one out of 13 cases in cirrhosis and in 12 out of nineteen patients with SLE. The results suggest that beta 2m and anti-beta 2m as in SLE may be further laboratory indicators of immunological activity in inflammatory hepatic disease.     

2型登革病毒通过线粒体途径诱导EA.hy926细胞凋亡

 目的：探讨登革病毒诱导EA.hy926细胞(人脐静脉内皮细胞融合细胞株)相对活力的变化与线粒体膜电位(mitochondrial membrane potential,Δψm)改变及线粒体凋亡途径的关系。方法：用2型登革病毒(dengue virus type 2, DENV-2)感染EA.hy926细胞,MTT法检测感染前后EA.hy926细胞的相对活力,荧光显微镜和流式细胞术分别观察感染前后JC-1在EA.hy926细胞线粒体内的聚集情况以检测Δψm的改变,通过比色法检测caspase-9的活性变化。结果：DENV-2感染EA.hy926细胞24 h、36 h及48 h后,细胞活性受到显著抑制,550 nm处的A值均低于未感染组,差异有统计学意义(P<0.05,P<0.01);JC-1染色显示,感染后各时点,代表正常线粒体的红色荧光均较未感染组减弱,而代表Δψm下降的绿色荧光较未感染组逐渐增强。流式细胞术检测Δψm平均荧光密度比未感染组减低,差异有统计学意义。DENV-2 感染后早期即可出现caspase-9活性的上升,与未感染组相比,各时点的活性差异均有统计学意义(P<0.01)。结论：DENV-2感染EA.hy926细胞后可诱发Δψm下降,增强caspase-9活性,进而启动线粒体的凋亡途径。     

High prevalence of anti-beta2 glycoprotein I antibodies in patients with ischemic heart disease.

Ischemic cardiac manifestations have been reported in a various percentage of patients with anti-phospholipid antibodies. As concerns the relationship between anti-beta2 glycoprotein I antibodies (anti-beta2-GPI) and ischemic heart disease (IHD), it was investigated in only one coronary primary prevention study. We investigated the prevalence of anti-beta2-GPI in a well characterized group of patients with different clinical manifestation of IHD. Sera from 37 patients (mean age 62.7 +/- 9.9) with IHD (20 with unstable angina-UA and 17 with effort angina-EA) and from 40 healthy subjects, matched for age and sex, were tested for the presence of IgG and IgM anti-beta2-GPI using an ELISA technique. Eleven/37 patients (29.7%) resulted positive for anti-beta2-GPI. A positivity for IgG anti-beta2-GPI was found in 10 patients, 1 patient was positive for IgM and 1 for both isotypes. The prevalence of anti-beta2-GPI in the control group resulted significantly lower (2.5%; p < 0.005) than in patients with IHD. Positivity for anti-beta2-GPI was found in 9/20 (45%) patients with UA and only in 2/17 patients (11.8%) with EA (p = 0.0365). IgG anti-beta2-GPI levels (median 7.7U/ml, range 2.6-24.1) were significantly higher in patients with UA compared to patients with EA (median 4.6 U/ml, range 2.3-11.5; p = 0.02) and controls (median 3.15 U/ml, range 2.3-9.0; p < 0.0001); also IgM levels resulted higher in patients with unstable angina. A positivity for anti-beta2-GPI was observed in 4/13 patients (30.8%) with a previous myocardial infarction (MI) and in 7/24 (29.2%) patients without a previous MI. Our findings suggest that anti-beta2-GPI could represent an expression of the T-cell activation detectable in patients with unstable angina. The lack of a significant difference in the prevalence of these antibodies in patients with or without a previous MI suggests that anti-beta2-GPI are not induced by tissue necrosis.     

Inhibitory Effects of Anti-HLA-A, B, C Heavy Chain and Anti-β2 Microglobulin Monoclonal Antibodies on Alloantigen and Microbial Antigen-Induced Immune Responses in Vitro

The role of HLA class I subunits in class II-restricted immune responses was investigated by means of a panel of monoclonal antibodies (MoAb) recognizing HLA-A,B,C heavy chain and different beta 2 microglobulin (beta 2m) epitopes. MoAb against either class I subunit strongly inhibited mixed lymphocyte cultures, generation of cytotoxic T lymphocyte cultures, generation of cytotoxic T lymphocytes or natural killer-like activity, and lymphoproliferation in response to soluble or particulate microbial antigens derived from Candida albicans. In general, anti-beta 2m MoAb were more efficient inhibitors than anti-HLA-A,B,C heavy chain MoAb. The inhibitory effects were specific, in that the parental myeloma ascitic fluid or a low-affinity MoAb against beta 2m, or MoAb directed against non-HLA surface structures did not affect any of the immune responses studied. The MoAb-induced inhibition could not be attributed to nonspecific toxic effects, since PHA-induced blastogenesis and IL-2-dependent proliferation of mixed lymphocyte culture (MLC) blasts were not inhibited. Furthermore, exogenous IL-2 did not reverse the block of MLC and microbial antigen-induced proliferative responses by MoAb. Taken together, these data suggest an involvement of both subunits of class I antigens in class II-restricted immune responses.     

Effect of anti-beta2glycoprotein I Lupus Anticoagulants on fibrin polymerization and fibrinolysis

Anti-beta2-Glycoprotein I (beta2GPI) autoantibodies are the prominent laboratory feature of Hughes syndrome. By prolonging some coagulation tests in the presence of exogenous phospholipids (PL), they behave as classical Lupus Anticoagulants (LA). We investigated the effect of 3 affinity-purified anti-beta2GPI IgG preparations from patients with Hughes syndrome on fibrin polymerization and fibrinolysis of normal plasma, measured by comparing the optical densities of assay mixtures in the presence of the autoantibodies or normal IgG. The presence of anti-beta2GPI IgG in diluted Russell Viper Venom Time (dRVVT) assays, carried out using a PL dilution of 1:8 or 1:64, resulted in a delay in the onset of polymerization by 30-40 and 60-70s, respectively. Fibrin polymerization was complete after 250s for both anti-beta2GPI IgG and normal IgG. The inhibitory effect of the anti-beta2GPI antibodies was not observed in the presence of excess PL, as expected for LA. Anti-beta2GPI IgG increased the plateau level of polymerization when dRVVT was performed in the presence of 1.5 nM recombinant tissue plasminogen activator, but did not impair the fibrinolytic process, which was almost complete after 250 min. The autoantibodies did not delay the onset of fibrin polymerization in tests carried out using recombinant tissue factor. On the contrary, the autoantibodies enhanced polymerization in prothrombin time assays, and accelerated it in tissue thromboplastin inhibition tests, with no effect on fibrinolysis. These data provide evidence that anti-beta2GPI LA may act as either anticoagulants or procoagulants in different in vitro coagulation tests.     

骨髓造血干/祖细胞自身抗体与免疫相关性全血细胞减少

背景：免疫相关性全血细胞减少涉及多个临床学科,近年来受到高度重视。骨髓造血早期细胞自身抗体及免疫调节功能可能是其症结所在。 目的：分析骨髓造血早期(干/祖)细胞自身抗体在免疫相关性全血细胞减少症发病机制中的作用及其进展。 方法：由作者电子检索1992年3月至2012年3月PubMed、万方医学网以及CHKD数据库中关于骨髓造血细胞相关抗体的文章,中文检索词为“免疫相关性全血细胞减少症、骨髓造血细胞、自身抗体、辅助性T细胞17”,英文检索词为“Immunorelated pancytopenia,Bone marrow hematopoietic cells,Autoantibody,Th17 cells”。排除重复性研究共保留30篇进行总结分析。 结果与结论：骨髓造血细胞自身抗体是由于T淋巴细胞调控失衡,导致B淋巴细胞及其亚群数量和功能异常而产生的一类抗体。它破坏或抑制骨髓早期造血细胞成熟分化,最终引起外周血细胞减少而致病。T辅助细胞17细胞数量增多、功能亢进可能与免疫相关性全血细胞减少症患者自身抗体产生呈一定相关性,是免疫相关性全血细胞减少症发病的重要因素。     

交感肾上腺素能神经在针刺治疗关节炎中的作用

以诱发荧光法研究了６－ＯＨＤＡ引起佐剂性关节炎大鼠的自然恢复减慢和电计可以促进的机理．证明：６－ＯＨＤＡ可破坏肾上腺素能神经末梢，在佐剂性关节炎组１周后虽有再生，但荧光膨体仍显著减少或不见，到８周还未恢复到正常水平．电针加６－ＯＨＤＡ的佐剂关节炎组与非电针组比较，荧光膨体明显增多增亮，说明电针有促进交感肾上腺素能神经末稍再生的作用．这是电针促进６－ＯＨＤＡ注入后佐剂性关节炎恢复的原因之一．