Drug modification of LPS-stimulated human monocyte-derived dendritic cells.

Many drugs have been reported to convert dendritic cells (DCs) into a tolerogenic phenotype in vitro. However, there is evidence that an additional stimulus, such as lipopolysaccharide (LPS), may also be necessary for tolerogenic function in vivo. Little is known concerning the effects of drug modification on LPS-prestimulated DCs. In this study, monocyte-derived immature DCs were stimulated with LPS first and the influence investigated of six different agents on surface antigen expression, cytokine production and lymphocyte proliferation and cytotoxicity. Mycophenolic acid- and rapamycin-exposed DCs had little effect on surface antigen expression or functional activity towards lymphocytes. In contrast, treatment of immature dendritic cells with aspirin, dexamethasone, 1alpha,25-dihydroxyvitamin D3 (VD3) or butyric acid was associated with diminished expression of CD1a, CD1c, CD40, CD80 and CD83. Dendritic cell modification by aspirin, dexamethasone and VD3 were all associated with decreased production of tumour necrosis factor-alpha (TNFalpha). Furthermore, VD3 treatment was associated with a consistent and significant elevation of IL-6 production. Aspirin-, dexamethasone- VD3- and butyric acid-modified DCs suppressed interferon-gamma production, proliferation and cytotoxicity in co-culture with allogeneic mononuclear cells, but inconsistent results were obtained with different allogeneic combinations. Different drugs show varying effects on DC phenotype. No single agent was consistently effective in suppressing the stimulation of allogeneic mononuclear cells and future work is needed to explore drug combinations.

Detection of an autologous ligand for mannan-binding lectin on human B lymphocytes.

Mannan-binding lectin (MBL) is a collectin and a major soluble pattern-recognition protein. MBL can distinguish self from nonself and altered self using its C-type carbohydrate recognition domain and may also interact via its collagen-like region with autologous cells. Recently, it was found that MBL could bind to adherent cells (monocytes) and dendritic cells in a specific and sugar-sensitive manner. We have now investigated the interaction of MBL with fresh human peripheral blood cells and report binding to B lymphocytes and natural killer cells. The binding to B lymphocytes was studied in detail and was compared with the binding of MBL to monocytes and dendritic cells. Binding of MBL to B cells was evident at physiological MBL and calcium concentrations but was optimal at supraphysiological MBL concentrations. It was readily inhibited by autologous serum, mannan, mannose, GlcNAc and (to a lesser extent) galactose but not by C1q. A similar, but not identical, inhibition profile was observed with dendritic cells, but monocytes were not sensitive to mannose or mannan. We conclude that MBL is capable of binding to differently glycosylated ligands on several autologous cell types via its carbohydrate-recognition domain. We speculate that this could have functional significance at extravascular sites, but perhaps only in individuals possessing MBL genotypes conferring MBL sufficiency.

Comparison of adult and fetal cytokine secretion during mixed lymphocyte reactions.

Umbilical cord blood transplants are associated with a lower incidence of graft-versus-host disease (GVHD) than adult marrow or peripheral blood stem cell transplants, and this could be related to a difference in cytokine production between fetal and adult mononuclear cells after allogeneic stimulation. Mixed lymphocyte reactions (MLRs) involving adult cells were associated with greater interferon-gamma (IFNgamma) secretion than MLRs between cord blood cells, although IL-2 secretion was similar. Experiments in which T cells were separated from accessory cells then recombined in artificial combinations indicated that differences in T cells were primarily responsible for the greater [IFNgamma]:[IL-2] ratios generally found after MLRs involving adult cells compared to fetal cells, but accessory cells also influenced this ratio. The cellular basis for the observed difference was not established, but mononuclear cell preparations from cord blood contained significantly higher proportions of CD16(+)56(-) NK-type cells and a CD19(+)1c(+) B cell subset, as well as more CD45 RA-expressing nai;ve T cells.

Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function.

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers. STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays. RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies. CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.

Variation in IgG1 heavy chain allotype does not contribute to differences in biological activity of two human anti-Rhesus (D) monoclonal antibodies.

BACKGROUND: Pooled human anti-Rhesus D antiserum is currently administered for the prevention of RhD alloimmunization. Increased demand, and decreased supply, of donated pooled antiserum has led to the investigation of the suitability of human monoclonal anti-RhD antibodies for use in its place. However, it is unclear which biological properties of monoclonal antibodies are important for function in RhD-positive foetal red cell clearance and the prevention of alloimmunization. Various antibodies behave differently in a number of in vitro assays of biological function. OBJECTIVES: To compare the function and structure of two human anti-RhD IgG1 monoclonal antibodies which differ in their ability to promote red cell lysis in vitro. In particular to examine whether the functional differences correlate to differences in the IgG1 heavy chain constant region (allotype). STUDY DESIGN: We report here the cloning, characterization and re-expression in stable myeloma cell transformants of cDNAs coding for two such antibodies, secreted by the heterohybridoma cell lines ESD-1 (THERAD 03) and LHM 70/45.3 (THERAD 06). The cDNAs were then recombined to exchange portions of the Fc encoding regions and the recombinant antibodies were assayed in vitro to determine RhD-positive red cell-dependent activity. RESULTS: Recombinant THERAD 03 and 06 antibodies behaved identically to the parent antibodies. The 'inactive' THERAD 06 did not have biological activity reconstituted by exchange with the THERAD 03 Fc regions, nor was THERAD 03 activity abolished by the reciprocal Fc region exchange. CONCLUSIONS: Human monoclonal anti-RhD antibodies can be cloned and re-expressed in stable cell lines, and exhibit identical properties to the parent antibodies. Differences in biological activity cannot be attributed to differences in IgG1 heavy chain allotype.

A retrospective study of red cell maternal antibodies by chemiluminescence.

Plasma samples from 109 patients with maternal IgG alloantibodies were investigated using a chemiluminescence (CL) assay, a functional test, to predict which antibodies were clinically significant. The CL assay was able to distinguish between those patients who were unaffected or mildly affected requiring only phototherapy, and those patients with moderate or severe haemolytic disease of the fetus or newborn (HDN) requiring transfusion therapy. The CL result was compared with the anti-D quantification result, the number of IgG molecules bound per red cell and, in 80% of the cases, the monocyte monolayer assay. If mothers carrying Rh-negative fetuses were ignored, then the CL assay correctly predicted the outcome for 93.4% of all cases (including those other than D), while the AutoAnalyzer and monocyte assay predicted correctly 92.7% (of the anti-D patients) and 81.5%, respectively. Greater than 80% of patients with severe or moderate HDN had both IgG1 + IgG3 subclasses in the maternal plasma, while those infants who were unaffected or only mildly affected had a greater chance of having IgG1 only (44%) in the maternal plasma, IgG3 only (27%) or both subclasses (29%).

A comparison of the functional activity of monoclonal and polyclonal Rh antibodies.

The functional activity of monoclonal anti-Ds has been compared with that of non-D monoclonal antibodies. Approximately 50% of anti-Ds were more active than a positive control while more than 75% of non-D antibodies failed to reach 10% activity of the positive control. These figures do not correlate with the IgG bound per red cell which, however, does correlate closely with the antigen site density as would be expected.

A case of unexplained mild Rh (D) haemolytic disease in utero.

This report describes the case of a patient with a history of HDN complicated by fetal losses, in which the alloantibody in this particular pregnancy did not appear to cause HDN in utero. No protective HLA-DR antibodies could be demonstrated, and transport of IgG across the placenta appeared to be normal. The infant's red cells possessed a normal D antigen and his mononuclear phagocyte system appeared unimpaired. However, the number of molecules of IgG bound in vivo per fetal red cell was below the level usually associated with significant haemolysis and HDN.

A chemiluminescence assay for erythrophagocytosis.

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 microliters, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35-45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.

The pathogenesis of emphysema.

Emphysema is an increase in size of the air spaces distal to the terminal bronchioles, and can thus only be diagnosed pathologically, but new quantitative CT methods hold promise, diagnosing, quantitating and locating the lesions in man, in life, non-invasively. The protease/antiprotease theory of the pathogenesis of emphysema proposes that cigarette smoke attracts alveolar macrophages to distal terminal bronchioles, these in turn releasing neutrophil chemotactic factors which attract circulating polymorphonuclear leucocytes, to release potent proteolytic enzymes (serine protease) in addition to the alveolar macrophage protease. These enzymes, which can cleave all the macromolecules of the lung interstitium, are antagonized (at least the serine elastase) in health by alpha 1-antitrypsin, a normal constituent of lung lining fluid. However, this can be oxidized by oxidants in cigarette smoke, and oxidants released by polymorphonuclear leucocytes in microbial killing. The role of these actions, and of antioxidants (both natural and therapeutic) and antielastases are reviewed, as well as the activities of lung defence cells in this process. Despite this explosion of recent knowledge, we are still unable to answer the all important question "Why don't all smokers develop emphysema?", and further research is needed into variability in these multiple factors involved in this important new theory of the pathogenesis of this, possibly the commonest of all respiratory disorders of a chronic disabling nature.

Estimation of phospholipase A2 activity in guinea-pig endometrium on days 7 and 16 of the estrous cycle.

Phospholipase A2 (PLA2) activity was detected in both the microsomal fraction and the supernatant fraction of guinea-pig endometrial homogenates following centrifugation at 100,000 x g for 60 min. Between 85% and 95% of the PLA2 activity was detected in the microsomal fraction. The PLA2 enzymes in both fractions had a pH optimum of 8.0. The microsomal PLA2 required 7 mM Ca2+ and the supernatant fraction PLA2 required 2 mM Ca2+ for optimum activity. The activities of the microsomal PLA2 and supernatant fraction PLA2 increased significantly from 662.6 +/- 74.5 and 42.0 +/- 7.6 pmol/mg protein/10 min respectively on Day 7 to 960.4 +/- 106.5 and 79.5 +/- 8.7 pmol/mg protein/10 min respectively on Day 16. There was, therefore, a 1.5- to 1.9-fold stimulation in the initial rate of PLA2 activity between Days 7 and 16 of the cycle. The activities of microsomal PLA2 in guinea-pig endometrium are more than adequate on both Days 7 and 16 of the cycle to account for the amounts of prostaglandin (PG)F2 alpha released from the guinea-pig uterus. It is considered that the activation of microsomal (presumably membrane-bound) PLA2 by increasing free intracellular Ca2+ levels is probably of more importance than the absolute activities of PLA2 in controlling the supply of free arachidonic acid for PGF2 alpha synthesis by the guinea-pig endometrium during the estrous cycle.

Uptake of [3H]-arachidonic acid by human endometrium. Differences between normal and menorrhagic tissue.

Human proliferative and secretory endometrium from normal women and from menorrhagic patients was maintained in culture for up to 24 h in the presence of [3H]-arachidonic acid (3H-AA). This prostaglandin (PG) precursor was incorporated into endometrial neutral lipids and phospholipids in a time-dependent manner. Uptake of 3H-AA into phospholipids was significantly higher in normal secretory endometrium than in normal proliferative endometrium. However, this increased uptake of 3H-AA into phospholipids between the 2 phases of the cycle did not occur in menorrhagic endometrium. In contrast, uptake of 3H-AA into neutral lipids (especially triglyceride) was approximately 2-fold higher in menorrhagic endometrium compared to normal endometrium at both stages of the cycle, particularly during the proliferative phase. Abnormalities apparently exist in menorrhagic endometrium in the uptake processes which control arachidonic acid (AA) turnover. These abnormalities may be responsible, in part for abnormal PG production by menorrhagic endometrium.

Kinetics of prostacyclin synthetase in umbilical artery microsomes from normal and pre-eclamptic pregnancies.

1 Prostacyclin synthetase in umbilical artery microsomes obeys Michaelis-Menten kinetics. 2 The maximum velocity (Vmax) of prostacyclin synthetase prepared from normal umbilical arteries was significantly higher than the Vmax of prostacyclin synthetase in umbilical arteries taken from pre-eclamptic pregnancies. 3 The Michaelis-Menten constant (Km) of the two prostacyclin synthetase preparations was significantly different with the synthetase from pre-eclamptic arteries have a higher affinity for substrate. 4 The limiting factor for prostacyclin synthesis is Vmax at high substrate concentrations which appears to be the case in umbilical arteries. However, at low substrate concentration there would be no difference in prostacyclin synthesis by the two forms of prostacyclin synthetase.

A comparison of arachidonic acid metabolism by microsomes from pregnant rat decidua and myometrium.

Microsomes were prepared from scraped decidua and myometrium of rat uteri on day 22 of pregnancy. When incubated with [1-14C] arachidonic acid decidual microsomes produced predominantly prostaglandin E2 with smaller quantities of prostaglandin F2 alpha, 6-oxo-prostaglandin F1 alpha, prostaglandin D2 and thromboxane B2. The major product from the incubation of the myometrial microsomes was 6-oxo-prostaglandin F1 alpha, with a small quantity of porstaglandin E2. Incubation conditions for the decidual and myometrial microsomes were characterized with respect to pH, incubation time, substrate, and co-factor concentrations for optimal prostaglandin biosynthesis. Biosynthesis was stimulated by reduced glutathione without altering the proportions of products synthesised. Hydroquinone was found to be essential for prostaglandin formation. The ability of several biogenic amines to substitute as co-factors for hydroquinone was investigated. There are major differences in the optimal incubation conditions for uterine microsomes and prostaglandin synthetase preparations from other tissues.

Prostaglandin and thromboxane production by rat decidual microsomes.

The preparation of a microsomal fraction from the decidual tissue of pregnant rat uteri is described. Incubation of such microsomes with a mixture of radiolabelled and cold arachidonic acid (51 micrometer) plus cofactors resulted in a 30% substrate conversion. Products were resolved into four peaks (A, B, C and D) by thin-layer chromatography. Combined gas-liquid chromatography-mass spectrometry and further thin-layer chromatography identified the products as PGF2 alpha (A); thromboxane B2 (B); a mixture of 6-OXO PGF1 alpha and PGE2 (C); PGD2 and PGE2 (D). PGE2 was the major product.