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Background Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites ( RHS), have been robustly associated with atopic traits in genome-wide association studies ( GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. Methods Differential transcription factor binding was analyzed by electrophoretic mobility shift assays ( EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry ( LC- MS/ MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and He La cells. Results The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. Conclusion The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding. [ABSTRACT FROM AUTHOR]

Background Regulatory B cells have been identified that strongly reduce allergic and auto-immune inflammation in experimental models by producing IL-10. Recently, several human regulatory B-cell subsets with an impaired function in auto-immunity have been described, but there is no information on regulatory B cells in allergic asthma. Objective In this study, the frequency and function of IL-10 producing B-cell subsets in allergic asthma were investigated. Methods Isolated peripheral blood B cells from 13 patients with allergic asthma and matched healthy controls were analyzed for the expression of different regulatory B-cell markers. Next, the B cells were activated by lipopolysaccharide ( LPS), CpG or through the B-cell receptor, followed by co-culture with endogenous memory CD4+ T cells and house dust mite allergen DerP1. Results Lower number of IL-10 producing B cells were found in patients in response to LPS, however, this was not the case when B cells were activated through the B-cell receptor or by CpG. Further dissection showed that only the CD24hi CD27+ B-cell subset was reduced in number and IL-10 production to LPS. In response to DerP1, CD4+ T cells from patients co-cultured with LPS-primed total B cells produced less IL-10 compared to similar cultures from controls. These results are in line with the finding that sorted CD24hi CD27+ B cells are responsible for the induction of IL-10+ CD4+ T cells. Conclusions Taken together, these data indicate that CD24hi CD27+ B cells from allergic asthma patients produce less IL-10 in response to LPS leading to a weaker IL-10 induction in T cells in response to DerP1, which may play a role in allergic asthma. [ABSTRACT FROM AUTHOR]

Background Metabolomics is a versatile unbiased method to search for biomarkers of human disease. In particular, one approach in cancer therapy is to promote apoptosis in tumour cells; this could be improved with specific biomarkers of apoptosis for monitoring treatment. We recently observed specific metabolic patterns in apoptotic cell lines; however, in that study, apoptosis was only induced with one pro-apoptotic agent, staurosporine. Objective The aim of this study was to find novel biomarkers of apoptosis by verifying our previous findings using two further pro-apoptotic agents, 5-fluorouracil and etoposide, that are commonly used in anticancer treatment. Methods Metabolic parameters were assessed in Hep G2 and HEK293 cells using the newborn screening assay adapted for cell culture approaches, quantifying the levels of amino acids and acylcarnitines with mass spectrometry. Results We were able to identify apoptosis-specific changes in the metabolite profile. Moreover, the amino acids alanine and glutamate were both significantly up-regulated in apoptotic Hep G2 and HEK293 cells irrespective of the apoptosis inducer. Conclusion Our observations clearly indicate the potential of metabolomics in detecting metabolic biomarkers applicable in theranostics and for monitoring drug efficacy. [ABSTRACT FROM AUTHOR]

The removal mechanism of a hydrous ferric oxide (HFO) reactive filtration (RF) process with coupled chemically enhanced secondary treatment (RECYCLE) for phosphorus removal from municipal wastewater (HFO-RF-RECYCLE) was examined. A 0.95-ML/d (0.25-mgd) demonstration of HFO-RF-RECYCLE was performed at a municipal wastewater treatment plant equipped with oxidation ditches and secondary clarifiers. Influent to the plant averaged 6.0 mg/L phosphorus, with a 3-month tertiary effluent average of 0.011 mg/L phosphorus. In addition to aqueous geochemical modeling, experiments with surface charge, scanning electron microscopy, adsorptive capacity, thermal desorption, and most probable number of iron(III)-reducing bacteria were performed on samples from the system, to determine the major phosphorus-removal pathways. Results suggest that, in addition to filtration of particulate phosphorus, the low tertiary effluent total phosphorus result was achieved by adsorption. [ABSTRACT FROM AUTHOR]

This work examines the performance of a hydrous ferric oxide (HFO) reactive filtration (RF) process with coupled chemically enhanced secondary treatment (RECYCLE) for phosphorus removal from municipal wastewater (HFO-RF-RECYCLE). A 3-month, 0.95-ML/d (0.25-mgd) demonstration of HFO-RF-RECYCLE was performed at a municipal wastewater treatment plant equipped with oxidation ditches and secondary clarifiers. Influent to the plant averaged 6.0 mg/L phosphorus, with a tertiary effluent average of 0.011 mg/L phosphorus. Iron doses to the plant were low, at 5 mg/L. Inline recycling of HFO solution rejects to the plant influent resulted in a maximum 90.3%, dose-dependent reduction of phosphorus in the secondary effluent at 4.5 ML/d (1.2 mgd). Other results included reduction of total suspended solids and turbidity. A mass balance analysis was performed. We conclude that HFO-RF-RECYCLE may allow very low levels of phosphorus discharge from municipal wastewater treatment plants with a ferric-iron-based tertiary filtration process and residual recycling. [ABSTRACT FROM AUTHOR]

Previous studies have shown that genetic factors control the susceptibility to mercury-induced immunoglobulin (Ig)G1 antibody formation, IgE synthesis, renal IgG deposits and antinucleolar autoantibodies (ANolA) production in the susceptible mice. In this study, we examined the genetic control of resistance to these characteristics after HgCl2 injection in F1 hybrid crosses between the highly mercury resistant DBA/2 and mercury susceptible NZB (H-2d), SJL (H-2 s), A.CA (H-2f) and DBA/1 (H-2q) mice and also in backcross hybrids between (DBA/2 × SJL)F1 and SJL mice. We observed that mercury-induced immune/autoimmune manifestations were profoundly downregulated in most (if not all) of the F1 hybrids, indicating that the resistance to mercury was a dominant trait. Analysis of mercury-induced immune/autoimmune responses in the (DBA/2 × SJL) × SJL backcross hybrids suggested that only one gene or a cluster of genes determined the resistance to the ANolA production, whereas the resistance to other characteristics was controlled by two and/or three gene loci. By H-2 genotyping the backcross mice, it was found that H-2d haplotype per se could confer resistance to ANolA production. However, we did not find any significant association between the H-2d haplotype and the resistance to increase of IgG1 and IgE synthesis and the development of renal IgG1 deposits. Thus, while in DBA/2 mice, gene(s) in the H-2 loci strictly contribute to the inheritance of resistance to ANolA production; non-H-2 genes mainly govern the inheritance of unresponsiveness regarding other characteristics. [ABSTRACT FROM AUTHOR]

SummaryThe objective of this study was to establish a suitable model for the prediction of the environmental stability of antibiotics in water and liquid manure. The model consists of incubation systems for solutions with installations for stirring and ventilation. It was used to examine the stability of tetracycline in ventilated and unventilated Ringer's solution and liquid pig manure for a period of 8 days. Tetracycline concentrations decreased significantly during the experiment. The fastest degradation was observed in ventilated liquid manure. The test might be used as a screening test for the determination of the degradation half-life of antibiotics in the first phase of an environmental safety study according to directive 92/18/EEC. [ABSTRACT FROM AUTHOR]

The binding properties of h. a. IgGa to the Fc-γ receptor on rat basophilic leukaemia cells and rat mast cells were analysed. When h. a. IgGa was coupled to SRBC, rosette formation was observed with normal mast cells as well as with cells obtained from Nippostronglyus brauliensis-infected rats. The binding of aggregates was detected by fluorescent analysis. With monomeric IgGa, no uptake was observed. Binding analysis was also performed with aggregates of different sedimentation coefficients varying from 10S to 130S. It was demonstrated that h. a. 125I-IgGa binds to rat mast cells as well as to RBL cells. The rate of binding is dependent on the number of Fc portions present within the aggregate. Pre-incubation of aggregates with protein A partially inhibited the subsequent binding. The binding of aggregates is more potent at 37°, increases by five-fold at pH 6 4 as compared to pH 7 4 and is reduced by 75% at pH 8·4, while the binding of 125I-IgE did not differ at the various pH values. In the presence of Ca2+ ions, a 30-40% increase in the uptake of aggregates was noted. Our results show the presence of Fc-γ-receptors on RBL-cells and rat mast cells by direct binding studies and suggest molecular differences of the Fc-γ and FC-ε receptors. [ABSTRACT FROM AUTHOR]

We here report the finding that the anti-inflammatory cytokine interleukin-10 (IL-10) inhibits motility of B lymphocytes. B cells were induced to display motile morphology and active migration by IL-4. IL-10 inhibited locomotor responses to IL-4, when B cells of both murine and human origin were used. The inhibitory effect of IL-10 was reversible, since washing of B cells preincubated in IL-10 restored the ability to respond to IL-4. Time-course experiments showed that IL-10 did not have to be present from the very onset of culture, but could be added as late as 5 hr after initiation. In addition, murine B cells stimulated with lipopolysaccharide (LPS) showed motile morphology, as well as cellular aggregation and proliferation. All these parameters were suppressed by IL-10. However, viability orb cells was not adversely affected by IL-10. Exposure to IL-10 did not result in any changes in the surface expression of molecules involved in adhesion, such as CD2, CD11a/CD18, CD44, CD54 or L-selectin, on B lymphocytes. [ABSTRACT FROM AUTHOR]

Injections of HgCl2 lead to autoimmune manifestations in genetically predisposed rats and mice. In this study, the authors examined the responsiveness of T subsets from different mouse strains to HgCl2 by tracing their expression of the very early activation antigen CD69. The authors found increased expression of the CD69 antigen on CD4+ T cells from the responder A.SW and BALB/c mice, but not on CD4+ T cells from the non-responder DBA/2 mice, indicating an activation of T helper cells in the responder strains. However, the CD69 antigen was induced on CD8+ T cells from all strains irrespective whether they were susceptible or resistant to mercury-induced autoimmunity. Since CD8+ T cells have been described as mediating immunosuppression and as being responsible for the resistance to autoimmune induction by mercury, the authors tested whether CD8+ T cells inhibited the activation of CD4+ T cells by HgCl2 in the non-responder strains. However, there was no evidence for a suppressive role of CD8+ T cells from the DBA/2 mice in the response to HgCl2. The findings indicate that T helper cells play a central role in the immunological effects of HgCl2 and unresponsiveness of T helper cells in the nonresponder strains is not due to CD8+-mediated immunosuppression. [ABSTRACT FROM AUTHOR]

Purified CD8+ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8+ cells would contribute to cytokine production in the presence of cytokine secreting CD4+ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4+ and CD8+ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8+ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4+ subset. When priming with SEB in vivo before subsequent restimulation in vitro, a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8+ cells and indicate that CD4+ and CD8+ T cells may, in a given situation, produce distinct cytokines. [ABSTRACT FROM AUTHOR]

Interleukin-4 (IL-4) mediated locomotor responses by murine B cells in vitro were examined in this paper. The IL-4 induced migration was found to act directly on purified, splenic B cells, without involvement of secondary mediators. It appeared that only a subpopulation of B cells was able to respond in migration assays. Flowcytometric analysis showed that the migrating cells had the characteristics of naive B cells: I-Alo, J11dhi, IgDhi. They also displayed high expression of the adhesion molecule L-selectin and made predominantly IgM antibodies. This is contrary to what has previously been observed regarding motile responses to chemotactic factors by T cells, which mostly affect memory or activated T cells. However, this is in accordance with other studies indicating that IL-4 is a cytokine that exerts its effect mainly on resting B cells. [ABSTRACT FROM AUTHOR]

High concentrations of thymus-independent (TI) antigens are capable of inducing polyclonal B cell activation by their intrinsic mitogenic properties, irrespective of the specificity of the Ig receptors. Due to a genetic defect on the 4th chromosome, B cells from C3H/HeJ mice do not respond to lipopolysaccharide (LPS). In order to define at which step the mutation affects the signalling pathway, we compared B cells from C3H/HeJ and CBA mice with regard to changes of three events, namely cell size, and MHC class I and II antigen expression after LPS stimulation. We found that cell size and expression of MHC antigens increase in B cells from CBA mice after LPS stimulation, whereas B cells from C3H/ HeJ mice do not respond at all. This suggests that the defect in C3H/HeJ mice interferes with early events in the signalling pathway, either due to the absence of a LPS receptor on B cell surface or the lack of an initial component necessary for effective signal transmission subsequent to LPS receptor binding. Our results also have shown that stimulation of anti-Ig antibodies and LPS differ in some signalling events and have different final effects on B cells, which suggests that they may function differently via distinct signalling pathways. [ABSTRACT FROM AUTHOR]

A palmitate-conjugate derivative of ovalbumin which can be inserted into the membrane of B cells has been prepared. The ability of these cells to act as antigen-presenting cells for specific T lymphocytes obtained from immunized mice was tested. It was found that the conjugates were more efficiently processed and presented than the naive form of the antigen. Palmitate-conjugated antibodies specific to ovalbumin were also inserted into the cell membrane of normal B lymphocytes. These cells were pulsed with the antigen and tested as antigen-presenting cells for T cells obtained from immunized mice. The antibody-decorated B cells presented ovalbumin more efficiently than non-decorated controls. Whether antibody-decorated, antigen-pulsed B cells could prime T cells in vivo was investigated. Some priming activity was found. [ABSTRACT FROM AUTHOR]

We have shown previously that different conditions in primary in vitro SEB stimulation of resting CD4+ T cells. could induce selectively production of either IFNγ or IL4 and IL10. The present investigation shows that the priming conditions also decide which lymphokines will be produced during restimulations. The cells that had been induced to produce mainly IFNγ during the primary SEB stimulation, or during stimulation with IL4 and IL10. synthesized the same lymphokines at restimulation, regardless of stimuli and were unaffected by exogenous IL4. Thus, at an early stage of primary SEB stimulation, cells became committed to produce a certain pattern of lymphokines which remained throughout the period of in vitro culture testing. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis synovial fluid (RA-SF) contains a novel biological activity, which selectively induces IgG2b antibody production in lipopolysaccharide (LPS)-activated mouse spleen cells in vitro and in vivo. Our previous studies have shown that this activity is not functionally identical to other well-known cytokines and interleukins. In this study we demonstrate the partial purification and biochemical characterization of the IgG2b inducing activity in RA-SF. Biochemical characterization revealed that the IgG2b inducing activity in RA-SF has the following properties: it is a protein, sensitive to pH » 11 and «4, which is precipitated by 50% of saturated ammonium sulphate and has a molecular weight of 50–70 kDa; it binds to Cibacron-blue and heparin and its activity is not mediated by immunoglobulins or immune complexes, which are present in RA-SF. Biochemical characteristics of the IgG2b inducing activity also differ from other cytokines and interleukins-The term IgG2b inducing factor is proposed for this novel activity. [ABSTRACT FROM AUTHOR]

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8+ cells and of exogenously added cytokines (interleukin (IL)-2, IL-4 and inierferon (IFN)-γ)on the cytokine production in splenic CD4+ T cells, IL-2, IL-4, IL-5 and IFN--γ production in CD4+ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8+ cells. On day 5 the cells were restimulated with mitogen alone and analysed lo evaluate the short-term development of cytokine-producing cells in such cultures, Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimulation, and the effects of both IL-4 and IFN-γ were augmented by IL-2, After preactivation in the presence of IL- 2 and IL-4, every third CD4+ cell could be induced to produce IL-4, Exogenous IL-4 or IFN--γ further decreased each other's production. Depiction of CD8+ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8+ cells will influence lymphokine production in CD4+ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4+ and CDS+ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines. [ABSTRACT FROM AUTHOR]

Lymphokine production. analysed at the single cell level, was compared in resting and primed T-cell populations. Cells were preactivated in vitro by repealed mitogen stimulations. or isolated as large, low density cells naturally activated in vivo, from normal spleens of unimmunized animals, A similar qualitative shift in the pattern of lymphokines synthesized after restimulation was found as a result of in vivo and in vitro preactivation of cells. Repeated stimulations in vitro resulted in a qualitative shift in the lymphokines produced in response to activation, from a dominance of IL-2 during the first and second culture, lo a dominance of IL-4 and IL-5 in the later stimulations. In vivo activation lead to a similar separation of lymphokine production as primarily IL-2 was made by small resting cells, while large cells preferentially produced IL-4 and IL-5, IFN-γ was produced by both small and large cells. Preactivation in vitro lead lo a more rapid appearance of lymphokines during restimulation. In contrast, the in vivo naturally activated cells responded with a slow onset of lymphokine production when stimulated in vitro. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies specific for ovalbumin were conjugated to palmitate and inserted into the membrane of normal spleen B cells. Their presence in the membrane, as well as their ability to bind ovalbumin, was established by immunofluorescence. The so called anti-ovalbumin-'decorated' B cells were tested for their ability to act as antigen-presenting cells for ovalbumin specific I-Ad-restricted T-cell hybridomas. It was found that the antibody-decorated B cells presented antigen more efficiently than non-decorated B cells. [ABSTRACT FROM AUTHOR]

We have investigated the ability of different cells from non-immunized mice of the BALB/c strain to present antigen to two ovalbumin-specific I-Ad-restricted T hybridomas. Lipopoiysaccbaride-activated B-cell blasts were found to be the most efficient antigen-presenting cells. Purified small and dense splenic B cells also stimulated the hybridomas, although not to the same extent as the activated blasts, but comparable to non-fractionated spleen cells, Glutaraldehyde-treated B cells failed to present antigen, whereas F(ab')2 anti-mouse IgM-treated B cells exhibited markedly increased ability to present antigen. Using flow cytometry, we further purified the resting B cells by sorting the small lymphocytes to ensure that the ability of these cells to activate the hybridomas was not due to contamination with large non-resting B ceils. The sorted small B cells retained the ability of antigen presentation. Their resting state was confirmed by the fact that they did not incorporate [3H]-thymidine as shown by autoradiographic analysis. [ABSTRACT FROM AUTHOR]

A collection of hybridomas from C57BL/6 mice producing antibodies to dextran B512 was analysed and found to reflect the immune response in vivo with regard to immunoglobulin class expression, T cell dependency und antibody affinity. IgM-, IgG-3, and IgG-2b, and IgA-producing hybridomas were found. IgG3-producing hybridomas were obtained from nude mice, indicating T cell independent IgG3 synthesis. All monoclonal antibodies were of κ light chain. A major antidextran idiotype was expressed in many monoclonals. Secondary immune responses to dextran were also suppressed at the hybridoma cell level. However, hybridomas from secondary responses produced antibodies expressing the major idiotype, suggesting that anti-idiotype mediated suppression was not responsible for the reduced secondary response. Most monoclonals belonged to the VHJ558 family, but the IgG3-producing hybridomas showed a preferential use of genes from the VHX24 family. All monoclonals were directed against internal structures of the dextran molecule. The affinity for dextran of the IgG antibodies produced in secondary immune responses was drastically increased, even when the mice were immunized with thymus-independent forms of dextran, indicating that T helper cells need not be involved in affinity maturation of the immune response. [ABSTRACT FROM AUTHOR]

We describe here that IL-4 and IL-5 together can induce the production of high amounts of IgG1 polyclonalantibodies by B cells from old (NZB × NZW)F1 miceeven in the absence of LPS. The effect was less marked in young mice from the same strain or in normal Ralb'ce mice. The cells sensitive to the treatment with the interleukins were the large B cells. Old mice had higher proportions of such cells than young mice or normal Balb'c mice, Synthesis of anti-DNA antibodies of the IgM class was induced. Although IL-4 plus IL.-5 induced a strong polyclonal IgG1 production, there were no IgG1 anti-DNA antibodies. [ABSTRACT FROM AUTHOR]

We do not agree with the analysis of Langman and Cohn on the function of Ig receptors. We have reviewed the available literature regarding anti-Ig activation of B cells and found it contradictory and unconvincing. We have presented experimental evidence on the inability of Ig receptors on B cells to mediate activation or tolerogenic signals. We suggest that the Ig receptors serve to focus antigen to specific B cells so the B cells can be activated by TI antigens or helper T cells. The Ig molecules also bind foreign antigen and thereby initiate internalization and antigen processing. The processed peptides are exported to the membrane, where they associate with MI-IC class!! antigens, thus transforming B cells into efficient antigen-presenting cells. [ABSTRACT FROM AUTHOR]

We have activated B cells with membrane-incorporated monoclonal antibodies against the hapten trinitrophenyl (TNP) using various concentrations of the polyclonal activator lipopolysaccharide (LPS) haptenated with TNP. We found that anti-TNP-decorated B cells were polyclonally activated by 1000-fold lower concentrations of TNP LPS than untreated B cells or B cells decorated with antibodies of other specificities, in order lo test whether the passively incorporated anti-TNP monoclonals could mediate activation signals or only served to focus the polyclonal activator TNP-LPS to the B cells, we compared the response of anti-TNP-decorated B cells from normal C3H/He mice and from the LPS-unresponsive strain C3H/HeJ. We found that B cells from C3H/HeJ mice could not be activated to polyclonal antibody synthesis, in contrast to B cells from the LPS-responsive strain C3H/He. Thus, contrary to what was previously suggested, the incorporated anti-TNP antibodies could not mediate activation signals, but only served to passively bind and concentrate TNP-LPS to the membrane of the B cells, thereby increasing the concentration of the polyclonal B-cell activator LPS to the B cells. [ABSTRACT FROM AUTHOR]

Synovial fluid from patients with rheumatoid arthritis (RA-SF) contains a biological activity which can replace T cells for activation of antibody secretion in human blood lymphoid cells and which can also induce the selective differentiation of IgG2b-secreting cells in lipopolysaccharide (LPS)-pre-activated mouse spleen cells. The B-cell activity of this factor was studied in CBA/N mice which have an X-linked B-cell immunodeficiency which manifests itself as a defective humeral response to certain thymus-independent antigens (TI-2). RA-SF has now been shown to reconstitute partly the B-cell deficiency in CBA/N splenic B cells in vitro. Addition of RA-SF to LPS-pretreated cell cultreuse results in IgG2b secretion in CBA/N spleen cells as well. In contrast to cells from normal CBA mice, cells from CBA/N mice cannot respond to interleukin 4(IL-4) after addition of LPS with production of IgG1 antibodies in vitro. However, the addition of RA-have been shown to allow the production of IgG antibody producing cell sin CBA/N splenic B cells. It is postulated that the xid immunodeficiency could be the result of a deficient production of biological activity which is abundant in RA-SF. [ABSTRACT FROM AUTHOR]

It is still not known how T cells are activated, which T-cell surface structures transmit activation signals, and if antigen-presenting ceils possess activation structures for T cells. We have studied whether the T-cell receptor (TcR) must be engaged for T-cell activation to occur. By using membrane-incorporated monoclonal antibodies, we artificially forced T cells to bind to antigen- presenting cells in a mixed lymphocyte reaction system and thereby bypassed the need for TcR engagement and also made it possible for any surface molecule on antigen-presenting cells to deliver a stimulatory signal to the T cells. Theoretically, T cells would become polyclonally activated by this procedure. However, we found that they did not, even though they were intimately bound to the antigen-presenting cell, thus demonstrating that the TcR must participate in antigen/MHC binding in order for the T cells to become activated. This study does not exclude the possibility that antigen-presenting cells possess structures that can activate T cells. [ABSTRACT FROM AUTHOR]

Mouse interleukin 4 (IL-4) has been shown to act on B cells as an induction factor for Ig class switch. We studied the characteristics of IL-4-regulated Ig isotype production in lipopolysaccharide (LPS)-stimulated splenic B-cell cultures with emphasis on the comparison between the IgGI and IgE responses. The results show that the kinetics for the appearance of IgGI and IgE isotypes are similar, but that the dose of IL-4 required for the induction of an IgE response is 3-10 times higher than that for an IgGI response. No requirement for T cells was found for the induction of either isotype. Pre-incubation of cells for 24 h with IL-4 alone was sufficient to induce an IgGI response when cells were recultured with LPS from days 1 to . However, the simultaneous presence of both IL-4 and LPS for at least 24 h was required for a detectable IgE response. For an optimal IgE response, IL-4 needed to be present for more than 72 h in LPS-activated cultures. The possible reasons for the different regulation of IgGI and IgE responses are discussed. [ABSTRACT FROM AUTHOR]

Human and mouse immunoglobulins (lg) or F(ab′)2 fragments of rabbit Ig were conjugated to lipids and the conjugates inserted into the membrane of mouse spleen cells. It was found that nearly all B cells, but not T cells, became decorated with lipid immunoglobulin. Both endogenous Is receptors and inserted Ig capped alter the addition of cross-linking F(ab′)2 antibodies and in both cases capping required energy. Capping or endogenous mouse Ig led to co-capping of inserted human Ig, but the reverse was not true. [ABSTRACT FROM AUTHOR]

Murine spleen cells were activated with concanavalin A (Con A), pokeweed mitogen (PWM), or phorbol myristate acetate (PMA) and the calcium ionophore A23187. Cells producing gamma interferon (IFN-γ) or interleukin 4 (IL-4) could be detected by lymphokine-specific monoclonal antibodies and indirect immunofluorescence. The frequency and kinetics of the lymphokine-producing cells were examined and were approximately the same after stimulation with Con A or PMA and A23187. Thirty hours after activation, 3-9% of the cells produced IFN-γ. There were few IL-4-producing cells, and the maximal frequency was 1 out of 400 spleen cells 48 h after activation. When the cells were activated with PWM, the frequency of IFN-γ-producing cells was still high 72 h after culture. The majority of the IFN-γ-producing cells were CD8+ and expressed receptors for IL-2. [ABSTRACT FROM AUTHOR]

The effect of trypsin on mouse spleen cells and enriched B cells, added alone or together with lipopolysaccharide (LPS), was investigated. With trypsin, proliferation in serum free spleen cell cultures was 2–6 times greater than the background using cells from LPS responder strains, and 2–4 times the background with cells from the C3H/HeJ strain. Trypsin also induced the formation of a low number of IgM plaque forming cells (PFC). When added together with LPS, trypsin increased the proliferation caused by LPS alone by 10–50% with cells from LPS responder strains and by 50–100% with cells from the LPS non-responder strain C3H/HeJ. Trypsin enhanced proliferation in cultures maximally stimulated by LPS. The increased proliferation obtained when trypsin was added to LPS-stimulation of cells from the C3H/HeJ strain, was therefore not interpreted as a reconstitution of the LPS response. We conclude that trypsin has a moderate mitogenic effect on mouse B cells, stimulating the cells to proliferate and secrete IgM. The mechanism of action is unknown, but is different and independent from the action of LPS. [ABSTRACT FROM AUTHOR]

Brain interstitial and cerebrospinal fluid drainage into the lymphatics was studied by injections of 5 μl of packed sheep red blood cells (SRBC) injected into the caudate nucleus, the occipital lobe, and the lateral ventricle of the brain in mice. The number of plaque-forming cells (PFC) was determined in the deep cervical lymph nodes, the axillar lymph nodes, and the spleen, and the number of PFC was compared with the response in the same tissues after intravenous immunization with 0.1 ml 10% SRBC. The weight of the deep cervical lymph nodes increased 3.0 times on day 3 after injection in the brain parenchyma compared with the weight of these nodes after intravenous immunization. The antigen-specific response peaked on day 5,392±37 PFC/106 for IgG in the deep cervical lymph nodes after antigen deposition in the caudate nucleus, whereas only a minor peak in the antigen-specific response was obtained after intraventricular antigen deposition, 127±79 PFC × 106 for IgG on day 6. There were no increased PFC in any of the lymph nodes after intravenous immunization. The experiments show an antigen-specific response in the deep cervical lymph nodes after intracerebral antigen deposition, whereas antigens deposited in the lateral ventricles drain preferentially to the blood, with a high response in the spleen. [ABSTRACT FROM AUTHOR]

The addition of Immune complexes (anti-horse red blood cell (HRC) antibodies plus HRC) to spleen cell cultures activated by lipopolysaccharide (LPS) selectively suppressed the anti-HRC plaque-forming cell (PFC) response, but did not affect the PFC response to sheep red blood cells (SRC). The degree of suppression was directly related to the concentration of immune complexes. F(ab′)2 preparations suppressed as efficiently as intact IgG, although the ability of the F(ab′)2 preparation to lyse the red cells was abolished. The addition of protein A to the immune complexes (using intact antibodies) did not affect the degree of suppression. The findings suggest that immune complex-induced suppression of polyclonal B cell activation is caused by constant pans of the light or heavy chains of the antibodies other than the Fc part. [ABSTRACT FROM AUTHOR]

We have found that some hybrids produced by fusing AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized CBA mice produce factors that affect the primary CBA IgM PFC response. By the use of IIPLC-DEAE chromatography, sometimes combined with affinity chromatography, we have purified from hyperimmune anti-Dx serum, from ascites-growing T hybrids, and from normal CBA serum a family of factors that specifically enhance the anti-Dx IgM response. They show specificity for either Dx or anti-Dx, they may contain an Iak determinant, and they are genetically restricted in their function. The factors from all three sources are found in the same fractions after separation and move in fractions without Ig contamination. The affinity-purified factor was eluted at an NaCl concentration of 0.15–0.20 M. On the basis of these characteristics we believe that we have separated a class of T cell-derived helper factors from serum. The concentration of the factor in scrum increases after hyperimmunization with Dx. As a specificity control we show that the same sera contain a natural anti-SRBC IgM response-enhancing factor. It co-separates with the anti-Dx-enhancing factor, but they tan be distinguished by their preferential binding to their own antigens. [ABSTRACT FROM AUTHOR]

When CBA × BALB/c mice are immunized with Th 1 hybrid prepared by fusing spleen cells from Dx-immunized mice with the AKR thymoma BW 5147. which secretes material that affects the anti-Dx response in CBA mice, antibodies arc produced that in the ELISA are shown to bind to the material secreted by Th 1 but not to the material secreted by parent BW 5147. A fraction of the Th 1 secreted material that starts eluting on DEAE chromatography in 20 mM Tris with 0,13 M NaC1 is shown to bind to Dx but not SRC or uncoated ELISA plates. This binding, like that of anti-Dx Ig, can be inhibited with 1–10 μg/ml concentrations of free Dx. However, the affinity of the product of Th 1 for Dx is apparently lower than that of anti-Dx Ig, since high concentrations of anti-Dx interfere with the binding of Th 1 products to Dx. On gel permeation chromatography, a similar Dx binding material elutes in two molecular weight areas, a high area of the molecular weight of IgG and less and a low area of the molecular weight of 43.0(H) and above, and both are found in samples of Th 1 ascites and hyperimmune CBA anti-Dx serum, but not (or less so) in normal CBA serum and not in the hyperimmune nude C57B1 anti-Dx serum. The Th 1-derived material that affected the anti-Dx response in previously shown experiments had similar elution characteristics. Since we also show that these fractions need not to contain Ig and that anti-Th 1 minimally cross-reacts with anti-Dx IgM or IgG, it is likely that that fraction of Th 1-secreted material represents a Dx-binding, non-Ig, anti-Dx response-regulating factor. [ABSTRACT FROM AUTHOR]

In vitro concanavalin A (Con A)-activated spleen cells suppressed the primary in vitro immune response of syngeneic spleen cells to sheep erythrocytes (SRBC) but enhanced the in vivo anti-SRBC responses when transferred to syngeneic untreated mice. Con-A-activated spleen cells injected into nude mice enabled these mice to respond to SRBC. [ABSTRACT FROM AUTHOR]

Dextranase mixed with dextran in vitro or injected into mice before the antigen abolished the immunogenicity of both thymus-dependent and thymus-independent forms of dextran. Dextranase could degrade dextran in vivo even when given 6-15 days after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 h, but not 48 h, after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. The induction of autoanti-idiotypic anti-dextran antibodies was abolished if dextranase was given 24 h, but not 48 h, after the antigen, indicating that anti-idiotypic antibodies can be induced in the absence of antigen or immune complexes once anti-dextran antibodies have been synthesized. [ABSTRACT FROM AUTHOR]

Bacterial lipoprotein (LP) and lipopolysaccharide (LPS) both activated an in viro line of the B-cell tumour BCL1 to IgM secretion, as determined by the protein A plaque assay. LPS but not LP activation was inhibited by polymyxin B. Activation with both LPS and LP resulted in a less than additive response. Several clones of BCL1 were tested, and all responded to both LPS and LP. Both LPS and LP induced broad dose-response curves in normal lymphocytes, recently cloned BCL1 cells, and cloned and synchronized (G1 phase) BCL1 cells. This suggests that the dose-response curve cannot be due to accumulation of responding cells with different threshold sensitivities for activation. We cannot exclude the possibility that the broad dose-response curve issue to a heterogeneity of the LPS or LP preparation. The results indicate that LPS and LP induce similar signals in BCL1 cells. Furthermore, binding to the cell membrane and activation of BCL1 cells by LPS or LP seem to be separate events. An anti-IgM antiserum inhibited spontaneous DNA synthesis and spontaneous and LP-induced IgM secretion of BCL1 cells. Equal inhibition was observed with F(ab′)2 fragments but not with Fab fragments of the antiserum. suggesting that cross-linking of IgM bound to the cell surface membrane-induced inhibition. Supernatants from concanavalin A (Con A)-activated spleen cells induced BCL1 cells to secrete IgM. Fab anti-IgM added alone to BCL1 cells did not induce IgM secretion. Furthermore. Fab anti-IgM plus Con A supernatant did not induce a higher response than the supernatant alone. This suggests that inductive signals via the IgM receptor do not occur in BCL1 cells. [ABSTRACT FROM AUTHOR]

Immunization with horse erythrocytes (HRBC) and a thymus-independent (TI) antigen of the TI-2 category, such as native dextran, dinitrophenyl-dextran (DNP-dextran), or DNP-Ficoll, resulted in a three- to four-fold enhancement of the plaque-forming cell response to the TI antigen. The effect was found to be T-cell-associated, since cyclosporin A abrogated enhancement. The T cells or T-cell factors Involved were elicited early in the response to HRBC and acted on the initial stages of the TI response. The HRBC-induced potentiation affected only B cells activated by the TI antigen; it could not induce a TI response when TI-2 antigen was given under nonresponsive conditions; and it had only a slight effect on the response to a thymus-dependent (TD) antigen. A soluble TD antigen, bovine serum albumin-coupled dextran (BSA-dextran), did not have potentiating ability equivalent to that of the particulate HRBC. It was concluded that non-specific T-cell help can influence TI-2 responses when they are present simultaneously. [ABSTRACT FROM AUTHOR]

The ontogenic development of the plaque-forming cell (PFC) response to the thymus-independent (TI) antigen alpha 1-6 native dextran B512 was studied to determine when the first minimal amounts of anti-dextran antibodies are formed. Substantial antibody responses to certain other TI polysaccharide antigens arise during the first month of life, whereas the development of the response to native dextran has been found to be conspicuously delayed in high-responder mice. Results indicated that CBA mice began to produce minimal amounts of anti-dextran antibodies between 15 and 21 days of age, and the development continued progressively until peak levels were reached at 90 days of age. The alpha 1-6 native dextran system is also one of the few murine models in which endogenous anti-idiotypic antibodies are formed subsequent to anti-dextran production. It has been shown that the anti-idiotypic antibodies are responsible for specific inhibition of secondary PFC responses to dextran. Suppression of the secondary response was used here to ascertain whether the initial low level of anti-dextran antibodies elicited in 15-day-old animals was sufficient to lead to inhibition of the secondary response. The approach confirmed the initiation of anti-dextran production at 15-21 days of age and indicated that the small amount of anti-dextran antibodies produced at this age was sufficient to induce the mechanism leading to suppression of the secondary response. [ABSTRACT FROM AUTHOR]

The effects of concanavalin A (Con A) on the effector phase of specific and nonspecific cytotoxicity were studied. The addition of the lectin to the cytotoxicity assay resulted in inhibition of specific cytotoxicity and induced the lysis of nonspecific targets only when the lectin was added after the target cells. Preincubation of the effector cells with the ligand strongly inhibited specific cytotoxicity and did not induce nonspecific cytotoxicity. However, preincubation of the target cells with Con A before addition to the assay had no effect on the specific lysis and strongly facilitated the lysis of nonspecific targets. The inhibitory effect was not due to the agglutinating property of the lectin, since another agglutinogenic and non-mitogenic lectin (Helix pomatia) did not inhibit cytotoxicity. Induction of effector-to-effector killing seemed unlikely, since the addition of Con A to 51Cr-labelled effector cells did not significantly enhance the release of isotope. The inhibitory effect could be reversed by a subsequent incubation of the Con-A-treated effectors with alpha-methyl- D-mannoside. We suggest that Con A inhibits specific alloreactive cytotoxicity by blocking the antigen-binding receptors of T cells and induces nonspecific cytotoxocity by already activated cytotoxic T lymphocytes (CTLs) by binding to the major histocompatibility complex (MHC) antigens of the targets and creating structures mimicking allogeneic MHC products that will be recognized by CTLs via the antigen-binding receptor. [ABSTRACT FROM AUTHOR]

We have compared previous studies of the immune response to dextran (Dx) B512 in thymus-deficient nu/nu and thymectomized, lethally irradiated, and bone marrow-reconstituted mice with those obtained with cyclosporin A (CyA) as a T-cell-inhibiting drug. Our data show that only immune responses to TD forms of Dx B512 are susceptible to suppression by CyA, whereas anti-alpha 1-6 and anti-DNP antibodies induced by high molecular weight dextran and DNP-native Dx, respectively, were not inhibited. Similar results were obtained when polyclonal responses were studied. The polyclonal response induced by another dextran preparation, the polyanion dextran sulphate (DxS), was inhibited by CyA to the same extent as polyclonal T-cell activation by T-cell mitogens. [ABSTRACT FROM AUTHOR]

Neonatal tolerance was induced in CBA mice to alloantigens expressed by lymphoid cells of the A/Sn strain. 15% of the grafted animals maintained viable skin grafts for greater than 120 days and thus were considered operationally tolerant. However, splenic lymphocytes from these animals were capable of in vitro activation of DNA synthesis in response to the tolerizing alloantigens. Likewise, the spleen contained precursors of cytotoxic effector cells capable, upon activation, of similarly recognizing the major histocompatibility complex alloantigens used for tolerance induction. In contrast, in vivo graft-versus-host (GVH) effector function was absent, suggesting a possible dissociation of cells involved in the in vitro cell-mediated lympholysis and the in vivo GVH. Determination of potential serum-blocking factors as a possible mechanism of graft survival in these tolerant animals proved negative. [ABSTRACT FROM AUTHOR]

The primary immune response to untreated sheep erythrocytes (SRBC) in vitro was suppressed by the addition of antibody-coated SRBC. A mixture of SRBC and antibody-coated SRBC also suppressed the induction of anti-SRBC plaque-forming cells by the polyclonal B cell activators lipopolysaccharide, purified protein derivative of tuberculin, and native dextran. Injection of a mixture of SRBC and antibody-coated SRBC into mice led to an increased response to SRBC. It seems plausible from the in vitro findings that the Fc part of antibodies complexed to an antigen can exert a negative signal on antigen-specific B cells that cannot be overcome by positive signals delivered by polyclonal B cell activators. [ABSTRACT FROM AUTHOR]

After a primary immune response to the alpha 1-6 epitope of dexiran B512, dextran high responder strains exhibit a specific inability to produce IgM und IgG antibodies against this epitope, although they gave unexpected secondary response to horse erythrncytes. Spleen cells from dextran-primed and-suppressed mice responded well to dextran after transfer to lethally irradiated previously untreated mice, indicating that tolerance or exhaustive proliferation of dextran reactive B cells is not responsible. Thymus-dependent dextran protein conjugates also induced specific suppression. Suppression to both dextran and horse erythrocytes could be passively transferred into untreated recipients with immune scrum. However, after absorption with horse erythrocyles and dextran, passive serum transfer only suppressed the response lo dexiran. It is suggested that the specific immunosuppression was caused by the appearance of autoanti-idiotypic antibodies directed against the immunoglobulin receptors of dextran-reactive B cells. [ABSTRACT FROM AUTHOR]

To test whether the polyclonal B cell activating property of protein A is due to its ability to bind to the Fc part of immunoglobulins, experiments were carried out with purified monomeric or dimeric Fc-binding fragments of protein A. Neither monomeric nor dimeric fragments induced polyclonal antibody synthesis in vitro, whereas intact protein A was a strong activator. It is concluded that the Fc binding part of protein A is not responsible for activation of polyclonal antibody synthesis. [ABSTRACT FROM AUTHOR]

Three different concentrations of horseradish peroxidase-labelled lipopolysaccharide (LPS-HRP) were added in vitro to spleen cells from the LPS high-responder strain C3H/Tif and to cells from the low-responder strain C3H/HeJ. After being washed and fixed the cells were exposed to the substrate and prepared for electron microscopy. After addition of 7 and 0.7 μg/ml of labelled LPS only lymphocytes from the high-responder strain were labelled. About 5–10 % of the cells from C3H/Tif bound LPS, which is in accordance with the known frequency of B cells possessing the genetically determined LPS receptor. At the highest dose of labelled LPS (70 μg/ml) a large proportion of lymphocytes from the low-responder strain also bound LPS. Erythrocytes from both strains bound LPS at all concentrations. It is concluded that LPS-HRP allows the detection at the cellular level of LPS binding to the genetically controlled membrane receptor for LPS. [ABSTRACT FROM AUTHOR]

CBA and C57BL mice both possess a VHdex gene coding for antibodies against the alpha 1–6 epitope of dextran B512. After immunization with dextran, CBA mice produce IgM plaque-forming cells (PFC) only, which show regular cyclical fluctuations. The second PFC peak disappeared after injection of dextranase, indicating that it is antigen-dependent. The anti-dextran response in C57BL mice is characterized by only one IgM peak, followed 1 day later by an IgG peak that may exceed the IgM response by a factor of 10. The IgG anti-dextran response in C57BL mice was thymus-independent. CBA mice gave an IgG response to the hapten fluorescein isothiocyanate (FITC) after immunization with FITC-dextran, indicating that dextran can function as a carrier for an IgG response in this strain. Attempts to induce IgG PFC against dextran by immunizing CBA mice with thymus-dependent dextran-protein conjugates consistently failed, although the conjugates induced IgG PFC in C57BL mice. Spleen cells from CBA mice failed to produce IgG antibodies against dextran after transfer into lethally irradiated C57BL mice. whereas the C57BL spleen cells produced IgG PFC after transfer into CBA mice. The lack of IgG synthesis against the alpha 1–6 epitope of dextran in CBA mice appears to be regulated exclusively at the B cell level and is restricted specifically to the VHdex gene product. [ABSTRACT FROM AUTHOR]