Structural studies have been carried out on the O-specific polysaccharide from the lipopolysaccharide of the reference strain (CDC 1604-55) for serogroup O8 of Serratia marcescens. The polymer has a branched, tetrasaccharide repeating unit of D-galactose(Gal),D-glucose(Glc), and 2-acetamido-2-deoxy-D-glucose(GlcNAc) with the following structure: (Formula: see text). The anomeric configuration assigned to the glucose residue differs from that (beta) previously proposed [Tarcsay, L., Wang, C. S., Li, S.-C. and Alaupovic, P. (1973) Biochemistry 12, 1948-1955]. The structure of the O8 polymer is identical with that of one of two polymers present in the cell envelope of a strain (CDC 1783-57) of S. marcescens O14.

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of the reference strain for Serratia marcescens serogroup O22. The neutral polymer has a linear structure with the repeating unit shown. The same tetrasaccharide unit also forms the backbone of the branched neutral polymer isolated from the reference strain for serogroup O10, which cross-reacts strongly with O22. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-L-+ ++Rhap-(1----3)-alpha- D-GlcpNAc-(1----

The B lymphoproliferative disorders B chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) produce a number of autocrine growth factors, including tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-1, all of which may induce positive feedback growth loops. If such malignancies depend on these autocrine growth loops for survival, their interruption may be therapeutically valuable. Interferon alpha (IFN-alpha) abrogates TNF- or IL-6-induced proliferation of HCL and B-CLL cells in vitro and has therapeutic activity in these diseases. We have investigated the possibility that IFN-alpha may act by interrupting autocrine growth factor loops. If purified B-CLL or HCL cells are cultured in the presence of TNF, there is induction of mRNA for TNF, IL-1 alpha, IL-1 beta, and IL-6. However, culture in the presence of IFN-alpha in addition to TNF reduced the level of mRNA for all these cytokines, compared with cells cultured in TNF alone. While cytokine mRNA levels were diminished, levels of mRNA for the ribonuclease activator 2-5A synthetase were increased. Analysis of the kinetics of cytokine mRNA production showed that levels fall shortly after the rise of 2-5A synthetase mRNA. IFN-alpha may produce these effects by shortening the half-life of cytokine mRNA, since TNF mRNA half-life in B-CLL and HCL cells is substantially reduced when the cells are cultured with IFN-alpha. These data suggest that IFN-alpha may mediate its therapeutic effects in these malignancies by blocking autocrine growth factor loops.

The presence of neutrophils in the synovial joint of patients with rheumatoid arthritis (RA) is thought to be due to the activity of chemotactic factors released by activated cells in the joint. We have shown in this report, for the first time, the abundance of one such factor, interleukin 8 (IL 8), in the synovial fluid of patients both with RA and other non-RA joint diseases, and the spontaneous production of IL 8 mRNA by RA synovial cells in culture. There was no correlation between the levels of chemotactic activity and IL 8 protein, suggesting that other factors with similar neutrophil chemotactic activity are also present in the synovial fluid exudate. In support of this concept neither the level of chemotactic activity nor IL 8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex. Such migration is likely to be due to a number of chemotactic signals in addition to IL 8, which may either synergize with, or inhibit, the action of IL 8.

The presence of transforming growth factor-beta (TGF-beta) in inflammatory joint disease was investigated. Synovial fluid from patients with rheumatoid arthritis (RA) and patients with other non-autoimmune inflammatory joint diseases contained high levels of both active and latent TGF-beta. Levels of active TGF-beta did not correlate with drug regimen in either patient group or with the recovery period in the individuals with non-RA joint disease. Freshly isolated synovial cells from individuals with RA were shown by Northern blotting to express the mRNA for TGF-beta 1 and to secrete latent TGF-beta protein which could be neutralized by antibodies to TGF-beta 1 and TGF-beta 2. Lipopolysaccharide-stimulated peripheral blood mononuclear cells from normal donors produced interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha) which was inhibited by pretreatment of these cells with recombinant TGF-beta. Cytokine production was not inhibited if the addition of TGF-beta was used after the inducing stimulus, suggesting that in activated cells cytokine production cannot be inhibited. This was confirmed by the observation that neither TGF-beta 1 or TGF-beta 2 inhibited spontaneous IL-1 or TNF-alpha production by rheumatoid synovial mononuclear cells in culture. These findings show that despite the presence of active TGF-beta in RA synovial joints and the spontaneous production of latent (potentially active) TGF-beta by RA cells in culture, additional TGF-beta did not inhibit ongoing cytokine synthesis in vitro. This suggests that TGF-beta may not inhibit cytokine production in the rheumatoid joint although it cannot be ruled out that in vivo TGF-beta already has an immunosuppressive effect which cannot be further increased in vitro by exogenous protein.

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNF alpha with an affinity of 2.5 x 10(-9) M. This binding can be competitively inhibited with unlabeled TNF alpha or lymphotoxin (TNF beta).

The effect of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) on the expression of HLA-DR, and the production of the cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) by human peripheral blood monocyte-enriched populations was investigated. GM-CSF was shown to induce both the expression of HLA-DR and the cytokines IL-1 and TNF alpha in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma), which induced major histocompatibility complex (MHC) class II expression, did not induce IL-1 or TNF alpha production. However, IFN-gamma enhanced the cell surface expression of HLA-DR and the production of IL-1 and TNF alpha on monocyte-enriched cells stimulated by GM-CSF. By itself, GM-CSF did not induce surface class II expression on the human monocytic tumour cell line THP-1, whereas it synergized with IFN-gamma to induce surface expression. These cells responded to GM-CSF by producing IL-1 and TNF alpha; Northern blotting showed that mRNA levels of IL-1 and TNF alpha were transiently induced, similar to other cytokines. Our results indicate that GM-CSF is a major macrophage activating factor that is capable of inducing both the expression of HLA-DR and the cytokines involved in T-cell activation by macrophages; therefore, GM-CSF may be of importance in potentiating antigen presenting function.

Previous studies have indicated that the cytokine transforming growth factor beta 1 (TGF beta 1) has immunosuppressive properties and can inhibit the production of tumor necrosis factor (TNF) and Interleukin 1 (IL 1) by human peripheral blood mononuclear cells. In this study, we have examined the effects of TGF beta 1 on the production of Interleukin 6 (IL 6) by human peripheral blood mononuclear cells. Treatment with only TGF beta 1 leads to the induction of IL 6, and this was both dose- and time-dependent. The effect of TGF beta 1 was evident at the level of IL 6 mRNA, suggesting TGF beta 1-induced de novo synthesis of IL 6. Induction of IL 6 by TGF beta 1 was specific, as other cytokines made by mononuclear cells (TNF and IL 1) were not induced by TGF beta 1. Furthermore, when a panel of stimuli were compared for their ability to induce IL 1, TNF and IL 6 in the presence or absence of TGF beta 1, IL 6 levels were augmented in the presence of TGF beta 1, while the induction of IL 1 and TNF was inhibited significantly. These results indicate that TGF beta 1 has complex effects on the production of cytokines by peripheral blood mononuclear cells and that TGF beta 1 is not inhibitory for all cytokine production. The ability of TGF beta 1 to induce IL 6 suggests that IL 6 may mediate some of the effects of TGF beta 1.

One of the major functions of cytokines is their ability to regulate cell growth and differentiation. The complexity of this process has been highlighted by recent studies on murine thymocytes; it has been shown that a number of cytokines interact to regulate thymocyte growth. We have investigated the effects of interleukin 4 (IL-4) and interleukin 7 (IL-7) on human thymocyte proliferation. Although maximal proliferation was dependent upon the presence of the mitogen phytohaemagglutinin (PHA), IL-7 alone stimulated thymocyte growth. In order to determine if this proliferation was due to the induction of IL-2, this pathway was inhibited by the addition of blocking antibody to the IL-2 receptor. Proliferation induced with IL-7 plus PHA, but not that induced by IL-7 alone, could be blocked by this treatment. In contrast, IL-4 stimulated thymocyte proliferation only in the presence of PHA; this proliferation was not inhibited by antibodies to the IL-2 receptor. Our findings show that both IL-7 and IL-4 can act as growth factors for human thymocytes, and that these cytokines stimulate proliferation through distinct mechanisms.

Cultured human keratinocytes and squamous cell carcinoma (SCC) cell lines were analyzed for the presence of ribonucleic acid (RNA) transcripts for the cytokines interleukin-1 and interleukin-6 and for these proteins. This study demonstrates that both cytokines are synthesized and secreted by both normal keratinocytes and SCC lines. The rate of secretion of these cytokines can be augmented in response to a variety of stimuli including tumor necrosis factor-alpha, granulocyte-macrophage colony stimulating factor, transforming growth factor-beta and the combination of lipopolysaccharide and phorbol myristate acetate. Interleukin-1 and interleukin-6 have been reported to influence the proliferation of cultured human fibroblasts. However, these cytokines had no significant effect on the proliferation of human keratinocytes or the SCC lines tested. Although it seems unlikely that interleukin-1 or interleukin-6 could directly influence keratinocyte proliferation in vivo, the capacity of these cells to synthesize and release these cytokines supports earlier observations that keratinocytes may play an important role in augmenting an immune or inflammatory response.

The major fraction of an acidic galactoglucomannan present in lipopolysaccharide extracts from cell walls of the O23 reference strain of Serratia marcescens has the tetrasaccharide repeating-unit shown. In a minor fraction, L-glutamic acid was amide-linked to about half of the D-glucuronic acid residues. The possible contributions of the acidic polymers and a neutral polymer produced by the organism to cross-reactions with other serogroups are discussed.

In order to define whether CD4+ T cells from autoimmune and non-autoimmune thyroid tissue could be classified according to their mediator production, lymphokine production was studied in 63 thyroid-derived CD4+ T-cell clones from four patients with Graves' disease, one with Hashimoto's thyroiditis, and one with non-toxic goitre (9-12 clones per patient). The production of interleukin 2 (IL-2), gamma interferon (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), lymphotoxin (LT), interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) was assessed at the mRNA level by slot-blot analysis in unstimulated clones as well as after activation with monoclonal anti-CD3 (OKT3) and IL-2. No lymphokine production was found in unstimulated clones, whereas 56% of the clones produced all six lymphokines simultaneously after stimulation. In the remaining 44% usually not more than one lymphokine was missing from the complete panel. Lymphokine mRNA concentrations varied between different clones and different patients, but, in this small sample, not between the diseases from which the clones were originated. There was a significant correlation between IL-6, LT, and IL-2 mRNA levels and T-cell helper function, which was estimated by the stimulation of thyroid microsomal autoantibody production using autologous peripheral B cells. TGF-beta and IFN-gamma mRNA expression was unrelated to T-cell help. The results demonstrate that intrathyroid T cells from autoimmune and non-autoimmune thyroid disorders cannot be classified according to their lymphokine production, unlike some results with in vitro-induced mouse T-cell clones, where two populations, Th1 and Th2, have been described. Single T cells are capable of producing a whole panel of lymphokines and thus are capable of triggering a multitude of different processes.

Both a neutral and an acidic polymer have been isolated from a lipopolysaccharide extract of Serratia marcescens strain S3255. The neutral polymer is a linear mannan with the repeating unit shown. The same repeating unit has been described for the O-specific polymers from Escherichia coli O8 and Klebsiella O5.

Simultaneous blood and cerebrospinal fluid (CSF) samples were taken from conscious sheep before, during and after parturition. Concentrations of plasma and CSF oxytocin were significantly elevated during contractions and particularly at birth. Mean prepartum CSF concentrations of oxytocin were around 55% of those found in plasma but postpartum they were up to 2-fold higher than those in plasma. Plasma concentrations of oxytocin were only significantly elevated, compared to prepartum levels, for 15 min postpartum whereas those in CSF were increased for the whole of the 120 min postpartum sampling period. Plasma, but not CSF, concentrations of arginine-vasopressin (AVP) were significantly raised during contractions and birth, and for 15 min postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were 10-fold higher than in CSF. In a separate experiment it was shown that 5 min of mechanical vaginocervical stimulation also stimulated significant increases in CSF and plasma oxytocin concentrations and in plasma vasopressin. Results support previous work suggesting an important role for central oxytocin release in the postpartum induction of maternal behavior and demonstrate that elevated concentrations of oxytocin in the CSF are present for a greater period than in blood. Elevated plasma AVP concentrations during contractions, birth or vaginocervical stimulation may be stimulated by stress associated with these stimuli.

In order to establish which neurotransmitters may influence the activity of zona incerta neurones in the sheep which respond selectively to the sight or ingestion of food, we have measured the release of amino acid and monoamine neurotransmitters from this region using microdialysis sampling. Co-ordinates for the placement of microdialysis probes in regions of the zona incerta where cells respond to the sight or ingestion of food were first established by making single-unit extracellular recordings. When animals were food-deprived results showed that release of gamma-aminobutyric acid (GABA) was increased in response to the sight and ingestion of food but not of aspartate, glutamate, taurine, noradrenaline, dopamine or serotonin. This release of GABA was absent when the animals were shown non-food objects or saw or ingested salt solutions. When the same animals were physiologically sodium-depleted GABA release was evoked by the sight and ingestion of salt solutions and release following the sight and ingestion of food was significantly reduced. These results provide further evidence that GABA is an important neurotransmitter in neural circuits controlling the regulation of food intake.

Transforming growth factor-beta 1 (TGF-beta 1) mediates many immunosuppressive effects on immune cells and can inhibit the production of tumor necrosis factor and interleukin 1 (IL 1). However, TGF-beta 1 can stimulate the production of IL 6 and platelet-derived growth factor, indicating that TGF-beta 1 initiates complex effects on the production of cytokines. In this report we show that treatment of peripheral blood monocytes with TGF-beta 1 leads to the induction of a recently described IL 1 receptor antagonist protein (IRAP). The effect of TGF-beta 1 was both dose and time dependent. TGF-beta 1 induced de novo synthesis of IRAP, as Northern blotting experiments indicated a rapid and transient induction of the mRNA encoding IRAP. The induction of IRAP suggests a potential mechanism by which some of the inhibitory effects of TGF-beta 1 are mediated.

The lipopolysaccharide extract from the cell wall of the reference strain for Serratia marcescens serogroup O18 contained, in addition to a neutral glycan characterised previously, an acidic glycan. Acidity was contributed both by D-glucuronic acid and by 4-O-[(R)-1-carboxyethyl]-D-glucose (4-O-Lac-D-Glc). By using n.m.r. spectroscopy, methylation analysis, and chemical degradations, the repeating unit of the acidic glycan was identified as a branched hexasaccharide having the structure shown; an O-acetyl group also present was not located. The glycan is believed to define the O18 serogroup, but is probably not an integral component of the lipopolysaccharide. [formula: see text].

The effect of monoclonal antibodies (MoAbs) recognizing idiotype, IgM heavy chain, and IgD heavy chain on the in vitro DNA synthesis of five randomly selected leukemic human low-malignancy B-cell lymphomas was investigated. In three lymphomas of different histologic subtype, low concentrations of anti-idiotypic (anti-Id) MoAb completely inhibited spontaneous 3H-thymidine uptake of T-cell-- and monocyte-depleted tumor cells, whereas two other tumors were not affected. Maximal inhibition of DNA synthesis was achieved at MoAb concentrations ranging from 0.5 to 250 micrograms/mL and required crosslinking by bivalent antibody but not Fc-mediated effects. While two anti-IgM MoAbs were similarly efficient as anti-Id MoAb in inhibition of DNA synthesis, two anti-IgD MoAbs had no effect. Thus, surface IgD molecules seemed to be neither able to deliver inhibitory signals themselves nor to antagonize IgM-mediated signals when simultaneously crosslinked by anti-Id MoAb. Leukocyte differentiation antigen expression, IgM density, and IgM/IgD ratio on the surface of lymphoma cells did not distinguish between sensitive and resistant tumors. In vitro tumor cell survival was differently affected by prolonged incubation with anti-Id antibody. In a centrocytic lymphoma and an immunocytoma, but not in a chronic lymphocytic leukemia, suppression of 3H-thymidine uptake persisted after removal of MoAb and tumor cell viability decreased during prolonged incubation with anti-Id MoAb. These results suggest that direct inhibitory signaling via surface IgM may contribute to anti-Id MoAb-mediated tumor regression in certain human B-cell lymphomas.

Lipopolysaccharide was isolated from both phases of an aqueous-phenol extraction of defatted cell walls from the reference strain for Serratia marcescens serogroup 021. The product from the aqueous phase was of the R type, lacking a polymeric side-chain. The polymeric fraction of the lipopolysaccharide from the phenolic phase (the 021 antigen) had a disaccharide repeating-unit with the following structure: ----4)-alpha-D-Glcp-(1----4)-beta-D-ManpNAc-(1----.

A neutral glycan containing L-rhamnose and 2-acetamido-2-deoxy-D-galactose is one of two polymers present in the lipopolysaccharide extract from the reference strain for Serratia marcescens serogroup O23. The glycan, which has the disaccharide repeating-unit shown, shares structural features with polymers from several other O serogroups. ----4)-alpha-L-Rhap-(1----4)-beta-D-GalpNAc-(1----.

Microdialysis sampling was used to measure the release of oxytocin (OXY) and monoamine and amino acid transmitters from the region of the medial preoptic area (MPOA) and the bed nucleus of the stria terminalis (BNST) during parturition and suckling in sheep. Results showed that OXY and gamma-aminobutyric acid release increased in both the MPOA and BNST during parturition and suckling. Noradrenaline (NA) release increased in both structures during parturition but not during suckling. Dopamine (DA) release increased in the MPOA and decreased in the BNST during both parturition and suckling. Aspartate release increased in the MPOA during parturition, and the BNST during suckling, and glutamate release increased in the MPOA and BNST at parturition and only in the BNST during suckling. No changes in the release of serotonin or taurine occurred in these structures during parturition or suckling. In a further experiment on 6 estrogen-primed sheep, OXY (10 micrograms/ml) was infused into the MPOA via bilaterally placed microdialysis probes. This treatment inhibited rejection behavior towards lambs, but did not activate positive maternal responses. These OXY infusions also stimulated release of NA. These results show that complex patterns of neurochemical release occur in two closely related areas of the brain, the BNST and MPOA, during parturition when maternal behavior is stimulated. However, while these patterns of release are similar in the two structures, particularly at birth when maternal behavior is stimulated, they are not identical during labor contractions and suckling. The release of oxytocin within the MPOA during parturition may be important for stimulating a reduction in aggression towards lambs, although this action might be mediated via the effect of OXY on NA release.

In addition to a neutral glycan, lipopolysaccharide extracts from the reference strain for Serratia marcescens serogroup O22 contain an acidic polymer which probably defines the serogroup and is of microcapsular origin. The polymer is doubly branched with a heptasaccharide repeating unit and a galactan backbone. By means of spectroscopic and degradative studies, the structure of the repeating unit was established as that shown. [formula: see text]