Binding of histamine and histamine analogs to lymphocyte subsets analyzed by flow cytometry

The binding of histamine, 4-methylhistamine (a histamine type 2 receptor agonist), cimetidine (a histamine type 2 receptor antagonist), and telemethylhistamine (an inactive analog) to human peripheral blood mononuclear cell subsets was investigated by flow cytometry by using conjugates of these ligands coupled to fluorescein-labeled human serum albumin. Our results indicate that binding of fluorescent protein conjugates of histamine and its analogs does not selectively identify a lymphocyte subset(s) that mediates the immunomodulatory effects of histaminergic ligands. Conjugates with both low (2.5 to 2.8:1) and high (28 to 57:1) ligand to protein coupling ratios were used. No binding above background could be detected for the low mole ratio reagents. The high mole ratio reagents were bound by 95 to 99% of all lymphocytes when used at ligand concentrations of 50 microM or greater. At lower ligand concentrations, the number of lymphocytes exceeding a set fluorescence threshold was decreased, but fluorescence distributions remained unimodal at all concentrations used (1 to 500 microM). Monocytes also bound the high mole ratio reagents and gave rise to a second high-intensity peak in the fluorescence distribution unless they were excluded by other means. Levels of conjugate binding detected by flow cytometry did not parallel ligand potencies at classical histamine type 2 receptors; at equivalent ligand concentrations, approximately equal amounts of histamine or 4-methylhistamine conjugate were bound per lymphocyte, and only 30% less telemethylhistamine conjugate was bound. Competition with free ligands (10(2)- to 10(4)-fold excess histamine, 4-methylhistamine, cimetidine, or telemethylhistamine) did not significantly decrease the level of binding observed for the high mole ratio reagents at bound ligand concentrations of 1 to 25 microM. Dual staining with fluorescein-labeled conjugate and phycoerythrin-labeled monoclonal antibodies Leu-3ab (anti-helper T), Leu-2a (anti-suppressor T), Leu-M3 (anti-monocyte), or anti-HLA-DR (B cells and monocytes) was also carried out. The extent of conjugate binding to helper and suppressor cells was identical for each of the ligands used, but higher levels of conjugate binding were seen for monocytes and B cells than for T cells in every case. Our data do not exclude the possibility of enhanced conjugate binding to small numbers of activated (HLA-DR positive) T cells that might be involved in mediation of histamine effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anti-stress effects of carnosine on restraint-evoked immunocompromise in mice through spleen lymphocyte number maintenance

Carnosine (β-alanyl-L-histidine), a naturally occurring dipeptide, has been characterized as a putative neurotransmitter and serves as a reservoir for brain histamine, which could act on histaminergic neurons system to relieve stress-induced damages. However, understanding of the role of carnosine in stress-evoked immunocompromise is limited. In this study, results showed that when mice were subjected to restraint stress, spleen index and the number of spleen lymphocytes including Natural Killer (NK) cells were obviously decreased. Results also demonstrated that restraint stress decreased the cytotoxic activity of NK cells per spleen (LU(10)/spleen) while the activity of a single NK cell (LU(10)/10(6) cells) was not changed. However, oral administration of carnosine (150 and 300 mg/kg) increased spleen index and number of spleen lymphocytes (including NK cells), and elevated the cytotoxic activity of NK cells per spleen in restraint-stressed mice. These results indicated that carnosine ameliorated stress-evoked immunocompromise through spleen lymphocyte number maintenance. Carnosine was further found to reduce stress-induced elevation of plasma corticosterone level. On the other hand, results showed that carnosine and RU486 (a glucocorticoids receptor antagonist) treatment prevented the reduction in mitochondrion membrane potential and the release of mitochondrial cytochrome c into cytoplasm, increased Bcl-2/Bax mRNA ratio, as well as decreased terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in spleen lymphocytes of stressed mice. The results above suggested that the maintenance of spleen lymphocyte number by carnosine was related with the inhibition of lymphocytes apoptosis caused by glucocorticoids overflow. The stimulation of lymphocyte proliferation by carnosine also contributed to the maintenance of spleen lymphocyte number in stressed mice. In view of the elevated histamine level, the anti-stress effects of carnosine on restraint-evoked immunocompromise might be via carnosine-histamine metabolic pathway. Taken together, carnosine maintained spleen lymphocyte number by inhibiting lymphocyte apoptosis and stimulating lymphocyte proliferation, thus prevented immunocompromise in restraint-stressed mice.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

Suppression of Graft versus Host Reaction by Univalent Anti-immunoglobulin Antibody

THE secondary response of spleen cells of mice immunized with hapten-protein conjugates can be inhibited if the spleen cells are incubated with rabbit serum to mouse immunoglobulin before stimulation by hapten-protein conjugate¹. These findings, together with evidence^2–4 for the presence of immunoglobulin on the surface of lymphocytes, have fostered the hypothesis of the immune receptor as an antibody-like molecule at the cell membrane of lymphocytes.     

Immune complexes and immunoconglutinin interactions associated with altered lymphocyte activity in Plasmodium chabaudi infections

Fresh plasma from rats infected with Plasmodium chabaudi, incubated with splenic lymphocytes from rats immunized 5 days previously with sheep blood cells, suppressed the capacity of the spleen cells to produce antibody against the sheep cells as was indicated by reductions in the numbers of hemolytic Jerne plaques formed by the treated cells. The effect was maximal in plasma of rats drawn on the 7th day of infection at a time the rats experienced a hemolytic crisis. Serologic studies indicated that the active plasma contained elevated titers of antibody against fibrinogen products, antibody against the soluble serum antigens elaborated during blood infections and antibody against the third component of fixed complement (C3) or immunoconglutinin. Titers of lytic complement were reduced and amounts of soluble immune complex precipitated with polyethylene glycol 6000 were elevated. The active plasma may have affected the antibody producing cells by one or both of two mechanisms. Soluble antigen-antibody complexes could have interacted with Fc receptors of activated lymphocytes to alter their function. Alternatively, the complexes may have fixed complement and interacted with receptors for fixed C3 on the lymphocyte membrane. Such cells, being coated with the antigen for immunoconglutinin, could be altered by immunoconglutination. Inasmuch as the immune complexes in the active plasma were generated in vivo, it would seem unlikely that the plasma would contain significant amounts of complex that had not fixed complement. With immunoconglutinin present in the plasma, alteration of the cells by immunoconglutination seems a more likely possibility.     

Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Modulation of granulomatous hypersensitivity. V. Participation of histamine receptor positive and negative lymphocytes in the granulomatous response of Schistosoma mansoni-infected mice

The possible role of histamine and histamine-receptored inflammatory cells in the granulomatous response of Schistosoma mansoni-infected mice was examined. Special staining revealed the presence of numerous mast cells, many partially degranulated within the liver granulomas. Treatment of infected mice with cimetidine (an H2 receptor antagonist) enhanced, and diphenyhydramine (an H1 receptor antagonist) decreased the granulomatous response. Fluorescein-labeled histamine-rabbit serum albumin conjugate (H-FRSA) and unlabeled conjugate (H-RSA)-coated culture plates were used to identify and isolate cells with histamine receptors. A large proportion of granuloma macrophages, lymphocytes, eosinophils, neutrophils, and splenic lymphocytes had histamine receptors. Elution of adherent cells from H-RSA-coated culture plates with H1 or H2 receptor antagonists suggested that receptors on granuloma cells were predominately H1 with some granuloma lymphocytes bearing H2-type receptors. Splenic lymphocytes from infected mice were functionally divided according to the presence or absence of histamine receptors on their cell surface. Receptor-negative lymphocytes appeared to mediate SEA-stimulated MIF production (TDH cells) and participated in the adoptive transfer of suppression of granulomas (TH cells). Whereas, TS cells appeared to have histamine receptors. Based on these data, it is inferred that lymphocytes that regulate lymphokine production (TS cells) within the granuloma may be triggered via their histamine receptors to exert suppressive activity.     

Effect of sera against aggregated mouse immunoglobulins on a population of lymphoid and hematopoietic cells]

The in vitro treatment of the mouse spleen cells immunized by the ram erythrocytes with the rabbit and mouse sera against the thermoaggregated mouse immunoglobulins resulted in the inhibition of antigen binding receptors of rosette forming cells. The mouse serum, unlike the rabbit one, induced the inactivation of receptors in rosette forming lymphocytes both in the non-immune and immune mice on the 8th day after the antigenic stimulation. The treatment of bone marrow cells from the intact mice with these sera increased insignificantly the number of hemopoietic colonies in the spleens of lethally irradiated syngenic recipients and stimulated markedly the migration of spleen cells. This may be due both to the direct effect of these sera and to their mediated (through the humoral factor) influence. The inactivation of antigen binding receptors in the spleen rosette forming cells suggests the presence of immunoglobulins on the membrane of B-lymphocytes in the aggregated state or in the form of antigen--antibody complexes.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Histamine and serotonin suppression of lymphocyte response to phytohemagglutinin and allogeneic cells

Histamine added to murine spleen cells suppressed in vitro proliferation of lymphocytes induced by PHA or allogeneic spleen cells. Another vasoactive amine, serotonin (5-hydroxytryptamine), exerted a similar inhibitory activity on PHA- or allogeneic cell-induced lymphocyte proliferation. Anti-H2 histamine antagonists, cimetidine, metiamide, and ranitidine, blocked the histamine and serotonin suppressive effect. Cyproheptadine, an anti-H1 histamine and anti-serotonin antagonist, and methysergide, an anti-serotonin antagonist, also blocked histamine and serotonin inhibitory activities. These data suggest the presence, on lymphocytes, of receptors for serotonin which might be related to histamine receptors.     

The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Immunoelectron microscopic observations

In an electron microscopic investigation of the entry of sporozoites of Theileria parva into bovine lymphocytes, the fate of the surface coat of the parasite was traced by immunocytochemical methods. A monoclonal antibody (MAbD1) raised in mice and directed against a surface antigen of sporozoites, was applied to ultrathin frozen sections of bovine lymphocytes infected in vitro. Sites of binding of MAbD1 were localized using a protein A-colloidal gold conjugate as an electron-dense label. The surface of all free sporozoites was labelled. Sporozoites in the process of entering were labelled only on that portion of the membrane not yet tightly bound to the lymphocyte membrane. No label was detected on sporozoites that had completed entry. After fixation with formaldehyde, but not with glutaraldehyde, local areas of labelling were found on lymphocytes in contact with sporozoites and on cells already invaded. The sporozoite organelles, called micronemes, occasionally appeared to contain labelled antigen. No label was found on sporozoites or lymphocytes in control preparations previously exposed to non-specific antibody or treated with protein A-colloidal gold alone. The findings support the conclusion that the sporozoite surface coat, containing the antigen recognized by MAbD1, is shed as the sporozoite enters the host cell.     

Modulation of cellular immune function in vitro by histamine receptor-bearing lymphocytes: mechanism of action.

When soluble histamine is added to guinea pig lymphocytes in vitro, antigen-induced cellular proliferation and the production of migration inhibitory factor is suppressed. The inhibitory effects that are produced by histamine have been shown to be mediated by the histamine-type 2 receptors of the involved cells, but the exact nature of this suppression has not been fully explored. The present studies have evaluated, following immunization, the effect of histamine on macrophage function in vitro, and affinity chromatography to delete a subpopulation of cells bearing histamine receptors. When we treated monolayers of peritoneal exudate cells with histamine (up to 10^?3M) we found that histamine did not interfere with antigen binding by macrophages, macro phage presentation of antigen to lymphocytes, nor the antigen-independent or antigen-dependent lymphocyte-macrophage rosetting. Columns containing insolubilized conjugates of histamine and rabbit serum albumin depleted a subpopulation of cells responsive to histamine i.e., the non-adherent cells made migration inhibitory factor and proliferated in the presence of histamine. The latter finding suggested that the retained cells might have suppressor function and if so, might mediate their effect through the release of a soluble factor. Preliminary data obtained in these studies supports this hypothesis. We conclude that cells bearing histamine receptors may serve a regulatory role in cellular immunity after their activation by histamine by producing a non-dialyzable factor with immunosuppressive properties.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.     

Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules

Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of approximately 0.05 to 0.15 micrometer Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy. The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values. A similar distribution was obtained after perfusion of H1 or H2 receptor agonists coupled to ferritin (2-pyridylethylamine- ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively). The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H1 and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H2 receptors.     

A monoclonal anti-HEBFPP antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat

Cell surface molecules involved in lymphocyte adhesion to high endothelial cell venules (HEV) of Peyer's patches (PP) have been studied in the rat by using a mouse monoclonal anti-HEBFPP (1B.2) antibody. We previously showed that rat thoracic duct lymph contains a high endothelial cell binding factor termed HEBFPP, which in vitro blocks lymphocyte binding sites of HEVPP but not HEVLN. Monoclonal 1B.2 antibody was produced by fusing P3U1 myeloma cells with spleen cells of a mouse immunized with this material. Immunoprecipitation studies with 125I surface-labeled rat thoracic duct lymphocytes (TDL) showed that the antibody recognized an 80-kilodalton protein. This antigen was present in the majority of TDL, spleen, LN, and PP cells but was found on few (5 to 10%) thymus and bone marrow cells (indirect immunofluorescence). Treatment of TDL with 1B.2 antibody blocked their ability to bind in vitro to HEVPP; antibody treatment did not interfere with TDL adhesion to HEVLN. Analysis of 1B.2 antigen isolated from lymph and detergent lysates of TDL by antibody-affinity chromatography showed that this material had the capacity to block lymphocyte binding sites of HEVPP but not HEVLN. In contrast, material with such blocking activity was not isolated from detergent lysates of thymocyte, a population deficient in HEV-binding cells. The results indicate that the 1B.2 antigen is a component of the lymphocyte surface recognition structure mediating adhesion to HEVPP and provide further evidence that distinct adhesion molecules of rat TDL mediate interaction with high endothelium of LN and PP.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBFLN (A.11) antibody

Lymphocyte entry into lymph nodes (LN) and Peyer's patches (PP) occurs specifically at high endothelial cell venules (HEV). We previously isolated a high endothelial binding factor (HEBFLN) from rat lymph that blocked the lymphocyte binding sites of HEVLN but not HEVPP. In this study, mouse monoclonal anti-HEBFLN antibody (A.11) was used to investigate rat lymphocyte surface structures mediating adhesion to high endothelium. The A.11 antigen was expressed on the majority of thoracic duct lymphocytes (TDL), spleen, LN, PP cells, but was only detected on few (1 to 10%) thymus and bone marrow cells (indirect immunofluorescence). The treatment of TDL with the A.11 IgG blocked their ability to bind to HEVLN. This effect was specific, inasmuch as A.11 antibody did not block lymphocyte binding to HEVPP, and an anti-leukocyte-common antigen monoclonal antibody, OX1, did not block lymphocyte binding to HEVLN. In addition, the A.11 antigen isolated from the lymph and detergent lysates of TDL by antibody affinity chromatography had the capacity to block the lymphocyte binding sites of HEVLN but not HEVPP. Immunoprecipitation studies revealed that the A.11 antibody recognized the radioiodinated surface membrane proteins of TDL and TDL-derived T cells and B cells, which resolved with SDS-PAGE autoradiography into three polypeptides with relative m.w. of approximately 135,000, 63,000, and 40,000. We conclude that the A.11 antigen is a component of the lymphocyte surface recognition structure that mediates adhesion to high endothelial cells of rat peripheral lymph nodes.     

Multivalent thioether-peptide conjugates: B cell tolerance of an anti-peptide immune response.

Antibodies which bind beta2-glycoprotein I (beta2GPI) are associated with antiphospholipid syndrome. Synthetic peptide mimotopes have been discovered which compete with beta2GPI for binding to selected anti-beta2GPI. A thiol-containing linker was attached to the N-terminus of two cyclic thioether peptide mimotopes, peptides 1a and 1b. The resulting peptides, with linker attached, were reacted with two different haloacetylated platforms to prepare four tetravalent peptide-platform conjugates to be tested as B cell toleragens. The linker-containing peptides were reacted with maleimide-derivatized keyhole limpet hemocyanin (KLH) to provide peptide-KLH conjugates. Peptides 1a and 1b were also modified by acylation with 3-(4'-hydroxyphenyl)propionic acid N-hydroxysuccinimidyl ester. The resulting hydroxyphenyl peptides were radioiodinated and used to measure anti-peptide antibody levels. The KLH conjugates were used to immunize mice to generate an anti-peptide immune response. The immunized mice were treated with the conjugates or saline solution and boosted with the appropriate peptide-KLH conjugate. Three of the four conjugates suppressed the formation of anti-peptide antibody. The stabilities of the conjugates in mouse serum were measured, and the relative stabilities did not correlate with ability to suppress antibody formation.     

Identification,isolation, and characterization of a human T cell population by a monoclonal antibody

The hybridization of spleen cells from mice immunized with mononuclear leukocytes with the HAT-sensitive nonsecreting myeloma, NS1, resulted in the production of hybrid cell lines secreting monoclonal antibodies to lymphocyte surface antigens. One of these, anti-Ta, was shown by fluorescence-activated cell sorter analysis to be specific for a subpopulation of peripheral human T cells. Anti-Ta did not react with peripheral human B cells. Immunoprecipitation followed by two-dimensional gel analysis demonstrated that the T cell subpopulation-specific antigen recognized by this monoclonal antibody is part of, or firmly associated with, a protein of the plasma membrane.     

Spontaneous adherence and rosette formation of lymphocytes to Leydig cells: an in vitro technique

A spontaneous rosette phenomenon occurs in vitro when suspensions of mouse testicular cells are mixed with suspensions of lymphocytes. The core cell of these rosettes are the Leydig cells, and the coronas are formed by lymphocytes. No other testicular cell, either seminal or Sertoli, participates in lymphocyte binding.Autogeneic, syngeneic, allogeneic, and xenogeneic lymphocytes from thymus, spleen, and peripheral blood similarly react with mouse Leydig cells. Lymphocytes from spleens of athymic nude mice and from chicken bursa also give Leydig rosettes. It is suggested that mouse Leydig cells carry membrane receptors spontaneously recognized by lymphocytes.