Localization of anti-mitochondrial antibody in experimental canine myocardial infarcts.

Alterations in cell and subcellular membrane integrity occur during evolving ischemic myocardial injury. We tested the hypothesis that an antibody against human liver mitochondria [anti-mitochondrial antibody developing in a patient with primary biliary cirrhosis] could identify altered cell membrane integrity in experimental canine myocardial infarcts. The proximal left anterior descending coronary arteries of 12 dogs were ligated and 1 hr later 131I-labeled F(ab')2 fragments from either a control human IgG (6 dogs) or anti-mitochondrial IgG (6 dogs) were injected. The 131I-labeled F(ab')2 anti-mitochondrial fragments concentrated maximally in the central infarct subendocardium [infarct-to-normal ratio of 9.2 +/- 3.5 (mean +/- SD) vs. 4.6 +/- 3.3 for control F(ab')2 IgG, P < 0.05]. There was also 1 1/2- to 2-fold greater anti-mitochondrial antibody F(ab')2 accumulation in the central infarct epicardium and the peripheral infarct subendocardium and subepicardium. Thus, an anti-mitochondrial antibody obtained from a patient with primary biliary cirrhosis concentrates in irreversibly damaged myocardium after experimental canine myocardial infarction. Presumably this occurs because of altered cell membrane integrity, which allows exposure of mitochondria to the anti-mitochondrial antibody. The F(ab')2 fragments of anti-mitochondrial antibodies labeled with suitable radionuclides should allow noninvasive scintigraphic detection of experimental acute myocardial infarcts.     

Effects of immunoglobulin on murine myocarditis caused by influenza A virus: experimental study

OBJECTIVES: Influenza A viruses play the largest role in the worldwide epidemiology of infectious diseases. We examined the effects of intact type and F(ab')2 type of immunoglobulin preparations on murine influenza A virus myocarditis in mice. METHODS AND RESULTS: In vitro study showed that intact type and F(ab')2 type of immunoglobulin preparations had antiviral activities against many substrains of influenza A virus and other cardiotropic viruses, and that dose-dependent suppression of an influenza A virus (NWS type) was demonstrated by the treatment of both intact immunoglobulin and F(ab')2 fragments of immunoglobulin. The dose inhibiting 50% of plaques was same between intact type and F(ab')2 type (both 0.0002 mg/dl). Intact immunoglobulin, but not F(ab')2 fragments of immunoglobulin, suppressed serum macrophage inflammatory protein-2 (MIP-2) production in influenza A virus infected macrophages in vitro, which is a murine counterpart of interleukin-8. This suppression of MIP-2 production by intact immunoglobulin treatment was blocked by a specific Fc receptor (Fc gamma III/II receptor) antibody pretreatment. Intact immunoglobulin (1 g/kg/day) or F(ab')2 fragments of immunoglobulin (1 g/kg/day) were administered to the virus-inoculated A/J mice intraperitoneally daily, starting simultaneously with virus inoculation (Experiment I ) and 2 days after the virus inoculation (Experiment II ), until 10th days after virus inoculation. In Experiment I, survival was higher in treated [intact (100%, 20/20), and F(ab')2 (100%, 20/20)] than in control (25%, 5/20) mice; intact type and F(ab')2 type immunoglobulin administration completely suppressed the development of myocarditis. In Experiment II, survival rate was significantly higher (75%, 15/20) and myocarditis was less severe in intact immunoglobulin treated mice, but not in F(ab')2 fragment treated mice (60%, 12/20), than in untreated mice (35%, 7/20). Serum neutralizing antibody titers in treated mice were significantly higher compared with untreated mice in Experiments I and II. In addition, serum MIP-2 concentrations in intact immunoglobulin treated mice, but not in F(ab')2 fragments treated mice, were lower compared with untreated mice in Experiment II. Immunoglobulin therapy suppresses influenza A virus myocarditis by increasing neutralizing antibody titers and the suppression of myocardial virus activities. From the stand-point of suppression of MIP-2 concentrations, intact type is superior to F(ab')2 type. CONCLUSIONS: Immunoglobulin treatment may be promising for the prevention of influenza A virus myocarditis.     

Further evidence for cross-linking as a protective factor in experimental cholera: properties of antibody fragments.

Enzymic fragments of IgG antibody were tested for their protective abilities in the infant mouse cholera model. F(ab')2 retained the full protective activity of the parent IgG molecule despite losses in complement fixation and opsonic functions. Fab' and Fab fragments also contained significant protective activity but at a level of only 10% of the intact IgG or F(ab')2. Self-recombinant univalent F(ab')2 also contained about 10% of the protective activity of the divalent F(ab')2 parent molecule. These results are interpreted as evidence that cross-linking is an important mechanism by which specific antibody protects mucosal surfaces against experimental infection with Vibrio cholerae.     

The specificity of antibodies to the F(ab')2 fragment of human IgG

The specificity of IgG anti-F(ab')2 antibodies was examined by unfractionated sera of patients with rheumatoid arthritis and also with affinity-purified antibody preparations. Examination of the sera by an enzyme-linked immunosorbent assay, using pooled human F(ab')2 fragments absorbed to microtiter plates, revealed that IgG anti-F(ab')2 antibodies cross-react with human and rabbit IgG and rabbit F(ab')2. IgG anti-F(ab')2 antibodies were purified by affinity chromatography and, when tested by a fluid-phase inhibition enzyme-linked immunosorbent assay, were found to be of 2 types. One fraction, similar to pepsin agglutinator, reacted with human F(ab')2 fragment alone. The other fraction was cross-reactive with human IgG and yet failed to react with idiotopes on Fab or epitopes on Fc fragments. The IgG anti-F(ab')2 antibodies we purified had no reactivity toward a human immune complex prepared from tetanus toxoid and antitoxoid.     

Recovery from anti-VIII:C (antihemophilic factor) autoimmune disease is dependent on generation of antiidiotypes against anti-VIII:C autoantibodies.

Plasma samples obtained from a patient 6 wk, 6 months, and 4 yr after recovery from anti-VIII:C (anti-hemophilic factor, where VIII:C = factor VIII procoagulant activity) autoimmune disease were found to contain antibodies that inhibited anti-VIII:C activity in the patient's prerecovery plasma and in the plasma of two other patients with anti-VIII:C autoantibodies. F(ab')2 fragments from postrecovery IgG suppressed anti-VIII:C activity in F(ab')2 fragments from prerecovery IgG within a narrow range of molar ratios. Anti-VIII:C activity in F(ab')2 autoantibodies was also inhibited by F(ab')2 fragments from polyspecific therapeutic immunoglobulins prepared from a large pool of normal donors (IVIg). IgG from prerecovery plasma bound to F(ab')2 from postrecovery IgG and to F(ab')2 from IVIg, as assessed by ELISA. Affinity chromatography experiments demonstrated that F(ab')2 from postrecovery IgG preferentially bound anti-VIII:C antibodies among F(ab')2 fragments from prerecovery plasma containing anti-VIII:C autoantibodies. F(ab')2 from prerecovery plasma bound in higher amounts to postrecovery F(ab')2 than to IVIg. Insolubilized F(ab')2 fragments from postrecovery plasma also bound F(ab')2 fragments prepared from the plasma of another patient with anti-VIII:C autoimmune disease, although in lesser amounts than the patient's own prerecovery anti-VIII:C F(ab')2 antibodies. These observations suggest that human anti-VIII:C autoantibodies share idiotypic determinants and that spontaneous recovery from anti-VIII:C autoimmune disease occurs through idiotypic suppression of autoantibodies. In patients who recover from autoimmune disease and in patients in whom autoantibodies have been suppressed by infusions of IVIg, antiidiotypic antibodies, possibly by providing internal images of the antigen, may have shifted the immune system toward the steady-state equilibrium that prevents autoimmunity in normal individuals.     

Regulation of autoimmune anti-platelet antibody-mediated adhesion of monocytes to platelet GPIIb/GPIIIa: effect of armed monocytes and the Mac-1 receptor

Platelet autoantigen-autoantibody-monocyte interaction was studied by utilization of a specific monoclonal antibody (MoAb) 10E5 to trap and immobilize the GPIIb-GPIIIa complex on microtiter plates. Peripheral blood mononuclear cells (PBMC) or purified monocytes formed distinct morphologic clusters after incubation with immobilized antigen for 18 hours at 37 degrees C. PBMC of 18 and 19 patients with autoimmune thrombocytopenic purpura (ATP) formed 48 +/- 6.8 (SEM) clusters/well compared with 7.4 +/- 1.0 for control subjects, P less than .001. The number of clusters per well correlated inversely and exponentially with platelet count, r = -.8, n = 21, indicating that the GPIIb-GPIIIa autoantigen is pathophysiologically relevant. Binding of ATP PBMC to immobilized GPIIb-GPIIIa could be inhibited by F(ab')2 fragments of immunoglobulin (Ig) G of ATP patients, indicating that monocyte IgG bound to autoantigen by its F(ab')2 domain. Optimal cluster formation could be obtained with normal monocytes if preincubated with ATP IgG but not with F(ab')2 fragments of ATP IgG, indicating that ATP IgG binds to monocytes by its Fc domain. Armed monocytes (ie, normal monocytes preincubated with ATP IgG) bound to immobilized autoantigen 5.8-fold greater than normal monocytes incubated with immobilized autoantigen opsonized with ATP IgG. Armed monocyte adhesion could be inhibited 81% from 18.9 +/- 1.6 to 3.6 +/- 0.5 clusters/well by prior fixation with 0.1% formalin, whereas fixation of IgG before arming of monocytes was not inhibitory. MoAb MM41, directed against the alpha m- chain of the Mac-1 adhesive protein receptor of monocytes, inhibited cluster formation by 79%. Thus, (1) armed monocyte interaction with autoantigen is considerably more effective than monocyte interaction with opsonized autoantigen; (2) armed monocyte interaction requires specific F(ab')2-antigen recognition; and (3) monocyte-autoantigen interaction requires a secondary nonimmunologic adhesive event.     

Serum platelet-reactive IgG of autoimmune thrombocytopenic purpura patients is not F(ab')2 mediated and a function of storage.

Serum platelet-reactive and glycoprotein (GP) IIb-GPIIIa-reactive IgG and F(ab')2 was examined in 39 patients with classic autoimmune thrombocytopenic purpura (ATP), two patients with anti-PLA1 antibody and 25 control subjects in an enzyme-linked immunosorbent assay. IgG was purified by diethyl aminoethyl chromatography and centrifuged at 100,000g before testing of the supernatant. Significant IgG binding (threefold to fourfold control IgG binding) was noted with 8 of 17 ATP patients' IgG, 2 anti-PLA1 IgGs, and 2 ATP patients with multiple platelet transfusions. However, F(ab')2 fragments of nine of nine positive ATP IgGs were nonreactive; F(ab')2 from the two anti-PLA1 and two multiply transfused ATP IgGs were as reactive as their intact IgG. Antiplatelet or anti-GPIIb-GPIIIa reactivity of ATP IgG could be adsorbed to fixed platelets or solid-phase GPIIb-GPIIIa and eluted with 0.1 mol/L glycine, pH 2.5. However, binding of IgG to GPIIb-GPIIIa could not be inhibited with F(ab')2 of ATP IgG or Fc fragments of control subjects. When platelet- or GPIIb-GPIIIa-reactive ATP IgG was applied to a Sephacryl 300 gel filtration column, no reactivity was noted in the 7S region, whereas anti-PLA1 localized to this region. Antiplatelet or anti-GPIIb-GPIIIa reactivity was noted in the void volume and accompanied by a high molecular weight protein region. An immunoblot of the void volume fraction with goat antihuman IgG (gamma chain) antibody showed high molecular weight bands greater than 250 Kd, which after reduction converted to a 55-Kd heavy-chain band. Fresh samples of ATP and control IgG processed within 1 to 2 days of blood withdrawal had no reactivity for GPIIb-GPIIIa. After storage at -20 degrees C for greater than 3 months, 5 of 19 ATP IgG became reactive, whereas 16 of 16 controls were nonreactive. Thus, platelet-reactive IgG of ATP sera appears to be caused by the development of IgG aggregates held together by disulfide bonds that develop on storage, and is not F(ab')2 mediated.     

Neutrophils do not bind to or phagocytize human immune complexes formed with influenza virus

Neutrophils appear to form the first line of defense against influenza virus, yet it is unclear how these leukocytes recognize influenza- infected cells. While demonstrating that neutrophils adhere specifically to the sialic acid-binding site on the hemagglutinin molecule (HA) on the surface of influenza-infected (WSN[H1N1]) epithelial cells and not to other viral or epithelial cell antigens, it was observed that human neutrophils do not recognize immune complexes formed with influenza virus. Intact antibodies (mouse monoclonal antibodies [MoAbs] IgG1 and IgG2b, human immune heat-inactivated serum [predominantly IgG1], and IgG purified from human immune serum) that block the sialic acid-binding site on HA significantly reduced (> 80%) neutrophil adherence to influenza-infected epithelial cells. Binding and phagocytosis of free influenza virions and neutrophil agglutination by influenza virus were completely prevented by these antibodies. Intact and F(ab')2 fragments of mouse MoAbs to other viral epitopes caused increased neutrophil adherence to infected cells. This binding was eliminated by F(ab'2) fragments of MoAbs against the sialic acid- binding site on HA, but not by saturating amounts of MoAbs, which block the neutrophil Fc receptors. Thus, it appears that human neutrophils show little ability to bind via their Fc receptors to the immune complexes formed with antibody and either influenza-infected epithelial cells or the free virion. These findings are in contrast to the general dogma, and are the first example of antibody opsonization reducing, rather than enhancing, neutrophil binding and phagocytosis of a pathogen.     

Lack of a requirement for the Fc region of IgG in restoring pneumococcal opsonization via the alternative complement pathway in sickle cell disease

Children with sickle cell disease have reduced serum opsonization of Streptococcus pneumoniae. Our previous studies have suggested that opsonization mediated by both the alternative and classic complement pathways is reduced because of a deficiency of IgG antibodies to pneumococcal capsular polysaccharide. This study compares the ability of purified IgG (fractionated from goat antiserum to pneumococcal capsular polysaccharide) and F(ab')2 fragments of the IgG preparation to restore alternative pathway-mediated opsonization of S. pneumoniae to sera from patients with sickle cell disease. Both the whole IgG preparation and F(ab')2 fragments of this preparation restored opsonization to normal levels and concomitantly increased alternative pathway-mediated deposition of C3 onto the pneumococci to a supranormal level. These results suggest that enhancement of opsonization is mediated by the F(ab')2 region of IgG antibody to capsular polysaccharide and is associated with an increase in complement deposition on the bacterial surface.     

Fc receptor-mediated inhibitory effect of immunoglobulin therapy on autoimmune giant cell myocarditis: concomitant suppression of the expression of dendritic cells

In the present study, the mechanisms and importance of the Fc portion of immunoglobulin in experimental giant cell myocarditis were examined. Giant cell myocarditis was induced in rats by immunization of porcine cardiac myosin. Human intact immunoglobulin (1 g. kg(-1). d(-1)) or F(ab')(2) fragments of human immunoglobulin (1 g. kg(-1). d(-1)) were administered intraperitoneally daily on days 1 to 21. Intact immunoglobulin administration significantly ameliorated myocarditis, but F(ab')(2) fragments did not. The ribonuclease protection assay revealed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed the mRNA expressions of inflammatory and proinflammatory cytokines. Immunohistochemical analysis showed that therapy with intact immunoglobulin, but not F(ab')(2) fragments, suppressed dendritic cell (DC) expression during both the early and the subsequent fulminant phases. Moreover, the early treatment of intact immunoglobulin until the 11th day or 14th day, when the expression of DCs was completely suppressed, ameliorated myocarditis. However, the late treatment of intact immunoglobulin beginning on day 15, when the expression of DCs had already been completed, failed to ameliorate the condition. An in vitro study showed that intact immunoglobulin, but not F(ab')(2) fragments, suppressed the lipopolysaccharide-induced interleukin-1beta production associated with the downregulation of CD32 antigen (Fcgamma receptor II) expression. Thus, intact immunoglobulin therapy markedly suppressed myocarditis as a result of Fc receptor-mediated anti-inflammatory action, and the suppression of the disease was associated with the suppression of DCs, ie, the suppression of the initial antigen-priming process in experimental giant cell myocarditis.     

IN-VITRO CONVERSION OF BLOCKING TYPE ANTI-TSH RECEPTOR ANTIBODY TO THE STIMULATING TYPE BY ANTI-HUMAN IgG ANTIBODIES

We examined the relationship between blocking type anti-TSH receptor antibody (TRAb) and stimulating type TRAb by trying to convert blocking type TRAb to the stimulatory type in vitro. Immunoglobulins (IgGs) purified from sera of six patients with primary hypothyroidism blocked bovine TSH (100 μU/ml)-induced cAMP production (58-3-82-1% inhibition) in cultured porcine thyroid cells. None of these IgGs showed stimulating activity even after their dilution. In the conversion experiment, thyroid cells were first incubated with these IgGs at 34°C for 30 min and then washed with incubation buffer. They were then incubated with various kinds of anti-human IgG antibodies first for 1 -5 h at 4°C and then for 18 h at 34°C, and the cAMP concentrations in the supernatants were measured. All six IgGs showed strong, dose-dependent thyroid-stimulating activity after addition of antibodies against human whole IgG, or Fab or Fc fragments of IgG. The Fab or F(ab')2 fragments of goat anti-human IgG antibody also had these converting activities, although less than whole IgG. Addition of normal IgG in the first incubation or anti-human IgG antibody alone had no thyroid-stimulating activity. Anti-human IgA or IgM antibodies did not have these converting activities. These results show that blocking type anti-TSH receptor antibodies can be converted to the stimulating type by anti-human IgG antibodies in vitro. The results suggest that the blocking and stimulating types bind to the same epitope(s) of TSH-receptor related antigens. The same anti-TSH receptor antibody may act as a stimulator or blocker by the influence of other factors, such as anti-idiotype antibody.     

Comparative effects of aspirin, a synthetic thrombin inhibitor and a monoclonal antiplatelet glycoprotein IIb/IIIa antibody on coronary artery reperfusion, reocclusion and bleeding with recombinant tissue-type plasminogen activator in a canine preparation

The comparative effects of intravenous aspirin, the synthetic thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (Argatroban) and F(ab')2 fragments of monoclonal antibody 7E3 against platelet glycoprotein IIb/IIIa (7E3-F[ab']2) on thrombolysis, reocclusion and bleeding associated with 0.45 mg/kg body weight bolus injections of recombinant tissue-type plasminogen activator (rt-PA) were studied in a canine coronary artery thrombosis model. Coronary patency was monitored for 2 h both by flow probe and by coronary angiography. Four groups were studied: Group I = pretreated with 17 mg/kg intravenous aspirin (n = 6), Group II = pretreated with 200 micrograms/kg per min intravenous Argatroban for 60 min (n = 5), Group III = pretreated with aspirin and Argatroban (n = 5) and Group IV = pretreated with 0.8 mg/kg intravenous 7E3-F(ab')2 (n = 5). In Group I, reflow occurred in four of six dogs, but did not persist; reflow was induced in Group II in four of five dogs, persisting in one; in Group III, reflow occurred in all five dogs, persisting in four; in Group IV reflow was achieved in four of five dogs, persisting in two. The frequency of persistent reflow in Group III was significantly higher than in the combined Groups I and II (p = 0.012), whereas the time to reflow was significantly shorter in the groups receiving Argatroban than in the aspirin group (median 25 versus 55 min, p = 0.04). There were no significant differences between Groups III and IV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adhesive protein expression on thrombin-stimulated platelets: time-dependent modulation of anti-fibrinogen, -fibronectin, and -von Willebrand factor antibody binding.

Platelets contain a pool of endogenous adhesive proteins that can be released and may bind to surface membrane receptors under appropriate conditions. Because the binding of exogenous fibrinogen to platelets was shown previously to be accompanied by a time-dependent decrease in fibrinogen accessibility to antibody and enzymes, studies were performed to evaluate changes in the expression of endogenous fibrinogen released from thrombin-stimulated platelets using monospecific polyclonal and monoclonal antibody F(ab')2 fragments. Parallel studies were performed to compare the expression of released fibronectin and von Willebrand factor (vWF). Binding of polyclonal antibody F(ab')2 fragments directed against individual adhesive proteins was inhibited by EDTA or the 10E5 monoclonal antibody, suggesting that fibrinogen, fibronectin, and vWF expression was mediated, in large part, by divalent cation-dependent interactions with the glycoprotein IIb-IIIa complex. Interestingly, when polyclonal antibody F(ab')2 fragments were added to platelet suspensions at discrete times after thrombin stimulation, antifibrinogen F(ab')2 binding decreased by 72% +/- 15% (mean +/- SD, n = 22) over a 60-minute time course, whereas antifibronectin and anti-vWF antibody F(ab')2 fragment binding changed minimally (6% +/- 23%, n = 22 and 3% +/- 26%, n = 14, respectively). Similar observations were made with monoclonal antibodies. Parallel experiments using 125I-labeled fibrinogen as a marker indicated that the observed decrease in antifibrinogen F(ab')2 binding was not accompanied by fibrinogen dissociation. Moreover, antibody accessibility to platelet-bound fibrinogen could be restored after Triton X-100 platelet lysis. The data suggest that fibrinogen, fibronectin, and vWF are not coordinately expressed on thrombin-stimulated platelets. Rather, fibrinogen expression appears transient compared with the expression of fibronectin and vWF. The ability of platelets to secrete and organize adhesive proteins on their surface is likely to have important implications for hemostasis and thrombosis.     

Interstitial dendritic cells of the heart harbor Trypanosoma cruzi antigens in experimentally infected dogs: importance for the pathogenesis of chagasic myocarditis

Heart sections from 16 mongrel dogs, two normal controls and 14 infected with Trypanosoma cruzi, were submitted to immunohistochemical staining with either rabbit anti-cow S100 Protein monoclonal antibody or rabbit anti-T. cruzi purified specific antibody, using the peroxidase technique to investigate the participation of the interstitial dendritic cells of the heart (IDCs) in myocarditis of Chagas disease. Trypanosoma cruzi antigens were revealed as granular and dense deposits in IDC membrane in the heart of infected dogs both during acute and chronic myocarditis, but not in normal controls. Anti-S100 Protein labeled the IDCs, both in normal and infected dogs and a significant increase in the numbers of IDCs occurred in the myocardium, proportionally to the intensity of the inflammatory infiltration. These findings suggest that IDCs, probably by presenting T. cruzi antigens to immune-competent cells, play an important role in the pathogenesis of Chagas disease.     

Identification of Fc and F(ab')2 IgG receptors on human platelets.

The human platelet receptors for normal, nonimmune IgG and its F(ab')2 and Fc fragments were studied by the use of a cleavable, bifunctional, photoactivable, 125I-labeled cross-linking agent. Derivatization of the ligands with N-[4-(p-azido-m-[125I]iodophenylazo) benzoyl]-3-aminopropyl-N'-oxysuccinimide ester (Denny-Jaffe reagent) reduced their binding to platelets by greater than or equal to 20%. Cleavage of the azo linkage of the Denny-Jaffe reagent, which splits the molecule so that its 125I-labeled portion becomes associated with the receptor half of the cross-linked ligand-receptor complex, was utilized to directly identify receptors for the various immunoglobulin ligands. Specificity of the binding reaction could be demonstrated by suppressing the iodination of the receptors with excess nonderivatized ligand. Two principal IgG-related receptors could be identified by high-resolution NaDodSO4/PAGE and subsequent analysis of the electrophoretically transferred peptides to nitrocellulose filters for localization of radioactivity and immunological characterization. Intact monomeric IgG and F(ab')2 fragments derived from it appeared to have the glycoprotein IIIa as the major receptor, whereas Fc fragments bound predominantly to a peptide of Mr approximately 200,000 (Mr, approximately 50,000 under rigorous reducing conditions).     

Preparation and purification of F(ab')(2) fragment from anti hepatoma mouse IgG(1) mAb

INTRODUCTION Since the advent of hybridoma technology[1], monoclonal antibodies have been widely used in basic studies and clinical application. F(ab')2 is a bivalent antibody fragment which is currently used for both diagnosis and treatment[2], and better than the original mAbs, because it does not retain complement-binding function due to lack of Fc regions and reduced interaction with non-specific proteins and the smaller molecular weight than the mAbs, furthermore, it can be digested by pepsin or papain and purified by size exclusion chromatography[4], ion-exchange chromatography[6] or hydrophobic interaction[7]. However, these methods are time-consuming, and cannot attain sufficient purity and recovery of F(ab')2 fragment. Development of efficient procedures for F(ab')2 preparation is an urgent necessity.     

Platelet activation by immobilized monoclonal antibody: evidence for a CD9 proximal signal.

Anti-CD9 monoclonal antibodies (MoAbs) are reported to activate human platelets through stimulation of the Fc gamma II receptor. We show here that nonstimulatory F(ab')2 fragments of the anti-CD9 MoAb 50H.19 induce dense-granule release and dose-dependent platelet aggregation when attached to polystyrene latex beads. Cross-linking F(ab')2 fragments of MoAb 50H.19 by F(ab')2 fragments of goat anti-mouse IgG does not result in platelet aggregation unless the second antibody is bound to latex beads, indicating that immobilization, and not cross-linking of the stimulus, is critical to the initiation of the CD9 signal. In contrast, F(ab')2 fragments of the second antibody readily induce the aggregation of platelets treated with the anti-Fc gamma II receptor MoAb IV.3. Immobilization of MoAb per se is insufficient to induce an activation signal because intact and F(ab')2 fragments of nonstimulatory MoAb directed to glycoprotein Ib and HLA class I do not become stimulatory when attached to beads. CD9-induced activation requires cytoskeletal rearrangement because it is inhibited by cytochalasin B. Aggregation is blocked by inhibitors of the thromboxane pathway, indicating that CD9 activates phospholipase C indirectly through prior activation of phospholipase A2.     

Effects of IgG and its F(ab')2 fragments of some patients with idiopathic thrombocytopenic purpura on platelet aggregation

OBJECTIVES: To make humanized monoclonal antibodies by phage surface display technology, we screened out the specific anti-platelet glycoproteins (GPs) IgG antibody from patients with chronic idiopathic thrombocytopenic purpura (ITP), which can inhibit platelet aggregation. METHODS: We studied plasmas from 68 patients with ITP for the presence of IgG antibodies specific for GPIIb/IIIa and/or GPIb/IX using modified monoclonal antibody immobilization of platelet antigen assays. The IgG antibody and its F(ab')(2) fragments of the positive plasmas which could inhibit platelet aggregation function were prepared and purified. Their immunoreactivity to platelet GPs and effects on platelet function were further analyzed. RESULTS: GPIIb/IIIa- and GPIb/IX-specific antibodies were found in 21 and 19 patients, respectively. Six of them had antibodies against both GP complexes. Among the 34 positive plasmas, four with positive anti-GPIIb/IIIa autoantibody showed significant inhibition of platelet aggregation induced by adenosine diphosphate (ADP), whereas one with GPIb/IX-specific antibody inhibited ristocetin-induced platelet aggregation. The purified IgG and its F(ab')(2) fragments from two patients not only retained the ability to bind to platelet GPs but also impaired the in vitro ADP-induced platelet aggregation. CONCLUSIONS: F(ab')(2) portion of the IgG is a functional fragment, which is responsible for the autoantibody interaction with platelet GPs in ITP, and some of them also affect platelet function, which can be used to develop completely humanized anti-GPIIb/IIIa small molecular phage antibody.     

IgG-mediated viral clearance in experimental infection with herpes simplex virus type 1: role for neutralization and Fc-dependent functions but not C' cytolysis and C5 chemotaxis

For determination of whether the Fc moiety is required for antibody effectiveness in models of herpes simplex virus type 1 (HSV-1) infection, the effects of immune IgG and F(ab')2 fragments were compared by using a passive transfer model of footpad infection. In the IgG- and the F(ab')2-treated groups illness developed in 2 (10%) of 20 and 6 (25%) of 24 mice, respectively, compared with 10 (63%) of 16 controls. IgG treatment markedly, and F(ab')2 treatment moderately, reduced footpad viral titer and viral spread to sciatic nerve and spinal cord. The marked viral clearance by IgG was not attributable to C'-dependent lysis because rapid viral clearance was observed in C5-deficient B10.D2/oSn mice. Viral latency as a consequence of acute infection occurred in 38 (63%) of 60 lumbosacral dorsal root ganglia in the control group, 5 (8%; P less than .001) of 60 in the IgG-treated group, and 26 (33%; P less than .01) of 78 in the F(ab')2-treated group.     

Gamma-globulins prepared from sera of multiparous women bind anti-HLA antibodies and inhibit an established in vivo human alloimmune response

It has previously been shown that sera from multiparous women have increased levels of anti-idiotypic antibodies specific for anti-HLA molecules. gamma-Globulins prepared from these sera may be superior to commercial preparations of intravenous gamma-globulin (IVIg) for inhibiting HLA alloimmunization. To test this, F(ab')2 fragments prepared from either commercial IVIg or from the sera of men or multiparous women were coupled to CNBr-Sepharose and tested for their ability to bind F(ab')2 fragments derived from polyspecific anti-HLA sera. As determined by flow cytometry, compared with columns coated with F(ab')2 derived from commercial IVIg or sera from men, columns coated with F(ab')2 prepared from the sera of multiparous women bound significantly more anti-HLA. In addition, intact IgG molecules prepared from the sera of multiparous women significantly neutralized the reactivity of the anti-HLA F(ab')2 fragments. To determine whether the intact IgG molecules or their corresponding F(ab')2 fragments could affect in vivo alloimmunity, they were tested for their ability to inhibit an established IgG human alloimmune response in humanized severe combined immunodeficient (SCID) mice. Compared with commercial IVIg, when intact IgG or F(ab')2 fragments derived from multiparous women were administered to SCID mice making human anti-HLA antibodies, a significant reduction in anti-HLA reactivity was observed. The findings suggest that IgG molecules prepared from the sera of multiparous women have increased anti-idiotypic reactivity against anti-HLA antibodies, which can significantly inhibit an established human IgG alloimmune response in an Fc-independent manner.