Binding of human C-reactive protein to bacteria.

The binding of C-reactive protein to a variety of species of bacteria with potential clinical significance was studied to assess the potential function of C-reactive protein in nonimmune defense against infection. Purified, radioiodinated human C-reactive protein bound to all Streptococcus pneumoniae tested and to some viridans streptococci, but not to group A or group B streptococci or to any of eight different gram-negative rods and cocci.     

Chemotaxis of human B lymphocytes to anti-IgD.

The resting population of small surface IgM+ and surface IgD+ B cells from the human tonsil can be preactivated by overnight culture in interleukin-4 (IL-4) to show locomotor responses to anti-IgM and anti-IgD at between 10 ng and 1 microgram/ml. Because this locomotion is activated through the antigen receptor and may simulate a response to antigen, we set out to establish whether this was a chemotactic response using a checkerboard filter assay with a range of concentrations and concentration gradients of anti-IgD. At high concentrations (100 ng/ml to 1 microgram/ml), a chemokinetic response, but no chemotaxis, to anti-IgD was seen. However, in concentration gradients set up at lower concentrations (0-50 ng/ml) a chemotactic response was demonstrable. During the period of culture in anti-IgD at 1 microgram/ml, a progressive loss of surface IgD from the cells was seen, but there was no loss at 10 ng/ml. This receptor loss from the cell surface may account for the lack of chemotactic effect of the anti-IgD at higher concentrations.     

Mitogenic activity of two Brucella fractions for murine B lymphocytes.

Two lipid A-free fractions extracted from Brucella melitensis (fractions PI and SF) were shown to behave as B-cell non-specific mitogens for murine lymphocytes: they stimulated the uptake of tritiated thymidine by normal unsensitized murine splenic lymphocytes, by spleen cells from nude mice and by T-cell depleted but not by T-cell enriched and B-cell-deprived splenic populations. Since depletion of adherent cells leads to a two- to three-fold depression of PI- or SF-induced mitogenic responses these two fractions were shown to require accessory adherent cell participation for an optimal mitogenicity. Moreover they behaved as polyclonal activators for murine spleen cells. These results are discussed in terms of a possible classification of PI and SF amongst other B-cell mitogens and of the respective role of the peptidoglycan and lipoprotein moieties in B-cell activation by Brucella fractions.     

Binding of mitogenic plant lectins to human lymphocytes. Flow cytometric analysis

Several plant lectins, such as phytohaemagglutinin (PHA), pokeweed mitogen (PWM), concanavalin A (ConA), Maclura pumifera (MPA) and Pisum sativum agglutinin (PSA), are potent mitogens for human lymphocytes. The pattern of activation induced, however, is not uniform for all mitogenic lectins. The different biological effects following lectin activation of human lymphocytes might be due at least in part to a differential binding of the various lectins to lymphocyte subsets. We have therefore studied the binding of five mitogenic plant lectins, namely PHA, PWM, ConA, MPA and PSA to three major human lymphocyte subsets as defined by anti-CD4, anti-CD8 and anti-CD16 monoclonal antibodies. Dual colour, flow cytometric analysis employing PE-conjugated monoclonal antibodies and FITC-conjugated lectins revealed that all subsets uniformly show high binding of PHA, whereas two different populations, one high binding and the other low binding, can be detected with PWM, ConA, MPA and PSA.     

Polyclonal activation of human lymphocytes by bacteria.

The proliferative response of human unseparated lymphocytes, T cells, and B cells to various bacterial strains was investigated. All the bacteria tested induced a proliferative response in unseparated lymphocytes and B cells. T cells were stimulated only after reconstitution with monocytes. Stimulation of umbilical cord blood lymphocytes suggests that the response is polyclonal. We interpret these and previous data as an indication of a common mechanism of resistance against infectious diseases.     

Binding of concanavalin-A to critical-point-dried and freeze-dried human lymphocytes

When human lymphocytes are treated with concanavalin-A (con A) and hemocyanin, the hemocyanin marker, which demonstrates con A binding sites, can be visualized by scanning (SEM) and transmission electron microscopy (TEM) on both critical-point-dried and freeze-dried cells. The ability to visualize the hemocyanin marker by SEM, its quantity and distribution, were all similar in lymphocytes prepared by both drying procedures. By TEM, hemocyanin was seen adjacent to the plasma membrane on critical-point-dried lymphocytes. In contrast, freeze-dried cells showed hemocyanin labeling at some distance from the plasma membrane (40–70 nm) as well as adjacent to it. The distribution of hemocyanin corresponded to the thickness of the amorphous coat seen on fixed, freeze-dried cells. Therefore, the extracellular coat on freeze-dried lymphocytes is a carbohydrate-containing glycocalyx.     

Blastogenic response of bovine lymphocytes to Brucella abortus lipopolysaccharide.

Brucella abortus lipopolysaccharide was tested in a blastogenesis assay with unfractionated and nylon wool-separated peripheral blood lymphocytes of Brucella-naive cattle and cattle immunized with B. abortus. Our results indicated that in cattle the lipopolysaccharide of B. abortus is not a B-cell mitogen. In immunized animals it stimulated predominantly nylon wool-adherent cells. The lipopolysaccharide of Escherichia coli O128:B12, in contrast, induced a substantially greater proliferative response in circulating lymphocytes, predominantly those adherent to nylon wool, of the Brucella-naive cattle.     

Binding of ovine uterine serpin to lymphocytes.

PROBLEM: The endometrium of the sheep produces a progesterone-induced member of the serpin superfamily of serine proteinase inhibitors that can inhibit lymphocyte proliferation and reduce natural killer cell activity. Present results indicate that this molecule, called ovine uterine serpin (OvUS), can bind specifically to lymphocytes. METHOD OF STUDY/RESULTS: Biotinylated OvUS bound to peripheral blood lymphocytes in a dose-dependent and saturable manner. Binding was inhibited by OvUS, but not by several other proteins, including serpin-enzyme complex (alpha 1-antitrypsin-trypsin). Heparin blocked binding when added to the binding reaction or when used to pretreat lymphocytes. Both lymphocytes and Madin-Darby bovine kidney (MDBK) cells also bound fluorescein isothiocyanate-labeled OvUS. CONCLUSIONS: Results suggest that OvUS can interact with lymphocytes and other cells through binding to a cell surface molecule. Such binding may indicate that inhibition of lymphocyte activation by OvUS involves 1) binding of OvUS to a cell surface receptor or 2) competitive inhibition of binding between OvUS and a co-activation molecule required for lymphocyte activation.     

Methods for the identification of human B lymphocytes.

Complement-receptor lymphocytes and monocytes were identified by a rosette method using trypsin treated sheep erythrocytes (Et), sensitized with affinity column purified IgM (19S) antibodies against sheep erythrocytes (E) and mouse complement deficient in C5. Fc receptor mononuclear cells were identified by a rosette method using Et and IgG (7S) antibodies against E. Identification of the cell-type rosetting was facilitated by myeloperoxidase staining of dry mounted rosetted preparations. Comparison of lymphocytes with receptors for activated mouse complement and lymphocytes with stable surface immunoglobulin detected by immunofluorescent assay, strongly suggests that these cells constitute the same population in the peripheral blood of healthy humans.     

Formalin-treated bacteria as selective B cell mitogens in the study of lymphocytes from patients with hypogammaglobulinaemia.

Staphylococcus aureus strain Cowan I, Haemophilus influenzae, and Branhamella catarrhalis that stimulate human B lymphocytes, but not T lymphocytes, were used to study the B lymphocyte function in patients with hypogammaglobulinaemia. Lymphocytes from two patients with no circulating B cells were not stimulated to increased DNA synthesis by these bacterial mitogens. The lymphocytes of six patients with hypogammaglobulinaemia and normal numbers of circulating B cells responded to the bacterial mitogens but in five patients the response was decreased in comparison with healthy controls.     

Differentiation of human peripheral blood B lymphocytes.

Human B-lymphocyte differentiation was studied by measuring the capacity of such cells, isolated from peripheral blood, to synthesize and secrete Ig after pokeweed stimulation. Results show that a maximum incorporation of [3H]-thymidine took place 2 days before the appearance of detectable Ig-secreting cells. On the 7th day after pokeweed stimulation, when Ig synthesis and secretion are at a maximum, [3H]-thymidine uptake was low. Since inhibition of DNA synthesis 3 days after pokeweed stimulation completely prevents the generation of Ig-secreting plasma cells, initial DNA synthesis is apparently essential before Ig-secreting plasma cells can develop in response to pokeweed stimulation.     

Co-operation between T and B lymphocytes from human tonsils in the response to mitogens and antigens.

Purified B lymphocytes obtained from human tonsil cell populations by removing E rosette-forming cells by density sedimentation did not proliferate at three days in response to PHA and Con A, but showed a significant 3H-labelled thymidine incorporation when the PHA response was assessed at day 6 of culture. The 6th-day responses, which was completely abolished by the reduction of T-cell contamination to less than 0-1% by re-rosetting and a second separation, was due in part to a direct activation by PHA of contaminating T cells and in part to a T cell-mediated B-cell response. When purified B cells were stimulated for 3 days by PHA in the presence of an equal number of autologous or homologous mitomycin-treated T lymphocytes a highly significant uptake of 3H-labelled thymidine was demonstrated. The majority of blast cells obtained at day 4 in these cultures were unable to form E rosettes and showed surface immunoglobulin by immunofluorescence stain. This response was markedly decreased by previous treatment of B cells with mitomycin C and it was abolished when B cells were killed by heating at 56degrees C for 1 hr. Purified B lymphocytes from human tonsils did not respond in vitro when cultured for 6 days in the presence of soluble antigens (PPD and Candida). However, a highly significant response to the same antigens could be demonstrated when B cells were cultured in the presence of autologous mitomycin-treated T cells. These models of T-B co-operation could provide an interesting tool for studying the differentiation and antibody production in vitro of human B lymphocytes.     

Binding of bacterial endotoxin to murine spleen lymphocytes.

The early events in lipopolysaccharide (LPS)-induced B-cell activation were investigated by studying the binding of 14C-labeled LPS to murine lymphocytes in vitro. In these studies we utilized intrinsically labeled 14C-labeled LPS from Salmonella minnesota or the 14C-labeled glycolipid derived from the Re mutant of S. minnesota (R595). Bone marrow-derived (B) lymphocytes bound more LPS than did thymus-derived (T) lymphocytes. Binding of LPS to murine spleen lymphocytes from strain C3H/HeN was compared with the binding to spleen lymphocytes from strain C3H/HeJ, a strain resistant to certain biological activities of LPS including mitogenesis. Spleen cells from both strains bound LPS equally well, suggesting that unresponsiveness of C3H/HeJ mice to LPS is due to factors other than a defect in binding of LPS. LPS binding to cells appeared to be due to a nonspecific interaction between the lipid moiety of LPS and the lipid components of the cell membrane. Thus, the highly lipophilic, polysaccharide-deficient glycolipid from R595 bound at least 20 times better than did LPS. Furthermore, partial removal of cell surface proteins with trypsin or sialic acids with neuraminidase enhanced glycolipid binding, suggesting that binding is not through a protein- or sialic acid-containing receptor. The binding of glycolipid to lymphocytes was only partially specific since unlabeled glycolipid R595, lipid A, and LPS did not completely inhibit the uptake of 14C-labeled glycolipid R595. In addition, binding could be inhibited by a nonmitogenic phospholipid (phosphatidyl ethanolamine), which also is consistent with a nonspecific lipid-lipid interaction. Experiments were performed to determine the relationship of LPS binding to lymphocyte activation in the lymphocytes. The process of activation of lymphocytes by LPS was a slow one, since LPS was required to be present in culture for at least 24 h in order to obtain significant lymphocyte activation, suggesting that the amounts of LPS bound earlier are either quantitatively or qualitatively insufficient to irreversibly activate the cell.     

Evidence for the existence of self-reactive human B lymphocytes.

Supernatants from human tonsil cells cultured in the presence of the PBAs, LPS and F (ab)2 monomers of rabbit anti-human beta2-microglobulin were found to have a complement-dependent cytotoxic activity to tonsil cells of the same donors. A direct toxic effect of the PBAs on the target cells was excluded by controls. Therefore the most likely explanation for this finding is that human lymphocytes contain a subpopulation of self-reactive B cells which can be triggered by PBAs to release antibodies to self-antigens. The implications of this finding for the understanding of infections accompanied by auto-immune phenomena are discussed.     

Binding of porcine ficolin-alpha to lipopolysaccharides from Gram-negative bacteria and lipoteichoic acids from Gram-positive bacteria

Protein(s) reactive with N-acetyl-D-glucosamine (GlcNAc) was isolated from porcine nonimmune serum. The molecular weight of the purified protein was found to be mainly 40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The N-terminal 10 amino acid sequence of the purified protein were found to be identical to that of porcine ficolin-alpha reported previously. In enzyme-linked immunosorbent assay, the purified protein was found to react with lipopolysaccharides (LPS) from different Gram-negative bacteria such as Esherichia coli, Salmonella typhimurium, Salmonella enteritidis, Salmonella abortus equi, Pseudomonas aeruginosa, Shigella flexeneri, and Serratia marcescens and with lipoteichoic acid (LTA) from Gram-positive bacteria such as Streptococcus sanguis, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. The purified protein also reacted with E. coli O26 isolated from food poisoning and bovine feces and heat-treated Gram-positive bacteria such as S. aureus, B. cereus, B. subtilis, Enterococcus faecium, and Corynebacterum bovis. On the other hand, porcine IgG isolated from nonimmune serum showed different reactivity with these LPS, LTA, and heat-treated bacterial cells. From the present findings, purified porcine serum protein reactive with GlcNAc is concluded to be ficolin-alpha playing an important role(s) in innate immunity against microbial infection with Gram-positive and -negative bacteria.     

Inhibitory effects of extracellular products from oral bacteria on human fibroblasts and stimulated lymphocytes.

Extracellular products of 12 strains of Streptococcus mutans and 5 additional species of oral bacteria were analyzed for their ability to inhibit proliferation of fibroblastoid cells (HeLa and AV3) and blast transformation of human peripheral blood lymphocytes obtained from normal individuals. Products from S. mutans strains AHT and BHT, Streptococcus intermedius, and Actinomyces viscosus inhibited [3H]thymidine uptake by fibroblastoid cells and phytohemagglutinin-stimulated lymphocytes. Products from S. mutans E49, Streptococcus salivarius, and Actinomyces naeslundii inhibited blast transformation of human lymphocytes but did not significantly inhibit the growth of fibroblastoid cells. Preparations from S. intermedius gave the greatest inhibitory activity against both target cell types; initial characterization of this preparation suggested a single factor active in both assays, in that the heat lability and Sephadex G-200 elution profile were similar for the inhibitory activity seen with the two cell types. The molecular weight of the inhibitor, estimated by gel filtration on Sephadex G-200 and Ultragel AcA34, was approximately 160,000. The results strongly suggest that oral bacteria produce heat-labile substances that interfere with fibroblast proliferation and alter the lymphocytic immunological response.     

人扁桃体B淋巴细胞mRNA的差异显示分析及活化B细胞表达的新EST的分离

目的分离新的Ｂ细胞活化基因。方法采用差异显示反转录ＰＣＲ技术（ＤＤＲＴ－ＰＣＲ）对人扁桃体活化和静止Ｂ细胞ｍＲＮＡ的差异显示情况进行分析，对显示片段进行克隆并行Ｎｏｒｔｈｅｒｎ分析，对Ｎｏｒｔｈｅｒｎ杂交阳性的ｃＤＮＡ片段进行测序并比较同源性。结果差异显示分析共获得明显差异表达的ｃＤＮＡ片段６２条，选择主要表达于活化Ｂ细胞上的２０条ｃＤＮＡ片段克隆到ｐＧＥＭ－Ｔ载体上，并逐一对静止和活化Ｂ细胞ＲＮＡ进行Ｎｏｒｔｈｅｒｎ分析，其中６条ｃＤＮＡ片段在活化Ｂ细胞中呈Ｎｏｒｔｈｅｒｎ杂交阳性且与差异显示情况相符，对它们进行测序，然后通过国际互联网与ＧｅｎＢａｎｋ，ＥＭＢＬ和ＤＤＢＪ的ＤＮＡ数据库比较序列同源性，发现其中４个克隆与已发现的基因具明显同源性，一个克隆是新的，另一个克隆虽然与人Ｔ细胞分泌的趋化因子Ｉ－３０９同源性达９５％，但二者转录本不同。结论通过ＤＤＲＴ－ＰＣＲ分离到两个可能为新的Ｂ细胞活化基因的ｃＤＮＡ片段，这为进一步克隆新的Ｂ细胞活化基因奠定了基础。     

Binding of cultured mammalian cells to immobilized bacteria.

The invasin protein of Yersinia pseudotuberculosis binds to integrin receptors on mammalian cells and promotes cellular penetration. We demonstrate here that the cell attachment activity of invasin can be detected in bacterial colonies that have been immobilized on filter membranes. Invasin expressed in either Escherichia coli K-12 or Y. pseudotuberculosis mediated binding to membranes, and as few as 10(5) Y. pseudotuberculosis resulted in detectable attachment of cultured epithelial cells. A similar binding activity was detected in clinical isolates of the related pathogen Y. enterocolitica but not in environmental isolates. Although there exist multiple mechanisms for the binding of wild-type organisms to host cells, efficient mammalian cell binding to immobilized Y. pseudotuberculosis required expression of a functional invasin protein. Several pathogens that are known to bind or penetrate mammalian cells were also tested, and only one of these bound cultured mammalian cells efficiently.