Anti-beta 2 glycoprotein I antibodies in systemic lupus erythematosus: a marker of thrombosis associated with a circulating anticoagulant]

An ELISA technique for the detection of anti-beta 2 glycoprotein I (beta 2gp I) antibodies was developed. Among 47 systemic lupus erythematosus patients, 17 had anti-beta 2gp I antibodies. These antibodies were statistically associated with anticardiolipin antibodies, lupus anticoagulant and thrombosis. Out of 18 patients with anticardiolipin antibodies without anti-beta 2gp I antibodies or lupus anticoagulant, only one had thrombosis (due to nephrotic syndrome). Therefore the presence of anti-beta 2gp I antibodies is a new immunologic marker of lupus patients with thrombosis. In addition, we propose that anti-beta 2gp I antibodies may be directly responsible for lupus anticoagulant activity.     

Immunoenzyme method in the diagnosis of human brucellosis

The optimum conditions for the determination of specific antibodies in the sera of brucellosis patients by means of enzyme immunoassay (EIA) have been selected. The comparative study of the specificity and sensitivity of EIA and other serological tests has demonstrated that EIA has high diagnostic effectiveness in the diagnosis of acute and chronic brucellosis. The presence of direct correlation between the results of EIA and Coombs' test is observed, which is indicative of the capacity of EIA for detecting both complete and incomplete specific antibodies. It should be pointed out that in all cases the titer of specific antibodies in EIA has been found to be 5-16 times higher than in Coombs' test, the passive hemagglutination test, and agglutination test.     

A rapid screening test for HIV-1 antibodies: application to biological studies on human tissue

Human umbilical cord vessels are commonly used as a source of human vascular tissue for physiological studies and as a source of endothelial and smooth muscle cells. Blood samples from 236 umbilical cords were tested for the presence of HIV-1 antibodies to access the prevalence of HIV-1 infection and to evaluate possible methods for screening umbilical cords. Ten of the 236 samples were HIV-1 antibody positive by ELISA whereas 3 were positive by Western blot and a new method, the Quick-Western blot. Two of the 3 positive samples contained antibody bands against gp160, gp120, gp41, and p24 HIV-1 proteins, and one sample had antibodies against only gp160, gp120 and gp41. The Quick-Western blot required only 45 minutes for the analysis while the ELISA and Western blot took 3 hours and 18 hours, respectively. These data indicate that HIV-1 infection in mothers may present a hazard to researchers using human umbilical cords as a source of vascular tissue. The Quick-Western blot method is a simple, portable, rapid and accurate method that may be used to screen blood. The short analysis time of the Quick-Western blot allows the identification of infected blood before the tissue deteriorates as a source of cells or vascular tissue for experimental studies.     

Human leukocytes kill varicella-zoster virus-infected fibroblasts in the presence of murine monoclonal antibodies to virus-specific glycoproteins.

Seven murine monoclonal antibodies reacting with major glycoproteins of varicella-zoster virus were tested for functional activity in assays for antibody-dependent cellular cytotoxicity (ADCC) and antibody-plus-complement-mediated lysis. Human peripheral blood mononuclear cells killed varicella-zoster virus-infected fibroblasts in the presence of three of four monoclonal antibodies directed against gp98/62 and a single monoclonal antibody directed against gp118. Neither of two monoclonal antibodies directed against gp66 was able to mediate ADCC. In 18-h assays, adherent effector cells were more active than nonadherent effector cells in mediating ADCC. Adherent cells treated with anti-Leu-11b and complement retained their cytotoxic activity, suggesting that monocytes are responsible for most of the adherent-cell-mediated cytotoxicity. Both immunoglobulin G1 and G2a murine monoclonal antibodies were able to participate in ADCC. Of the two immunoglobulin G2a monoclonal antibodies tested, both of which reacted with gp98/62, only one mediated lysis in the presence of complement. These results indicate that some murine monoclonal antibodies against major glycoproteins of varicella-zoster virus have functional activity in cytotoxicity assays.     

Chicken thrombocytes in culture: lymphocyte-conditioned medium delays apoptosis.

Chicken thrombocytes are nucleated cells, analogs to mammalian platelets. These cells are involved in hemostasis, phagocytosis and secretion of specific products. Most of the properties of avian thrombocytes have been established in experiments that employed recently isolated blood cells. Attempts to cultivate these cells for a long period of time under optimal culture conditions for peripheral blood cells were unsuccessful; thrombocytes died after 24 h of cultivation unlike macrophages cocultured with them. Here we investigate the reasons and type of thrombocyte death in culture. Thrombocytes were separated from peripheral blood of roosters and cultured for 48 h. The influence of different culture conditions on thrombocyte viability was studied. Cells were cultured as adherent cell monolayers or under agitation (preventing adherence), in the presence or lack of lymphocytes or their soluble factors, and various concentrations of fetal bovine serum. After 24 h in standard culture thrombocytes displayed cytoplasm and chromatin condensation, DNA cleaved into oligonucleosomal fragments and unaltered mitochondria. These results strongly suggest that thrombocytes suffer an apoptotic cell death in culture. Apoptosis could be delayed by culturing thrombocytes in the presence of lymphocytes or their soluble factors.     

Extracellular matrix of lymphoid tissues in the chick

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.     

Experimental erythrocyte autoantibodies. V. Induction and suppression of red blood cell autoantibodies in mice injected with rat bromelain-treated red blood cells

Mice injected with rat red blood cells (RBC), or rat bromelain-treated (brom) RBC, produce RBC autoantibodies and suppressor cells that specifically inhibit the autoimmune response without inhibiting the net production of antibodies against rat RBC. It has been investigated whether suppressor cells induced by injections of rat RBC are effective in preventing autoantibody production induced by rat brom RBC and vice versa. Autoantibodies were induced in C3H mice by weekly ip injections, each 0.2 ml, of a 6% suspension of rat RBC or rat brom RBC. Autoantibody production was assayed using Coombs' test. Suppressor cells were present in the spleens of mice positive in Coombs' tests and were shown by intravenous injections of 40 X 10(6) viable cells per mouse into untreated syngeneic mice 18 hr before the first injection of rat RBC or rat brom RBC. Autoantibodies eluted from mice positive in Coombs' tests after injections of rat RBC or brom RBC were absorbed by either type of rat RBC but not by RBC from sheep. This suggests that rat RBC and rat brom RBC display antigens that are similar, if not identical, to autoantigens on the mouse RBC. Spleen cells from mice injected with rat RBC suppressed autoantibodies induced by both rat RBC and rat brom RBC. In contrast, spleen cells from mice injected with rat brom RBC suppressed autoantibodies induced by rat brom RBC but not those induced by unmodified rat RBC. This differential suppression may be due to the removal from rat RBC, by bromelain, of a suppressor site and/or autoantigens of some specificities. Thus rat brom RBC may not induce the total range of specificities of autoantibodies, and of suppressor cells, induced by rat RBC.     

Production of interleukin 2 (IL 2) by salivary gland lymphocytes in Sj?gren's syndrome. Detection of reactive cells by using antibody directed to synthetic peptides of IL 2

Because abnormalities in interleukin 2 (IL 2) production have been reported in the blood of patients with certain autoimmune diseases, we have examined the lymphocytes from patients with Sj?gren's syndrome (SS) in which it is possible to obtain simultaneous samples of inflammatory site (i.e., salivary gland) lymphocytes and blood lymphocytes. We found that IL 2 production by peripheral blood lymphocytes (PBL) after mitogen stimulation was markedly diminished (4 +/- 2 U/ml) in 8/32 SS patients. However, salivary gland lymphocytes (SGL) from six out of six SS patients (including three patients with low IL 2 production by their PBL) had a high level of IL 2 production (97 +/- 32 U/ml), suggesting that IL 2 production by inflammatory site lymphocytes may differ from blood lymphocytes in the same patients. Low IL 2 production by a patient's PBL was not correlated with the patient's age, duration of disease, immunoglobulin level, or presence of antinuclear antibodies. Low IL 2 production was associated with a decreased ratio of Leu-3a/Leu-2a positive cells (p less than 0.05) and with an increased proportion of "activated" T cells expressing HLA-DR and gp140 (p less than 0.05). To determine the proportion of PBL and SGL containing cytoplasmic IL 2-like material, we used affinity-purified rabbit antibodies prepared against chemically synthesized peptides of human IL 2. Before mitogen stimulation, PBL were not stained by these antibodies (less than 1% reactive cells), whereas SGL T cells eluted from the salivary gland of SS patients contained a small (3.4% +/- 1.8) proportion of reactive cells. A similar proportion (2.4% +/- 1.2) of reactive cells was noted when frozen tissue sections of salivary gland biopsies were examined with these antibodies. After mitogen stimulation, 35% +/- 17 of PBL and 56% +/- 18 of SS SGL were specifically stained with anti-IL 2 peptide antibodies. In summary, these studies demonstrate a significant difference in IL 2 production between PBL and SGL of the same patients. Furthermore, antibodies against IL 2 peptides provide a powerful tool for detection of T cells producing IL 2 in vitro and in situ, and for understanding the role of this lymphokine in pathogenesis.     

Cell types and interactions in the spleen of the dogfish Scyliorhinus canicula L.: an electron microscopic study

The cell composition of the spleen of the dogfish, Scyliorhinus canicula L. was investigated by electron microscopy. It comprised areas of red and white blood cells. The red cells were observed in various stages of development and the spleen is probably the main erythropoietic organ, it is likely that thrombocytes are also produced in the spleen. The presence of plasma cells, and the appearance of rosette-like contacts between lymphocytes and phagocytic cells, probably macrophages, indicated that immunological processes were taking place. Destruction of effete blood cells, primarily erythrocytes, was indicated by the presence of macrophages containing residues of ingested blood cells.     

The murine gammaherpesvirus 68 ORF27 gene product contributes to intercellular viral spread

Herpesviruses remain predominantly cell associated within their hosts, implying that they spread between cells by a mechanism distinct from free virion release. We previously identified the efficient release of murine gammaherpesvirus 68 (MHV-68) virions as a function of the viral gp150 protein. Here we show that the MHV-68 ORF27 gene product, gp48, contributes to the direct spread of viruses from lytically infected to uninfected cells. Monoclonal antibodies to gp48 identified it on infected cell surfaces and in virions. gp48-deficient viruses showed no obvious deficit in virion cell binding, single-cycle replication, or virion release but had reduced lytic propagation between cells. After intranasal infection of mice, ORF27-deficient viruses were impaired predominantly in lytic replication in the lungs. There was a small deficit in latency establishment, but long-term latency appeared normal. Since ORF27 has homologs in both Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, it is likely part of a conserved mechanism employed by gammaherpesviruses to disseminate lytically in their hosts.     

Postoperative changes of the thrombocyte number and function]

In 30 patients the number of thrombocytes was determined 24-48 hours after surgical interventions and compared with the normal range. The function of thrombocytes was determined by the method of pressure registration in combined thrombocyte-aggregation-adhesion (DKTA method). In spite of the occurring thrombocytosis there is a tendency towards a decrease of response in most cases and thus of haemostatic function of blood platelets. The influence of the thrombocyte function caused by fibrinolytic split products or by a change of prostaglandine metabolism is discussed.     

Composition of main haematopoietic compartments in normal and bled channel catfish

Differential cell counts showed that the head and trunk kidney of control and bled channel catfish Ictalurus punctatus had myeloid characteristics. They contained lymphoid and granuloid cells, thrombocytes, erythroid and agranular cells in decreasing order of abundance (%). Among the blast and precursor cells, the most numerous erythroid ones were followed by granuloid, lymphoid and agranular ones. The main changes after blood withdrawal were the decrease of thrombocytes and the increase of precursor cells in both kidney parts. In the group examined 7 days after bleeding the head kidney had a higher percentage of erythroid cells and lymphocyte precursors than the trunk kidney while the latter had more granuloid cells and their precursors. Basophils were present ( c . 1%) in both regions of the kidney of all groups. The spleen was predominantly a lymphatic organ. It contained c . 80% lymphoid cells, a higher incidence of granulated lymphocytes than in kidneys, 15% thrombocytes and 1.4% agranular cells. Blood withdrawal caused an increase of thrombocytes, a decrease of lymphoid cells and an increase of erythroid precursors in the spleen. The last probably stemmed from the circulation. While haematocrit values failed to indicate the anaemic state in the bled groups, the differential red blood cell count showed dramatic differences between the control and bled groups as well as between the two groups in different stages of recuperation from the blood loss.     

Conformational and structural features of HIV-1 gp120 underlying the dual receptor antagonism by cross-reactive neutralizing antibody m18

We investigated the interaction between cross-reactive HIV-1 neutralizing human monoclonal antibody m18 and HIV-1YU-2 gp120 in an effort to understand how this antibody inhibits the entry of virus into cells. m18 binds to gp120 with high affinity (KD≈5 nM) as measured by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR analysis further showed that m18 inhibits interactions of gp120 with both soluble CD4 and CD4-induced antibodies that have epitopes overlapping the coreceptor binding site. This dual receptor site antagonism, which occurs with equal potency for both inhibition effects, argues that m18 is not functioning as a mimic of CD4, in spite of the presence of a putative CD4-like loop formed by HCDR3 in the antibody. Consistent with this view, m18 was found to interact with gp120 in the presence of saturating concentrations of a CD4-mimicking small molecule gp120 inhibitor, suggesting that m18 does not require unoccupied CD4 Phe43 binding cavity residues of gp120. Thermodynamic analysis of the m18-gp120 interaction suggests that m18 stabilizes a conformation of gp120 that is unique from and less structured than the CD4-stabilized conformation. Conformational mutants of gp120 were studied for their impact on m18 interaction. Mutations known to disrupt the coreceptor binding region and to lead to complete suppression of 17b binding had minimal effects on m18 binding. This argues that energetically important epitopes for m18 binding lie outside the disrupted bridging sheet region used for 17b and coreceptor binding. In contrast, mutations in the CD4 region strongly affected m18 binding. Overall, the results obtained in this work argue that m18, rather than mimicking CD4 directly, suppresses both receptor binding site functions of HIV-1 gp120 by stabilizing a nonproductive conformation of the envelope protein. These results can be related to prior findings about the importance of conformational entrapment as a common mode of action for neutralizing CD4bs antibodies, with differences mainly in epitope utilization and the extent of gp120 structuring.     

Reticulocytopenia and "absence" of red cell autoantibodies in immune haemolytic anaemia.

A raised reticulocyte count is common in patients with immune or autoimmune haemolytic anaemia, and the result of the direct antiglobulin test (DAGT) is usually positive because of IgG or IgG and complement components on the red cells. We report on three patients who had low reticulocyte counts when they were most anaemic, and in whom no red cell autoantibodies could be detected by the DAGT. We postulate that reticulocytes may be selectively destroyed if antibodies are directed against antigenic sites on these young red cells, thus giving rise to a population of cells whose antigenic sites are poorly expressed. This theory might explain the low reticulocyte counts and the "absence" of antibodies (as tested by the DAGT) in such patients. Radioisotopic studies with 51Cr and 59Fe may provide useful information on the rate and sites of red cell destruction.     

Development of the anti-gp120 antibody response during seroconversion to human immunodeficiency virus type 1.

We have studied the development of the antibody response to the surface glycoprotein gp120 of human immunodeficiency virus type 1 in three individuals who presented with primary human immunodeficiency virus type 1 infection syndrome. Serum anti-gp120 antibodies were first detected 4 to 23 days after presentation, after p24 antigen and infectious-virus titers in the peripheral blood had declined manyfold from their highest values. Whether anti-gp120 antibodies present at undetectable levels are involved in clearance of viremia remains unresolved. Among the earliest detectable anti-gp120 antibodies were those to conformationally sensitive epitopes; these antibodies were able to block the binding of gp120 monomers to soluble CD4 or to a human monoclonal antibody to a discontinuous epitope overlapping the CD4-binding site. Some of these antibodies were type specific to a degree, in that they were more effective at blocking ligand binding to autologous gp120 than to heterologous gp120. However, the appearance of these antibodies did not correlate with that of antibodies able to neutralize the autologous virus in vitro by a peripheral blood mononuclear cell-based assay. Antibodies to the V3 loop were detected at about the same time as, or slightly later than, those to the CD4-binding site. There was a weak correlation between the presence of antibodies to the V3 loop and autologous virus-neutralizing activity in two of three individuals studied. However, serum from the third individual contained V3 antibodies but lacked the ability to neutralize the autologous virus in vitro, even immediately after seroconversion. Thus, no simple, universal correlate of autologous virus-neutralizing activity in a peripheral blood mononuclear cell-based assay is apparent from in vitro assays that rely on detecting antibody interactions with monomeric gp120 or fragments thereof.     

Prevalence and specificity of some rare red cell pan-reactive alloantibodies against high frequency red cell antigens among hospitalized Saudi patients

Some patients'' sera react with all available donors'' red cells and a compatible donor is difficult or impossible to be found. These may either be due to a complex mixture of antibodies or the presence of alloantibodies against high-frequency antigens (HFAs). The aim of this study is to identify the prevalence and characteristics of antibodies to HFAs in Saudi Arabian patients. A total of 23 out of 172 000 patients who received blood transfusions had rare alloantibodies to HFAs at an incidence of 0.013%. Twenty-three patients suspected with pan-reactive alloantibodies against HFAs had their red cells tested using antisera to HFAs, while their plasma was tested against a selected panel of red blood cells with rare phenotypes. Anti-Ge2 antibody was found in the highest number of patients (56.5%), whereas anti-U, anti JK3, anti H, anti-RH 29, anti-hr^s, anti-Kn^a, anti-Ch, anti-Rg, anti-Yt^a, and anti-Cr^a antibodies were found in the remaining patients (43.5%). This study suggests that although antibodies such as anti-Ge2, anti- Kn^a, anti-Ch, anti-Rg, anti-Yt^a , and anti-Cr^a are not clinically significant, they cause a delay in the provision of compatible blood. Whereas, anti-U, anti JK3, anti H, anti-RH29 and anti-hr^s are clinically significant antibodies. An understanding of antibody characteristics to HFAs and the widespread use of the extended red cell phenotype and antibody identification panel will both be helpful for the diagnosis of these HFAs.     

The Breddin test in various forms of idiopathic thrombocytopenic purpura]

In 100 healthy persons and 38 patients with idiopathic thrombocytopenic purpura (ITP) the morphology of thrombocytes was examined according to BREDDIN. The survival time of the marked thrombocytes and their place of sequestration was determined in the patients and in 17 voluntary persons. A correlation was found to exist between the morphology of thrombocytes and those four forms of ITP which could be limited by means of radiometric methods. Moreover, a certain degree of dependence was found between the thrombocyte sequestration place and the presence of antibodies.     

Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears.

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a variety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefore, phenotypic characterization of disseminated carcinoma cells for expression of protease activators might define the invasive potential of the cells. We present an immunocytochemically enhanced staining method that allows phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytokeratin and of u-PAR intracellularly and on the surface of carcinoma cells. This novel approach can be used for detection and phenotyping of carcinoma cells in blood smears for u-PAR or, presumably, for any other heterogeneously expressed antigen on the surface of the detected cells.     

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.     

共表达HIV-1中国流行株gp120与IL-18重组鸡痘病毒的构建及其免疫原性观察

为筛选我国的HIV-1疫苗候选株,以鸡痘病毒282E4中国疫苗株为载体,构建了共表达中国流行株HIV-1外膜蛋白gp120和IL-18的重组鸡痘病毒,并将该重组鸡痘病毒免疫BALB/c小鼠,检测免疫小鼠脾特异性CTL杀伤活性和血清抗体水平。结果显示,HIV_1外膜蛋白gp120和IL-18不但可在重组鸡痘病毒感染的鸡胚成纤维细胞中表达,而且可在重组鸡痘病毒感染的哺乳动物细胞中表达。重组病毒具有良好的免疫原性,可诱导小鼠产生特异性抗体和脾特异性CTL反应,且IL_18发挥了免疫佐剂的作用。本研究结果为制备安全、有效的HIV-1基因工程活载体疫苗奠定了基础。