Comparison of 16S rRNA sequences from the family Pasteurellaceae: phylogenetic relatedness by cluster analysis

The taxonomy of the family Pasteurellaceae has remained controversial despite investigations of biochemistry, serology, and nucleic acid relatedness. In an attempt to resolve some of this confusion, we have partially sequenced the 16S rRNAs of seven members of the family, representing all three genera. The sequences were aligned, similarity scores calculated, and single, average and complete linkage cluster analysis of the resulting distance matrix performed. In this way, an evolutionary branching pattern of these closely related species was reconstructed, and the approximate phylogenetic position of the family determined. Actinobacillus (Haemophilus) actinomycetemcomitans clustered with Haemophilus instead of Actinobacillus, supporting transfer of this species to the genus Haemophilus. Thus cluster analysis of phylogenetic relatedness was found to be particularly useful for studying closely related organisms, and could be performed using a microcomputer.     

IFN-gamma treatment of K562 cells inhibits natural killer cell triggering and decreases the susceptibility to lysis by cytoplasmic granules from large granular lymphocytes

Pretreatment of human K562 leukemia cells with rIFN-alpha and rIFN-gamma resulted in decreased susceptibility to lysis by human peripheral blood NK cells. The reduction of NK-susceptibility after IFN treatment was not due to a general effect of IFN on the stability of the cell membrane because the susceptibility of K562 cells to lysis by antibodies plus C, distilled water, or lysolecithin was unaffected. Binding studies with effector cell preparations enriched for NK cells with large granular lymphocyte morphology revealed no difference in binding to control and IFN-gamma-treated target cells. The sensitivity to soluble NK cytotoxic factors was not affected significantly by the IFN treatment. In contrast, the susceptibility of IFN-treated target cells to the cytotoxic activity of purified cytoplasmic granules from a rat large granular lymphocyte tumor was significantly reduced, indicating that the IFN-induced resistance acted at the level of susceptibility to the lytic mechanism of NK cells. However, IFN-alpha was more effective than IFN-gamma in inducing resistance to the cytoplasmic granules although resulting in only a weak resistance in the cell-mediated cytotoxic assay. IFN-gamma but not IFN-alpha caused a reduction in the frequency of effector cells that had reoriented their Golgi apparatus toward their bound target cell. In addition, IFN-gamma treated K562 cells failed to elicit an influx of Ca2+ into effector cells. Taken together, the results suggest that IFN-gamma in addition to an increased resistance to the lytic molecules released by NK cells can also induce changes in the target cells which prevent the triggering and activation of the effector cell.     

Role of the CD22 human B cell antigen in B cell triggering by anti-immunoglobulin

As B cells mature during ontogeny the CD22 human differentiation Ag is exported from the cytoplasm onto the membrane. Surface expression is lost in terminal differentiation and after activation. In tonsils, CD22 is expressed on the surface of 60 to 80% of the dense B cells. Some IgM+ dense cells, however, and buoyant in vivo activated B cells are CD22-. This differential expression of CD22 and the finding that an anti-CD22 mAb augmented anti-Ig induced B cell proliferation suggested that CD22 may play a role in B cell activation. In this study we have found that CD22+ but not CD22- B cells could be triggered by anti-IgM or anti-IgD to have increased free intracellular calcium ([Ca2+]i). The presence of CD22 rather than of IgD seems to determine the ability of B cells to respond to anti-Ig with a [Ca2+]i flux. Also the proliferative response to anti-Ig or anti-Ig + B cell growth factor was restricted to the CD22+ population. Anti-CD22 mAb, although not inducing [Ca2+]i on their own after binding to B cells, did augment [Ca2+]i fluxes by anti-Ig when cross-linked. Together these results suggest that CD22 may regulate triggering of B cells through surface Ig perhaps by acting as a "bridge" to transmit an early signal into the cytoplasm.     

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Characterization of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima

Lesion-free areas of aortic intimas from seven men, 30 to 49 years old, were extracted with aqueous buffer within a few hours after an accidental or sudden death. Two lipoprotein fractions could be isolated by density gradient ultracentrifugation from all cases. The mean composition of fraction I (d less than 1.012 g/ml) resembled that reported for the cholesteryl ester-rich, beta-migrating very low density lipoprotein (beta-VLDL); the composition of fraction II (d 1.021-1.046 g/ml) resembled that of plasma low density lipoprotein (LDL). Mean diameter of the particles was 35 +/- 8 nm in fraction I and 25 +/- 5 nm in fraction II (22 +/- 2 nm in plasma LDL). Both fractions contained apolipoproteins B (apoB) and E (apoE), and had increased electrophoretic mobilities and reduced contents of linoleic acid. The immunoreactivity of apoB to a polyclonal and two monoclonal antibodies in both fractions was not different from that of plasma lipoproteins. The apoE isoform patterns in both fractions were similar to those obtained from the respective postmortem plasmas. When incubated with mouse peritoneal macrophages, fractions I and II enhanced the incorporation of radioactive oleate into cholesteryl esters by 10- to 20-fold and 3- to 4-fold, respectively, in comparison to plasma LDL. In conclusion, our results indicate that lesion-free human aortic intima contains two types of apoB- and apoE-containing lipoprotein particles, both of which might be potentially atherogenic.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-Ig-mediated proliferation of human B cells in the absence of protein kinase C

Cross-linking of surface Ig has been shown to stimulate phosphatidylinositol hydrolysis in murine B cells, leading to increases in [Ca2+]i and activation of protein kinase C (PKC). Preliminary evidence suggests that a similar activation mechanism occurs in human B cells. We wished to examine whether anti-Ig antibody-stimulated human B cell proliferation is as dependent upon the presence of PKC as is anti-Ig-mediated murine B cell proliferation. Using highly purified, small, dense peripheral-blood B lymphocytes from healthy adult donors, we confirmed that PMA, a direct activator of PKC, is a potent mitogen for human B cells that synergizes with anti-mu antibody. Furthermore, we demonstrated that PMA treatment abolishes detectable cellular stores of immunoreactive PKC. However, after such depletion of cellular PKC, anti-mu antibody is still capable of delivering a proliferative signal to human B cells. It is unlikely that this signal occurs solely on the basis of increases in [Ca2+]i, because the calcium ionophore A23187 does not induce a proliferative response in PMA-treated B cells similar in magnitude to that seen with anti-mu. Additionally, the finding that pretreatment of B cells with PMA ablates the ability of anti-Ig antibody to mobilize intracellular and extracellular calcium also suggests that the ability of PMA to enhance anti-Ig mediated stimulation does not depend on elevations of [Ca2+]i induced by anti-Ig. Together, these observations suggest that anti-Ig signaling of human B cells may occur via other pathways in addition to the phosphatidylinositol system of calcium influx and PKC activation.     

Protein L. A novel bacterial cell wall protein with affinity for Ig L chains

A novel Ig-binding protein has been isolated from the surface of bacteria belonging to the anaerobic species Peptococcus magnus. To solubilize the protein, peptococci were treated with different proteolytic enzymes (papain, pepsin, and trypsin) or with mutanolysin, a bacteriolytic agent known to digest the cell walls of streptococci. Papain, trypsin, and mutanolysin all solubilized peptides showing affinity for radiolabeled human IgG in Western blot analysis. Compared with papain and trypsin, mutanolysin liberated a more homogeneous material, which also had a higher m.w. This mutanolysin-solubilized protein (Mr 95 kDa) was obtained highly purified by a single isolation step on IgG-Sepharose, and the molecule was found to exhibit unique Ig-binding properties. Thus, in dot blots and in Western blots, human IgG, F(ab')2 and Fab fragments of IgG, and human kappa and lambda L chains all showed affinity for the protein. Moreover, the molecule also bound human IgM and IgA, whereas no binding was recorded for IgG-Fc fragments or IgG H chains. Finally, the protein bound to human polyclonal Ig L chains immobilized on polyacrylamide beads. These different data demonstrate that the isolated peptococcal protein binds Ig through L chain interaction. The name protein L is therefore suggested for this novel Ig-binding bacterial cell wall protein.