Characterization of monoclonal lipid A antibodies with synthetic lipid A analogues

Abstract The specificity of monoclonal antibodies directed against the Salmonella minnesota R595 lipid A and the structural requirements of lipid A epitopes were studied with chemically synthesized lipid A analogues by enzyme-linked immunosorbent assay and its inhibition test. The results suggest that lipid A has specific and common epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups.     

Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis

A synthetic lipid A of Porphyromonas gingivalis strain 381 (compound PG-381), which is similar to its natural lipid A, demonstrated no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). On the other hand, compound PG-381 had stronger hemagglutinating activities on rabbit erythrocytes than compound 506. Compound PG-381 also induced mitogenic responses in spleen cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice, as well as LPS-responsive C3H/HeN mice. The addition of polymyxin B resulted in the inhibition of mitogenic activities, however, compound 506 did not show these capacities. Additionally, compound PG-381 showed a lower level of activity in inducing cytokine production in peritoneal macrophages and gingival fibroblasts from C3H/HeN mice, but not C3H/HeJ mice, in comparison to compound 506. Thus, this study demonstrates that the chemical synthesis of lipid A, mimicking the natural lipid A portion of LPS from P. gingivalis, confirms its low endotoxic potency and immunobiological activity.     

Biological activities of chemically synthesized partial structure analogues of lipid A

Analogues of the nonreducing sugar part of lipid A were chemically synthesized and tested for biological activities such as Limulus amebocyte lysate gelation, interferon- and tumor necrosis factor-induction, lethal toxicity in galactosamine-sensitized mice, and pyrogenicity. A 4-O-monophosphorylglucosamine derivative possessing 2-N-3-tetradecanoyl-oxytetradecanoyl and 3-O-tetradecanoyl groups (GLA-27) exhibited all activities tested except for pyrogenicity. Alteration of the acyl substituents or dephosphorylation as well as acylation or phosphorylation of the 6-OH caused most activities of GLA-27 to diminish or disappear altogether. On the other hand, the biological activities expressed by GLA-27 were not significantly affected even when the glucosamine backbone was changed to 1-deoxy type, epimer type at C-3 (allose form), or 3-amino type. These results indicate that the acyl substituents and the phosphorylation positions rather than the backbone structures in these partial structure analogues of lipid A affect the expression of biological activities of endotoxin. The results also clearly indicate that some biological activities of endotoxin can be expressed separately from pyrogenicity.     

Endotoxic and immunobiological activities of a chemically synthesized lipid A of Helicobacter pylori strain 206-1

A synthetic lipid A of Helicobacter pylori strain 206-1 (compound HP206-1), which is similar to its natural lipid A, exhibited no or very low endotoxic activities as compared to Escherichia coli-type synthetic lipid A (compound 506). Furthermore, compound HP206-1 as well as its natural lipid A demonstrated no or very low mitogenic responses in murine spleen cell. On the other hand, compound HP206-1 showed a weaker but significant production of interleukin-8 in a gastric cancer cell line, MKN-1, in comparison with compound 506. Furthermore, compound HP206-1 exhibited induction of tumor necrosis factor-alpha production in human peripheral blood mononuclear cells and the cytokine production was clearly inhibited by mouse anti-human Toll-like receptor (TLR) 4 monoclonal antibody HTA125. Our findings indicate that the chemically synthesized lipid A, mimicking the natural lipid A portion of lipopolysaccharide from H. pylori strain 206-1, has a low endotoxic potency and immunobiological activities, and is recognized by TLR4.     

Synthesis of lipid A type carboxymethyl derivatives with ether chains instead of ester chains and their LPS-antagonistic activities

Synthesis of lipid A type carboxymethyl derivatives having ether chains at both the C-3 and C-3' positions and their LPS-antagonistic activities toward human U937 cells are described.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Early embryonic lethality caused by targeted disruption of the TRAF-interacting protein (TRIP) gene

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are key adaptor molecules in the TNFR-signaling complexes that promote a wide variety of signaling cascades including cell proliferation, activation, differentiation, and apoptosis. TRAF-interacting protein (TRIP) is required for the inhibitory regulation of TNF-induced NF-κB signaling via the TNFR/TRAF-signaling complexes in vitro. TRIP also directly interacts with the familial cylindromatosis tumor suppressor gene (CYLD) and negatively regulates NF-κB activation in vitro. However, although there appears to be a relationship between TRIP, the TRAFs and also CYLD as modulators of NF-κB signaling in vitro, the functional role of TRIP in vivo is still unclear. To identify the role of TRIP in vivo, we have generated TRIP-deficient mice. Homozygous mouse embryos were found to die shortly after implantation due to proliferation defects and excessive cell death. These results indicate that TRIP is an essential factor during early mouse embryonic development in vivo.     

The structure of lipid A from the lipopolysaccharide of Rhodopseudomonas gelatinosa 29/1

The structure of the lipid A component of Rhodopseudomonas gelatinosa 29/1 lipopolysaccharide was established. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester-and glycosidically-bound phosphate residues. Both phosphate groups are in turn nonstoichiometrically substituted by ethanolamine. The amino groups of the disaccharide are N-acylated by 3-acyloxyacyl residues: that at the reducing glucosamine by 3-O-(14:0) 10:0, and that at the non-reducing one by 3-O-(12:0)10:0. Hydroxyl groups at C-3 and C-3 are esterified by hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free lipid A were shown to be unoccupied by methylation with diazomethane. A similar methylation of the intact lipopolysaccharide revealed a free hydroxyl group only at C-4, indicating that C-6 is the attachment site of 3-deoxy-d-anno-octulosonic acid.By preparative thin-layer chromatography free lipid A could be resolved into at least two major and one minor fractions. Lipid A of R. gelatinosa 29/1 shows high lethal toxicity, comparable to that of Salmonella lipid A.Abbreviations GlcN d-Glucosamine - dOclA 2-keto-3-deoxy-d-manno-octonate - GC-MS combined gas liquid chromatographymass spectrometry - LPS lipopolysaccharide Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r=0.993, P<0.001). Similar ML levels were measured in drug-induced apoptotic cells (A≈30–40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter≤1.0 μm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, ‘biochemically active’ phase of programmed cell death.     

Association study of TRAF1/C5 polymorphism (rs10818488) with susceptibility to rheumatoid arthritis and systemic lupus erythematosus: A meta-analysis

To determine whether the tumor necrosis factor (TNF)-receptor associated factor 1/complement component 5 (TRAF1/C5) polymorphism (rs10818488) confers susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematous (SLE), a meta-analysis was performed. A total of 11 studies with 17 comparisons (11 for RA, 6 for SLE) were available for this meta-analysis, which consisted of 13,456 patients, 12,259 controls for RA and 1,894 patients, 6,729 controls for SLE. A significant association of the A allele of TRAF1/C5 polymorphism (rs10818488) with RA susceptibility was detected in the North Africa population (OR = 1.557, 95% CI: 1.225–1.977). Furthermore, the association between this allelic variant and SLE risk was additionally found in population of European (OR = 1.247, 95% CI: 1.060–1.466). Analysis also showed the A/G allelic frequency of TRAF1/C5 variant (rs10818488), in different healthy populations, had a different distribution (χ² = 269.41, P < 0.001). Taken together, our study demonstrates that the TRAF1/C5 polymorphism (rs10818488) may confer susceptibility to RA in North Africa population, and in European population, it might be a contributory factor towards SLE.     

Association between IL-4 polymorphism and acute rejection of solid organ allograft: A meta-analysis

Background

Cytokines have been implicated in the acute rejection of solid organ transplantation. Many studies have investigated the association between recipient or donor IL-4 polymorphism and acute rejection, with different studies reporting inconclusive results.

Methods

We searched PUBMED and EMBASE until June 2012 to identify eligible studies investigating the association between IL-4 polymorphism with acute rejection after solid organ transplantation. Statistical analysis was performed using STATA10.0.

Results

A total of 12 studies were included. Pooled ORs suggested 1) no significant association was detected between recipient or donor IL-4 − 590C/T polymorphism and acute rejection of solid allograft; 2) no significant association was detected between recipient IL-4 − 33C/T polymorphism and acute rejection of solid allograft; 3) when stratified by transplantation type, IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation (T/T + C/T vs. C/C: OR = 0.36, 95%CI = 0.14–0.90); 4) significantly decreased risk of acute rejection was detected in recipient IL-4 − 590*T-negative/donor T-positive genotype pairs than all other recipient–donor IL-4 − 590T/C pairs (OR = 0.14, 95%CI = 0.03–0.66).

Conclusions

Our meta-analysis suggested that recipient IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation, but nor renal or heart transplantation. It was also suggested that combined recipient IL-4 − 590*T-negative/donor T-positive genotype may suffer decreased risk of acute rejection of solid allograft. Further well-designed studies with larger sample size were required to verify our findings, with focus on the association of IL-4 polymorphism with acute rejection in patients with liver transplantation and studies investigating combined recipient–donor genotype.     

The p76 and p100 truncated forms of the Rb protein exert antagonistic roles on cell death regulation in human cell lines

Several caspase-cleaved forms of the retinoblastoma protein have been described. Here, we compared the effect of full-length Rb versus the truncated p76^Rb and p100^Rb proteins on cell death regulation in five human cell lines. Interestingly, we observed that p76^Rb triggers cell death in all tested cell lines and that p100^Rb protects two cell lines against etoposide or TNF-α-induced cell death, whereas full-length Rb has no apoptotic effect. These results show that truncated forms of Rb can have specific activities in the regulation of cell death. They also suggest that caspase cleavage of Rb should not be simply assimilated to a degradation process. Finally, we show that cell death induced by p76^Rb is Bax-dependent and is diminished by Bcl-2 overexpression or by caspase inhibition and that p100^Rb could inhibit cell death by decreasing both p53 stability and caspase activity.     

Structure of the sugar-phosphate moiety of lipid A from lipooligosaccharide of Neisseria meningitidis group B,strain BC5S No. 125

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BC₅S No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)--gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)--glucopyranose carrying the -(2-aminoethyl)pyrophosphate residue at 0–4 and the pyrophosphate or phosphate residue at 0–1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0–1 was practically completely removed whereas that at 0–4 was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0–4. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues.Abbreviations KDO 2-keto-3-deoxyoctulosonic acid - LA-I, LA-II preparations of lipid A - LOS lipooligosaccharide - LOS-H⁺ the acidic form of LOS - OS oligosaccharide - TLC thin-layer chromatography - GLC-MS gas-liquid chromatography/mass spectrometry     

Dietary phytosterols modulate T-helper immune response but do not induce apparent anti-inflammatory effects in a mouse model of acute, aseptic inflammation

Although most studies have focused on the cholesterol-lowering activity of phytosterols, other biological actions have been ascribed to these plant sterol compounds, one of which is a potential immune modulatory effect. To gain insight into this issue, we used a mouse model of acute, aseptic inflammation induced by a single subcutaneous turpentine injection. Hypercholesterolemic apolipoprotein E-deficient (apoE(-/-)) mice, fed with or without a 2% phytosterol supplement, were treated with turpentine or saline and euthanized 48 h later. No differences were observed in spleen lymphocyte subsets between phytosterol- and control-fed apoE(-/-) mice. However, cultured spleen lymphocytes of apoE(-/-) mice fed with phytosterols and treated with turpentine showed increased IL-2 and IFN-gamma secretion (T-helper type1, Th1 lymphocyte cytokines) compared with turpentine-treated, control-fed animals. In contrast, there was no change in Th2 cytokines IL-4 and IL-10. Phytosterols also inhibit intestinal cholesterol absorption in wild-type C57BL/6J mice but, in this case, without decreasing plasma cholesterol. Spleen lymphocytes of turpentine-treated C57BL/6J mice fed with phytosterols also showed increased IL-2 production, but IFN-gamma, IL-4 and IL-10 production was unchanged. The Th1/Th2 ratio was significantly increased both in phytosterol-fed apoE(-/-) and C57BL/6J mice. We conclude that phytosterols modulate the T-helper immune response in vivo, in part independently of their hypocholesterolemic effect in a setting of acute, aseptic inflammation. Further study of phytosterol effects on immune-based diseases characterized by an exacerbated Th2 response is thus of interest.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.