Suppression of B-cell activation and IgE, IgA, IgG1 and IgG4 production by mammalian telomeric oligonucleotides.

BACKGROUND: The fine balance of immunoglobulins (Ig) E, IgG1, IgG4 and IgA in healthy production is maintained by the interaction of B cells with adaptive and innate immune response. The regulation of toll-like receptors (TLRs)-driven innate and adaptive immune effector B-cell response and the role of mammalian telomeric TTAGGG repeat elements represent an important research area. METHODS: Human PBMC and purified naive and memory B cells were stimulated with specific ligands for TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9 in the presence or absence of telomeric oligonucleotides. B-cell proliferation, differentiation and antibody production were determined. RESULTS: TLR9 ligand directly activates naive and memory B cells, whereas TLR7 can stimulate them in the presence of plasmacytoid dendritic cells. Human B cells proliferate and turn into antibody-secreting cells in response to TLR3, TLR7 and TLR9, but not to TLR2, TLR4, TLR5 and TLR8 ligands. Stimulation of B cells with intracellular TLR3, TLR7 and TLR9 induced an activation cascade leading to memory B-cell generation and particularly IgG1, but also IgA, IgG4 and very low levels of IgE production. Mammalian telomeric oligodeoxynucleotide (ODN) significantly inhibited all features of TLR ligand-induced events in B cells including B-cell proliferation, IgE, IgG1, IgG4, IgA production, class switch recombination, plasma cell differentiation induced by TLR3, TLR7 and TLR9 ligands. CONCLUSION: B cells require specific TLR stimulation, T-cell and plasmacytoid dendritic cell help for distinct activation and Ig production profiles. Host-derived telomeric ODN suppress B-cell activation and antibody production demonstrating a natural mechanism for the control of overexuberant B-cell activation, antibody production and generation of memory.

Synthesis of the HSA-conjugate of the S-linked thiomimetic of the branched tetrasaccharide repeating unit of the immunostimulant polysaccharide, schizophyllan. Evaluation as potential immunomodulator.

The conjugate with human serum albumin (HSA) of the S-linked thioanalogue of the branched tetrasaccharide repeating unit of the polysaccharide, schizophyllan, was synthesized from 1,2,4,6-tetra-O-acetyl-3-S-[2,4-di-O-acetyl-3,6-di-S-(2,3,4,6-tetra-O-ac etyl-beta-D-glucopyranosyl)-3,6-dithio-beta-D-glucopyranosyl]-3-thio-bet a-D-glucopyranose [M.O. Contour-Galcera et al., Carbohydr. Res., 281 (1996) 119-128] in five steps, and its potential immunomodulatory activity was evaluated in human blood mononuclear cells. The protein glycoconjugate did not effect proliferation or production of IL-4, IL-5 and IFN-g in a significant way.

Glucocorticoids inhibit human antigen-specific and enhance total IgE and IgG4 production due to differential effects on T and B cells in vitro.

Although anti-inflammatory properties of glucocorticoids (GC) are well documented, their activity in allergic diseases is still controversial. Recently, it has been reported that GC can increase, both in vivo and in vitro, the polyclonal production of total IgE. In this study we investigated the effects of GC on the antigen (Ag)-specific IgE response in a human in vitro system with peripheral blood mononuclear cells or B cells of bee venom-sensitized individuals that allows the production of bee venom phospholipase A2 (PLA)-specific IgE and IgG4 antibodies (Ab). PLA-specific Ab were induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand (sCD40L) in the presence of interleukin (IL)-4. Indeed, dexamethasone and prednisolone enhanced the formation of total IgE and IgG4 in PBMC, while the production of PLA-specific IgE and IgG4 Ab was selectively inhibited in a dose-dependent manner. The suppressive effect of GC was mediated during Ag-specific stimulation and T cell-B cell interaction. This was due to GC suppressing specific T cell proliferation and cytokine production, whereas neither allergen-specific nor total IgE and IgG4 production by sCD40L/IL-4-stimulated pure B cells was affected. In contrast to GC, cyclosporine A inhibited both total and PLA-specific IgE and IgG4 secretion in peripheral blood mononuclear cells and B cell cultures. Further experiments showed that increase in nonspecific total isotype response resulted from inhibition of IL-4 uptake by cells other than B cells and sufficient availability of IL-4 to B cells for isotype switch and synthesis. Furthermore, demonstration of opposite regulatory effects of GC on specific and total isotype formation in vitro, including the inhibition of allergy-relevant Ag-specific IgE response, may contribute to a better understanding of apparently controversial observations, and explain why most allergic patients benefit from GC therapy.

Induction and differential regulation of bee venom phospholipase A2-specific human IgE and IgG4 antibodies in vitro requires allergen-specific and nonspecific activation of T and B cells.

Investigations on the mechanisms of IgE regulation in vitro have been conducted thus far in systems that allow the synthesis of total rather than specific IgE. To study the regulatory prerequisites of antigen-specific IgE antibody production, we have established a culture system that allows the generation of bee venom phospholipase A2-specific IgE and IgG4 antibodies. Allergen-specific IgE was induced by simultaneously activating T cells and B cells specifically with allergen and polyclonally with anti-CD2 and soluble CD40 ligand in the presence of IL-4. Additional stimulation of T cells through the CD2 activation pathway by two different anti-CD2 monoclonal antibodies enhanced both the allergen-specific and the total IgE and IgG4 responses. An optimal amount of allergen (0.1 ng/ml) resulted in the induction of both allergen-specific IgE and IgG4 antibodies. Higher antigen doses reduced allergen-specific antibodies and enhanced total isotype production. This differential regulation of allergen-specific and total isotypes reflects different allergen dose-dependent mechanisms in specific and polyclonal activation of T and B cells. Although both isotypes require IL-4 for initial induction, opposite regulatory effects by T cells were observed for IgE and IgG4 antibody expression. In peripheral blood mononuclear cell cultures stimulated with soluble CD40 ligand, IL-4, and phospholipase A2, stimulation of T cells with higher amounts of anti-CD2 enhanced IgG4 in parallel to increased IL-2 and interferon-gamma secretion but inhibited IgE synthesis. These results provide evidence for differential regulation of allergen-specific and total IgE and IgG4 by antigen concentration and demonstrate the pivotal role of T cells controlling the synthesis of the IgE and IgG4 antibody isotypes.

Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

Bee venom phospholipase A2 (PLA) is the major allergen in bee sting allergy. It displays three peptide and a glycopeptide T cell epitopes, which are recognized by both allergic and non-allergic bee venom sensitized subjects. In this study PLA- and PLA epitope-specific T cell and cytokine responses in PBMC of bee sting allergic patients were investigated before and after 2 mo of rush immunotherapy with whole bee venom. After successful immunotherapy, PLA and T cell epitope peptide-specific T cell proliferation was suppressed. In addition the PLA- and peptide-induced secretion of type 2 (IL-4, IL-5, and IL-13), as well as type 1 (IL-2 and IFN-gamma) cytokines were abolished, whereas tetanus toxoid-induced cytokine production and proliferation remained unchanged. By culturing PBMC with Ag in the presence of IL-2 or IL-15 the specifically tolerized T cell response could be restored with respect to specific proliferation and secretion of the type 1 T cell cytokines, IL-2 and IFN-gamma. In contrast, IL-4, IL-5, and IL-13 remained suppressed. Treatment of tolerized T cells with IL-4 only partially restored proliferation and induced formation of distinct type 2 cytokine pattern. In spite of the allergen-specific tolerance in T cells, in vitro produced anti-PLA IgE and IgG4 Ab and their corresponding serum levels slightly increased during immunotherapy, while the PLA-specific IgE/IgG4 ratio changed in favor of IgG4. These findings indicate that bee venom immunotherapy induces a state of peripheral tolerance in allergen-specific T cells, but not in specific B cells. The state of T cell tolerance and cytokine pattern can be in vitro modulated by the cytokines IL-2, IL-4, and IL-15, suggesting the importance of microenvironmental cytokines leading to success or failure in immunotherapy.

Direct measurement of cytokines (IFN-gamma, IL-4, -5, and -6) from organs after antigenic challenge.

A mouse in vivo model has been developed in which cytokines produced within the regional lymph nodes and other organs after an antigenic challenge were measured directly without the need of an in vitro reculturing step. Mice were subcutaneously (s.c.) immunized with antigens such as keyhole limpet hemocyanin (KLH) in aluminium hydroxide (alum) at the base of the tail. After defined periods of time lymph nodes, spleen, liver, and lung were collected and frozen. The organs were then homogenized (or sonicated), centrifuged, and the cytokine levels, for example, IFN-gamma, IL-4, IL-5, and IL-6 were determined by ELISA. It was found that injection of alum alone caused measurable cytokine production in draining lymph nodes, but inoculation of an antigen-alum mixture, such as KLH and dust-mite antigen (DMA), significantly increased in vivo cytokine production over that of alum alone. In the dose range tested there appeared to be an inverse relationship between the dose of antigen and cytokine levels. Thus, 0.1-microgram KLH induced more IL-4 in vivo than 10-micrograms KLH. Kinetic studies showed that for IL-4 and IFN-gamma, peak production occurred around days 5 and 6, respectively. Route of immunization and the dose of antigen were found to be very critical, in that injection of 10-micrograms DMA intraperitoneally induced significant levels of cytokines in the lung, while the same dose of antigen given at the base of tail, s.c., did not induce appreciable levels of cytokines in any organ tested. Cyclosporin A inhibited in vivo production of IFN-gamma, IL-4, and IL-5 by approximately 80%, but not that of IL-6. Surprisingly, dexamethasone enhanced the production of IL-6 while inhibiting all the other cytokines. In conclusion, these results suggest that direct determination of cytokines in the organs may provide an easy readout to assess differential effects of immunoregulatory molecules on the production of Th1- and Th2-type cytokines in vivo.

Mice lacking the IFN-gamma receptor have impaired ability to resolve a lung eosinophilic inflammatory response associated with a prolonged capacity of T cells to exhibit a Th2 cytokine profile.

To investigate the modulatory role of IFN-gamma on the induction and maintenance of Th2 mucosal immunity in vivo, experiments were performed in mice lacking the IFN-gamma R. Aerosol OVA challenge of immunized wild-type mice resulted in an infiltration of eosinophils into the lung, associated with the ex vivo production of Th2 cytokines (IL-4 and IL-5) from purified lung Thy1.2+ cells stimulated via the CD3/TCR complex. However, while immunized IFN-gamma R-deficient mice exhibited elevated levels of IgE, IgG1, and reduced levels of IgG2a compared with wild-type mice, there was no difference in the recruitment of eosinophils into the lung or the production of IL-4 and IL-5 from lung T cells on day 3. In contrast, up to 2 mo after a single Ag challenge, eosinophils were still present in the lungs of IFN-gamma R-deficient, but not wild-type, mice. Likewise, lung-derived T cells from IFN-gamma R-deficient mice produced higher levels of IL-4 and IL-5, both at 1 and 2 mo after OVA challenge compared with T cells from wild-type mice. We conclude that endogenous IFN-gamma regulates the humoral isotype Ab pattern, but does not modulate the commitment of T cells to a Th2 phenotype in vivo or the acute infiltration of eosinophils to the lung. However, in the absence of IFN-gamma-mediated signaling, there is a transition from a spontaneously resolving to a persisting eosinophilic inflammation of the lungs, associated with a sustained capacity of lung T cells to secrete a Th2 cytokine profile.

Cytokine immunotrapping: an assay to study the kinetics of production and consumption or degradation of human interferon-gamma.

With the aim of determining the rate of cytokine production, we have investigated immunoassay conditions which prevent consumption/degradation. These assays, termed cytokine immunotrapping assays (CITA), are based on early capturing of cytokines secreted during cell culture by immobilised or soluble mAbs and a recently described chemiluminescent immunoassay. Here we describe assay conditions using IFN-gamma as a prototype cytokine. For production of IFN-gamma, PBMC, purified CD4+ or CD8+ T cells, or diluted whole blood were cultured with different T cell stimulating agents. Polystyrene macrobeads precoated with an anti-IFN-gamma mAb were put in culture and after a defined incubation period, a dimethyl acridinium ester (DMAE)-labelled second anti-IFN-gamma mAb and sodium azide were added into the culture for additional 24 h. The beads were washed and chemiluminescence signals determined in a luminometer. Trapping experiments were also performed with the beads or the soluble mAbs alone. Irrespective of the configuration, IFN-gamma concentrations measured in trapping conditions were always higher (3-20-fold) than in conventional cultures. By using the best trapping combination, i.e. both bead-mAb1 and DMAE-mAb2 added at the start of culture (single step), it was possible to detect IFN-gamma production as early as 2 h. Also, IFN-gamma secreted by less than 500 PBMC or whole blood cells could be detected within 24 h. When purified CD4+ or CD8+ cells were used instead of PBMC, a reduction of the trapping effect was observed. Conversely, addition of monocytes to purified T cells increased the trapping factor suggesting that a substantial amount of IFN-gamma was consumed or degraded both by CD14+ cells as well as T cells in culture. Preliminary results show that this assay is also suitable for the early detection of IL-1 and IL-4 which are known to be more tightly regulated. Thus, the new principle described here is expected to be useful in clinical settings where both the time and amounts of material are limited to investigate the role of cytokines in particular disease.

Effects of a new protein kinase C inhibitor CGP 41251 on T cell functions: inhibition of activation, growth, and target cell killing.

The effects of CGP 41251, a specific inhibitor of protein kinase C (PKC), its inactive derivative CGP 42700, and of staurosporine have been analyzed in vitro on T lymphocyte functions. The proliferation of fresh human peripheral blood lymphocytes stimulated with antigen (PPD) or anti-CD3 mAb was strongly inhibited by both staurosporine (IC50 < 0.01 microM) and CGP 41251 (IC50 = 0.092 microM) but not by the PKC inactive compound CGP 42700 (IC50 > 10 microM). Antigen-specific activation and proliferation of mouse lymph node T cells was inhibited by staurosporine and CGP 41251 with IC50 values of 0.008 and 0.05 microM, respectively. The inactive derivative caused 50% inhibition in this mouse T cell assay only at concentrations of 25 microM. In order to evaluate possible differential effects of PKC inhibitors on CD4+ T cell subsets, murine T helper cell type 1 (Th1) and type 2 (Th2) clones were used. The KLH-specific clone 9/6 secretes IL-2 and IFN-gamma (Th1), whereas clone 9A/B does not secrete these lymphokines but secretes IL-4 and IL-5 (Th2). It was found that CGP 41251 inhibited antigen-induced proliferation of both Th1 and Th2 equally well with an IC50 of 0.02 microM. Furthermore, CGP 41251 inhibited the IL-2 or IL-2 and IL-4-mediated growth of Th1 and Th2 cells (IC50, 0.08 and 0.02 microM, respectively). Moreover, CGP 41251, but not CGP 42700, inhibited antigen-specific killing of target cells by Th1 clones (IC50 = 0.2 microM), a phenomenon which does not require cell proliferation. When Th1 cells were preincubated with the compound, washed, and rested for 24 hr, they killed the target cells, whereas killing by similarly preincubated, washed, but not rested Th1 cells was inhibited. Thus, the inhibitory effect of CGP 41251 is reversible and excludes the possibility of inhibition due to toxicity at the IC50 dose given. The comparison of CGP 41251 and staurosporine showed that although CGP 41251 has lower activity, it is more specific and much less toxic than staurosporine. Thus, CGP 41251 is more suitable for PKC studies.

Monoclonal anti-gamma interferon antibodies enhance experimental allergic encephalomyelitis.

Interferon-gamma (IFN-gamma) is a cytokine with multiple activities on a variety of cells. Under various circumstances, IFN-gamma can exhibit either pro-inflammatory or inhibitory actions. Treatment of SJL/J mice with a monoclonal antibody (Mab) to IFN-gamma during the afferent limb of the immune response to myelin protein produced an enhancement of acute experimental allergic encephalomyelitis (EAE), with increased morbidity, mortality and earlier onset of disease. Systemic administration of IFN-gamma did not improve or worsen clinical outcome, but delayed disease onset. Passive transfer of immune lymph node cells co-activated with MBP and anti-IFN-gamma Mab resulted in more sever disease than that induced by MBP stimulated cells or MBP and IFN-gamma co-stimulated cells. However, in vitro proliferation of an MBP specific T cell line was not influenced by IFN-gamma nor anti-IFN-gamma treatment. Mab to IFN-gamma inhibited suppressor function, in a non-specific assay. These in vivo and in vitro results suggest that systemic IFN-gamma serves as a physiological regulator of a suppressor mechanism in EAE. The abrogation of this regulatory mechanism by anti-IFN-gamma administration contributes to a more severe form of experimental allergic encephalomyelitis.

Functional heterogeneity of CD4-positive T-cell subsets: the correlation between effector functions and lymphokine secretion is limited.

Several effector functions and the lymphokine secretion pattern of 30 antigen-specific CD4+ T-cell clones have been investigated. The clones were generated directly by limiting dilution cloning of nylon wool-purified T-cells obtained from KLH immunized BALB/c mice and avoiding an initial bulk culture phase. Using this approach the CD4+ T-cell clones were grouped into helper and nonhelper subsets. Among the helper subset, clones which helped B-cells for specific antibody production by either cognate or noncognate recognition were identified. Some but not all of these helper clones fitted into the Th1 and Th2 scheme, if the lymphokine secretion pattern was evaluated. Among the nonhelper subset CD4+ clones which killed activated APC in a MHC class II-restricted and antigen-specific manner were identified. In addition, one clone which suppressed B-cell antibody production mediated by helper clones was found. However, neither the suppression of antibody responses nor the inability of the nonhelper clones to help B-cells is due to the killing of B-cells. Various attempts were made to convert nonhelper into helper clones and helper into killer clones, without success. Thus, the functional properties of these clones are stable traits and not convertible by varying the experimental conditions.

Three human interferon-alpha 2 subvariants disclose structural and functional differences.

The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.

Tenascin, an extracellular matrix protein, exerts immunomodulatory activities.

Tenascin is a nonubiquitous extracellular matrix protein mainly expressed during morphogenesis in embryonal life. In adults it reappears in malignant tumors and during inflammation and tissue repair. Extracellular matrix proteins can alter cell morphology, adhesion, motility, differentiation, and growth. Since cells of the immune system can express receptors for extracellular matrix, we investigated the effects of tenascin on human monocytes and T and B lymphocytes. Tenascin inhibited monocyte adhesion to fibronectin and enhanced the LFA-1 (lymphocyte function-associated antigen 1)-dependent clustering of Epstein-Barr virus-transformed B cells. The physiological consequences of the effects of tenascin were studied in several T-cell activation models. Tenascin inhibited T-cell activation induced by a soluble antigen (tetanus toxoid), alloantigens, or the mitogen concanavalin A. However, T-cell activation with phytohemagglutinin, crosslinked anti-CD3 antibody, or a mixture of ionomycin and phorbol ester was not inhibited by tenascin. Tenascin did not prevent interleukin 2-dependent T-cell growth or the cytolytic activity of an antigen-specific CD4+ T-cell clone. These results suggest that tenascin alters the adhesion properties of human monocytes, B cells, and T cells. The in vitro immunosuppressive activity of tenascin might be due to abrogation of an accessory cell function at an early stage of the interaction between antigen-presenting cells and T cells.

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Characterization of a monoclonal antibody against infective larvae of Brugia malayi.

Monoclonal antibodies were produced following immunization of mice with live infective larvae of Brugia malayi. One of these, 46.08.76, is an antibody that promotes adherence of mouse peritoneal macrophages and human peripheral blood leucocytes to the infective larvae of B. malayi and Wuchereria bancrofti, respectively, and kills them. Fresh normal serum, as a source of complement, augments this effect. The same monoclonal antibody conferred 89% protection to jirds (Meriones unguiculatus) against challenge infection of B. malayi stage-three larvae. This monoclonal antibody recognizes antigens of 80,000, 67,000, 52,000 and 36,000 MW proteins present among the antigens of larvae, as detected by an immunoblotting technique. The antibody also reacts with antigens of infective larvae of Litomosoides carinii, Dipetalonema viteae and B. pahangi, but to a smaller extent.

Enhanced antiproliferative action of interferon targeted by bispecific monoclonal antibodies.

It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.

Estimation of heterokaryon formation and hybridoma growth in murine and human cell fusions.

Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions.

Prostaglandin E2 (PGE2)-dependent suppression of interleukin alpha (IL-2) production in patients with major trauma.

The depression of interleukin-2 synthesis represents a major dysfunction within the cascade of immunologic defects induced by mechanical and thermal trauma. This study was undertaken to elucidate the negative control mechanisms that were responsible for the deficiency of IL-2 production in polytraumatized patients. Peripheral blood mononuclear cells (PBMC's) from 29 patients (average age, 35.8 years; average ISS, 35) were separated on post-trauma days 1, 3, 5, 7, 10, 14, and 21 and cultured as untreated cells (C), cells treated with indomethacin (C + INDO), and cells depleted of adherent cells (C-AC). Cell cultures were assayed for proliferative responses to PHA, IL-2 synthesis, PGE2 production, gamma-interferon levels, and phenotyping studies. On all days post-trauma there was found a marked reduction of IL-2 production compared to controls with a highly significant nadir from day 5 to day 10 with an almost 80% inhibition of IL-2 (p less than 0.005). C + INDO cells showed increases of IL-2 synthesis over untreated cells ranging from 48% (Day 1) to 220% (Day 7). Removal of adherent cells (C-AC) did not reverse the suppression of IL-2 production. gamma-interferon levels were depressed in parallel with IL-2 levels but did not increase with C + INDO. The phenotyping of the PBMC's showed highly significant suppression of OKT3+, OKT4+, and IL-2R+ lymphocytes as well as a highly significant elevation of the monocyte (p less than 0.005) count. There was a highly significant increase of PGE2 synthesis from monocytes, due to the monocytosis and to a higher capacity of synthesis of the individual cells following trauma. PGE2 levels peaked on Day 5 and 7 post-trauma at 400% of control (p less than 0.005). These data suggest that the suppression of IL-2 synthesis post trauma is caused mainly by two factors: the excessive PGE2 output of inhibitory monocytes and inadequate function in immature and/or blocked lymphocytes. The partial restoration of IL-2 synthesis by indomethacin suggests that blockade of the cyclo-oxygenase pathway as an immunomodulating therapy may reverse some of the immunologic abnormalities in multiple trauma patients.

Monoclonal antibodies to human renin: properties and applications.

A series of 11 different monoclonal antibodies generated against human kidney renin have been characterised. Their binding affinity, inhibition of renin activity, epitope distribution, crossreactivity with related enzymes and finally in vivo pharmacological effects were analysed. All antibodies were found to be specific for primate renin recognising 6 independent antigenic structures on the renin molecule. They expressed different effects on renin activity namely (1) no inhibition, (2) only partial, or (3) complete inhibition. Partially inhibiting antibodies demonstrated specific degrees of inhibition (30, 60 or 80%). One antibody, R-36-16, demonstrated an IC 50 of 1.3 X 10(-11) M/L and, when injected into marmosets, induced complete inhibition of plasma renin activity and reduction of blood pressure. Using a selected pair of antibodies a radioimmunoassay has been established providing a fast and highly reproducible determination of human and marmoset immunoreactive renin, detecting both active and inactive renin down to concentrations of 10 pg/ml (1.25 X 10(-17) moles of renin per 50 microliter sample).