Guanidino compounds as cause of cardiovascular damage in chronic kidney disease: an in vitro evaluation.

Chronic kidney disease is considered a major cause of cardiovascular risk and non-traditional risk factors remain largely unknown. The in vitro toxicity of 10 guanidino compounds (GCs) was evaluated via a standardized approach on different cell systems of relevance in cardiovascular disease. The parameters evaluated were production of reactive oxygen species, expression of surface molecules, cell proliferation, cytotoxicity and calcification. Several GCs had a stimulatory effect on monocytes and granulocytes (SDMA, creatine and guanidinobutyric acid (GBA)). Some GCs (guandine (G), guanidinosuccinic acid (GSA) and SDMA) inhibited endothelial cell proliferation or reduced calcification in osteoblast-like human VSMC (ADMA, GSA and SDMA). Stimulation of osteoclastogenesis could be demonstrated for ADMA, G, guanidinoacetic acid and GBA in a RAW264.7 cell line. No compounds were cytotoxic to AoSMC or endothelial cells, nor influenced their viability. GCs, especially SDMA, likely contribute to cardiovascular complications in uremia, mainly those related to microinflammation and leukocyte activation.

Monoclonal antibodies that neutralize HEV recognize an antigenic site at the carboxyterminus of an ORF2 protein vaccine.

In order to obtain monoclonal antibodies that might have prophylactic applications and to understand better the immune response to hepatitis E virus (HEV), we used phage display to isolate chimpanzee antibodies to HEV. The panning antigen was an two open reading frame (ORF2) recombinant protein that elicits a broadly protective immune response in vaccinated monkeys. Two major antigenic sites were identified on the ORF2 protein: one site was not accessible on the surface of infectious virions but the other site was accessible to antibodies and was recognized specifically by antibodies that neutralize the virus.

Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation.

We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hyaluronan, can induce angiogenesis in vivo and, as such, o-HA is an important regulator of the neovascularization process. As a continuation of this work, we have studied the cytoplasmic signal transduction pathways responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 microg/ml) to bovine aortic endothelial cells induces tyrosine phosphorylation of multiple proteins within 1 minute and that the activity remains above basal levels for at least 24 hours. Increased phosphorylation of the CD44 receptor was also observed. Pretreatment of cells with an anti-CD44-receptor antibody (5 microg/ml) or the tyrosine kinase inhibitor genistein (10 microM) inhibited both o-HA-induced proliferation (p < 0.05) and protein tyrosine phosphorylation. In comparison, native hyaluronan had little effect on tyrosine phosphorylation across the same time period. Protein kinase C (PKC) activity was increased 2- to 3-fold in the membranes of cells treated with o-HA, and a pretreatment with phorbol 12,13-dibutyrate (PDBu) to down-regulate PKC significantly inhibited o-HA-induced cell proliferation (p < 0.05). Examination by Western blotting showed that only the betaI and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating the proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furthermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-regulated kinase [ERK-1]) by o-HA culminated in the nuclear translocation of ERK-1. This pathway is essentially linear, as shown by the ability of specific enzyme inhibitors (PDBu and PD98059) to prevent both activation of ERK-1- and o-HA-induced proliferation. We conclude that phosphorylation of the CD44 receptor results in an increase in tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed that o-HA-induced proliferation of endothelial cells is CD44-receptor-mediated and accompanied by early response gene activation.

Regulation of antigen receptor signal transduction by protein tyrosine kinases.

The past two years have seen further clarification of the early events occurring in antigen receptor signal transduction that are mediated by the immunoreceptor tyrosine-based activation motif (ITAM). The ITAM was shown to be a specific binding site for the ZAP-70/Syk protein tyrosine kinases and the structure of this complex was solved. In addition, possible mechanisms of activation and functions for these kinases were reported. Lastly, genetic studies established the critical importance of these kinases in antigen-receptor signaling and lymphocyte development.

Phenylarsine oxide (PAO) blocks antigen receptor-induced calcium response and tyrosine phosphorylation of a distinct group of proteins.

Antigen receptor (AgR) crosslinking by antigens or AgR-specific antibodies induces a cascade of enzymatic events in lymphocytes which involves activation of several non-receptor tyrosine- and serine/threonine kinases, phosphatases, phospholipases, etc. Here we show data demonstrating that a thiol group-reactive protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide (PAO), uncouples a crucial part of the signaling events induced by anti-IgM or anti-Leu-4 (CD3) in human tonsil B lymphocytes, BL41 and Daudi B cell lines and Jurkat T lymphoma cells. PAO treatment (10 microM) resulting in distinct modification of AgR-induced tyrosine phosphorylation pattern inhibited the AgR-mediated calcium response (Ca++ release and influx) of all of these cells completely. Since this treatment did not alter the cell viability and the binding capacity of the AgR crosslinking antibodies, alteration of the tyrosine phosphorylation pattern and blockage of the calcium response indicate prompt inactivation of essential signal transduction element(s).

Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide: effect of ebrotidine.

The effect of lipopolysaccharide from H. pylori, a bacteria implicated in the etiology of gastric disease, on the gastric mucosal laminin-receptor interaction was investigated. The receptor protein, prepared from rat gastric epithelial cell membrane by affinity chromatography on laminin-coupled Sepharose, was radioiodinated, and incorporated into liposomes which exhibited specific affinity towards laminin-coated surface. The binding was inhibited by H. pylori lipopolysaccharide, which caused a maximum inhibition of 96% at 50 micrograms/ml. The inhibitory effect of the lipopolysaccharide was prevented by an antiulcer agent, ebrotidine that evoked essentially complete restoration in binding at 6-8 micrograms/ml. The results demonstrate that H. pylori through its lipopolysaccharide interferes with gastric epithelial cell-laminin binding, and that this disruptive effect could be successfully countered by ebrotidine.

Increasing the efficiency of lipid-conjugated antibodies incorporation into the membrane of antigen presenting B cells.

We have introduced some modifications in the technique called "cell decoration" in order to increase the amount of lipid-conjugated antibodies which can be incorporated into the membrane of B cells. As shown by FACS analysis, we have obtained an approximately 4-fold increment in the amount of specific antibodies incorporated into the cell membrane. The procedure, which consists of successive changes of the medium that contains the lipid-conjugated antibodies, avoided changes on parameters that interfere with cell viability. The proposed modification resulted in an approximately 2-fold enhancement of the ability of decorated B cells to act as antigen presenting cells for specific T hybridomas.

Physiology and function of the vero cell receptor for the hepatitis B virus small S protein.

The African green monkey kidney-derived Vero cell line expresses a receptor activity for noninfectious hepatitis B surface antigen (HBsAg) particles containing the small S protein. (M.E. Peeples, K. Komai, R. Radek, and M.J. Bankowski, 1987, Virology 160, 135-142). In this report, the binding characteristics, the physiological requirements, and the functions of this receptor are further characterized. The association rate constant (ka) was determined by measuring binding during a short (10-min) incubation period to avoid the complication of dissociation. The results indicated an extremely high affinity binding: ka = 2.0 x 10(10) M-1 min-1. HBsAg particle binding to the Vero cells was also found to be slowly reversible, and dependent on temperature, pH, and Ca2+. After binding to Vero cells. HBsAg particles were quickly internalized as measured by trypsin removal from the cell surface. Once removed from the cell surface by proteolysis, regeneration of receptor activity required protein synthesis, indicating that there is no significant receptor pool within the cell. Receptor activity was also found to recycle to the cell surface after HBsAg particles were internalized.

[Lymphocytes with antigen receptors in drug allergy]. / Limfotsity s retseptorami dlia antigena pri lekarstvennoi allergii.

The data on the possibility of using the rosette-formation tests for the diagnosis of drug allergy are presented. Tests based on changes in the levels of activated T- and B-lymphocytes after their incubation with allergenic drugs have proved to be low informative. The test found to be highly informative is the antigen-specific rosette-formation test based on the detection of lymphocytes, capable of binding allogeneic erythrocytes loaded with antibiotics causing allergy in patients, in the peripheral blood. This test may be of importance not only in diagnosis, but also for prognosis, as it permits the detection of sensitization to a drug before the clinical manifestations of allergy.

Morphine depression of T cell E-rosetting: definition of the process.

Two kinetic assays were developed to assess opiate effects on rates of T cell E-rosetting. The first adopted the thermal conditions of active E-rosetting assays (varying between 37 and 23 C) whereas the second incorporated cooler thermal conditions (varying between 0 and 29 C). In vitro treatment of lymphocytes with morphine depressed E-receptor levels and E-rosetting in both assays. With the 0-29 C procedure early stages of E-rosette formation were characterized by phase transition kinetics indicative of sequential gain and loss of E-rosettes. Assay thermal and erythrocyte (E) to T cell contact conditions, and the inclusion of morphine during E-rosetting, were independent variables that coordinately modulated the expression of phase transitions. Phase transitions were also noted during capping of total T cell E-rosettes at 37 C. The reason for phase transitions appears to be that T cells undergo sequential cycling of E-receptors, increasing because of the new expression of dormant E-receptors as the result of E-receptor microdisplacement and decreasing because E-rosettes are lost owing to patching and capping processes. According to this construction of the E-rosetting process, morphine inhibits E-rosetting and modulates expression of phase transitions by interfering with E-receptor microdisplacement processes. Presumably this interference by morphine is mediated through alteration of membrane fluidity and promotion of E-receptor coupling (and/or inhibition of uncoupling) to a transducer-effector component within the cell membrane. These findings and conclusions are specifically relevant to immunoregulatory processes and are also helpful for understanding the general nature of biological and physiological responses associated with receptor-ligand interactions.

Interaction of hapten-sensitized liposomes with cells bearing hapten-specific receptors.

The binding of liposomes sensitized with 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) to MOPC-315 cells, which secrete and bear on their surfaces anti-DNP immunoglobulins, was studied. The binding was affected by cholesterol content, phospholipid composition and hapten density of liposomes: The binding of distearoylphosphatidylcholine liposomes sensitized with 5 mol% hapten to the cells increased with increasing cholesterol content in liposomes. The amount of liposomes composed of phospholipid with a higher transition temperature (such as distearoylphosphatidylcholine), which bound to MOPC-315 cells, was much higher than that of liposomes composed of phospholipid with a lower transition temperature (such as egg yolk phosphatidylcholine). The amount of distearoylphosphatidylcholine liposomes containing equimolar cholesterol, which bound to the cells at 0 degrees C, increased with increasing amount of the hapten in liposomes up to 2.5 mol%. The binding became maximum at 2.5 mol% and decreased with higher hapten density in liposomes. The immunogenicity of hapten-sensitized liposomes is known to be affected by the liposomal composition such as cholesterol content, phospholipid composition and hapten density. This model study suggests that the binding of liposomes to cells is important for expressing the immunogenicity of hapten-sensitized liposomes.

Persistent expression of Ia-antigen on a subpopulation of murine resident peritoneal macrophages.

The stability of Ia-antigen expression on murine resident peritoneal macrophages was assessed during the course of in vitro culture. Contrary to published findings with radioimmunoassays and immunofluorescence assays, the cultured cells bore Ia-antigen, as shown by their rosetting with sheep erythrocytes coupled with anti-Ia.2 monoclonal antibody. In support of this finding, cultured cells presented the copolymer of glutamine, alanine, and tyrosine (GAT) to GAT-primed T lymphocytes in an Ia-dependent manner. Thus, functional Ia antigen is present on cultured macrophages. Disappearance of the antigen after fixation of macrophages with either glutaraldehyde or paraformaldehyde, a routine procedure in the radioimmunoassays and immunofluorescence assays, explains its presumed absence on cultured cells.

The Sdx antigen and antibody: biochemical studies on the inhibitory property of human urine.

Anti-Sdx is an IgM complement-binding autoantibody that defines a red cell antigen which is independent of I, i, Sp1 (Pr) and Gd. Hemagglutination by the antibody is unusually sensitive to variation in pH, salt, or other charged molecular species. The antibody is inhibited by urine from Sd(a+) persons, but inhibition is a nonspecific effect caused by charged molecules. No specific Sdx substance could be demonstrated, and Sdx antigen does not appear to be directly associated with the Sid blood group. In view of these findings we propose that this antibody should be renamed anti-Rx.

Mechanisms of idiotype suppression. V. Anti-idiotype antibody inhibits early B cell triggering induced by antigen.

To investigate the relationship between antigen-mediated B cell commitment and induction of idiotype (id) suppression, anti-id antibody directed against the major id (TEPC-15 idiotype or T15id) of the anti-phosphorylcholine (PC) antibody was added at various time intervals to BALB/c spleen cell cultures stimulated with a T-independent PC antigen, R36a. The suppressive effect of anti-T15id antibody on the anti-PC response was rapidly decreased as addition of the antibody was delayed; when anti-T15id antibody was added 6 hr after the initiation of the cultures, only partial suppression was induced, whereas the addition of anti-id antibody after 24 hr did not result in significant suppression of the anti-PC response when compared with similar cultures treated with mock anti-id antibody. This acquisition of resistance to id suppression was completely inhibited by treatment with either sodium azide or colchicine, as well as at temperatures below 20 degrees C. The induction of resistance to id suppression during the preincubation period was dependent on the presence of an immunogenic level of specific antigen. This antigen-mediated B cell commitment did not appear to require macrophages because preincubation of macrophages with antigen did not affect the sensitivity of the B cells to anti-id antibody. These results support the possibility that anti-id antibody inhibits early B cell triggering, which involves an energy-dependent, epitope-mediated, lateral mobility of antigen receptors possibly followed by repolymerization of microtubules.