Production of auto-anti-idiotypic antibody during the normal immune response. XII. An enzyme-linked immunosorbent assay for auto-anti-idiotype antibody

An enzyme-linked immunosorbent assay (ELISA) to detect anti-idiotype (Id) antibody is described. Using this assay auto-anti-Id was detected in the serum of aged mice immunized with 2,4,6-trinitrophenylated-Ficoll (TNP-F). Hapten eluates from anti-TNP-F immune spleen cells also contained readily detectable auto-anti-Id.     

Different neuronal localization of aspartate-like and glutamate-like immunoreactivities in the hippocampus of rat, guinea-pig and Senegalese baboon (Papio papio), with a note on the distribution of gamma-aminobutyrate

Antisera were raised in rabbits against aspartate or glutamate conjugated to bovine serum albumin by glutaraldehyde. After immunosorbent purification the antisera reacted selectively with brain protein-glutaraldehyde conjugates of the respective amino acids. These results from model systems encouraged us to employ the antisera to study the distribution of free aspartate and glutamate in brain tissue. The aspartate antiserum produced intense staining of interneurons and deep hilar neurons and modest labelling of pyramidal and granular cells in the hippocampal formation of rats, guinea-pigs and baboons perfusion-fixed with glutaraldehyde. In contrast, glutamate-like immunoreactivity was generally high in pyramidal and granular cells and low in interneurons. In hippocampal slices immersion fixed in glutaraldehyde after being soaked in Krebs' solution aspartate-like and glutamate-like immunoreactivities were lost from perikarya and dendrites. The staining that remained occurred in nerve terminal-like dots and matched the distribution of the major excitatory fiber systems, except that only glutamate-like immunoreactivity, and not aspartate-like immunoreactivity, was concentrated at the site of the mossy fiber terminals, and that aspartate-like but not glutamate-like immunoreactivity occurred between the granular and pyramidal cell bodies. The present technique specifically demonstrates aspartate and glutamate in glutaraldehyde-fixed tissue. We suggest that in perfusion-fixed material the staining intensities reflect the total concentrations of the amino acids (i.e. the "metabolic pool" plus the "transmitter pool"). In immersion-fixed hippocampal slices the "transmitter pool" may be preferentially visualized.     

Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to study (i) binding of rabbit antibodies (raised against litter mate liver plasma membrane fraction) to the immunizing membrane fraction, and (ii) binding of human antibodies to liver membrane fractions and to liver-specific lipoprotein (a liver membrane-derived antigen complex). When assays were conducted using the non-ionic detergent Tween 20 as blocking agent, high non-specific binding was encountered. With the low titre rabbit antisera high binding of non-immune test antibody and of second antibody (anti-rabbit IgG) to the immunogen, and also directly to the solid phase, was found. This was abolished by replacement of Tween 20 in the antibody diluent buffers by a non-reactive protein, casein proving to be a more effective blocking agent than either bovine serum albumin or gelatin. With human sera, high binding of human IgG to the solid phase was noted. This too was blocked by casein, but only when the anti-microbial agent Thimerosal was included in the casein buffer, and when Tween 20 in the wash buffer was replaced by casein-Thimerosal so that the solid phase was exposed to casein before incubation with the test serum. The casein buffers described may prove of general value in solid-phase assays where high non-specific binding is encountered.     

3种成软骨诱导培养基对单层培养和微球培养的人脂肪干细胞成软骨效果比较

目的比较3种成软骨诱导培养基对单层培养和微球培养的人脂肪干细胞(human adipose mesenchymal stem cells,hASCs)体外成软骨分化的效果。方法从人脂肪组织中提取hASCs,分别采用单层培养和微球培养,加入3种不同成分的成软骨诱导培养基,A组:胎牛血清(FBS)+10 ng/ml转化生长因子3(TGF-β3);B组:胰岛素-转铁蛋白-亚硒酸钠(ITS)+1 ng/ml TGF-β3;C组:ITS+10 ng/mL TGF-β3。3周后提取单层培养的细胞蛋白进行成软骨相关标志物检测,并对细胞微球进行阿尔新蓝、甲苯胺蓝、天狼星红染色,比较成软骨效果。结果3周后单层培养体系C组的软骨标志物表达最高,微球培养阿尔新蓝、甲苯胺蓝、天狼星红染色C组着色最深。结论作为培养基添加物,ITS在诱导hASCs成软骨分化时的效果优于FBS,而作为刺激hASCs成软骨的重要生长因子TGF-β3,10 ng/ml的浓度比1 ng/ml作用更强。     

Homogeneous colorimetric enzyme inhibition immunoassay for cortisol in human serum with Fab anti-glucose 6-phosphate dehydrogenase as a label modulator

The Fab fragment of rabbit IgG antibody to bacterial glucose 6-phosphate dehydrogenase covalently linked to the cortisol retained the capacity to inhibit the enzyme completely. In optimal conditions the antibody to cortisol effectively bound the cortisol residues of the cortisol-Fab conjugate, making it incapable of inhibiting the enzyme. The enzyme modulatory properties of the cortisol-Fab conjugate were exploited to set up a direct competitive homogeneous enzyme immunoassay for cortisol in human serum. The procedure involved the use of the auxiliary enzyme diaphorase, specific for NADH, which converts the nitro blue tetrazolium salt to a colored formazan. The procedure detects modulated glucose 6-phosphate dehydrogenase activity by a single-point measurement without serum interference. The assay working range was between 20 and 640 micrograms/1 of cortisol and used 50 microliters of sample.     

Effect of reduction and alkylation on structure and function of rabbit IgG antibody--II. Effects on classical pathway C3 convertase formation

Anaerobic reduction of purified rabbit IgG antibody (Ab) with 1.5 moles of dithiothreitol per mole of Ab at pH 8.0, followed by alkylation, cleaves 39% of the inter-heavy-chain (H-H) disulfide (SS) bonds. This treatment has the following effects on the ability of the Ab to activate the classical pathway of complement. Compared to control Ab, reduced and alkylated (RA) Ab retained 4-5.6% of overall hemolytic activity and 55% of complement-fixing activity at 0 degrees C. Complexes of RA Ab and equivalent amounts of soluble Ag consumed C4, C2 and C3 at 37, 51 and 44%, respectively, of the rate at which these components were consumed by equal concns of complexes containing control Ab. Complexes made with RA Ab bound 18% as much C-1 as those made with native Ab. These data indicate that the principal, if not the only, effect of RA is on C-1 binding. Measurements of the ability of complexes of Ab with cell-bound Ag to bind C-1 showed at most a 20% loss of C-1 binding sites and a ca two-fold decrease in affinity for C-1. Similar results were obtained with purified (activated) C-1 and with native C1 in serum. No significant difference could be detected in the rate of activation of bound C1. Normal rabbit IgG which was reduced and alkylated under the same conditions retained 52% of its H-H SS bonds and 30% of its ability to bind C-1. This finding suggests that the impairment in C-1 binding results from an effect on the C1 binding site itself, rather than from an effect on the ability of the RA Ab to transmit a putative conformational "signal" from the Ag-binding site to the C1 binding site. Finally, our data show that the observed functional effect of reduction and alkylation depends strongly on the assay used to evaluate that effect.     

Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response

As an alternative to the measurement of radiolabeled thymidine incorporated into DNA, a method is presented in which thymidine has been replaced by its analogue, 5-bromo-2-deoxyuridine (BUdR). BUdR incorporated into DNA (BUdR-DNA) is measured by a sandwich-type enzyme immunoassay using a monoclonal anti-BUdR antibody. This method allows the quantitation of 4 ng of BUdR-DNA. Comparative experiments with myeloma cells and LPS stimulated spleen B-cells have shown that this technique is at least as sensitive as the traditional counting of [3H]thymidine.     

Macrophage handling of soluble immune complexes: Evaluation of mechanisms involved in the selective clearance of complexes from the circulation

The binding and release of soluble guinea pig IgG2-containing DNPBSA-anti-DNP complexes and antigen-free, covalently-linked anti-DNP IgG2 oligomers of similar size, by guinea pig peritoneal macrophages, has been examined in the absence and presence of monomeric IgG2, of unrelated antibody specificity, or the monovalent hapten, DNP lysine. Complex binding was found to differ from the binding of the oligomers in that it was about twice as efficient and was essentially irreversible even in the presence of an inhibitor of ingestion, cytochalasin B. On the other hand, quantitative complex release could be achieved, in the presence of the ingestion inhibitor, by including 1.5mM DNP lysine in the medium. Complex handling by macrophages at 37°C was also examined in the presence of monomeric IgG2, at its serum concn, and in the absence and presence of cytochalasin B. Inhibiting ingestion did not impair the capacity of the macrophages to take up complexes under these conditions. On the basis of these findings and previous reports that complexes bound to a receptor-bearing membrane undergo additional antibody-antigen bond formation [Dower et al, Biochemistry20, 6326–6334 (1981a) and Leslie, Protides biol. Fluids29, 431–434 (1982)] it is proposed that complex aggregation at the phagocyte surface may constitute the critical irreversible event required for the selective clearance of complexes in vivo. Other biological implications of receptor-mediated complex aggregation are also discussed.     

In vivo Effects of Interleukin 2 on Lymphocyte Subpopulations in a Patient with a Combined Immunodeficiency

This report describes a clinical trial with Interleukin 2 (IL-2) on a 17-month old male child with combined immunodeficiency (Nezelof's syndrome). IL-2 was prepared from conditioned media of phytohemagglutinin-stimulated leukocytes from buffy coats. The purification of IL-2 involved chromatography on Matrex Blue A sepharose and gel filtration chromatography. The preparation was free of macrophage cytotoxicity factor, macrophage migration inhibition factor and colony-stimulating factor. It contained negligible activity of interferon-γ. IL-2 activity was adjusted to 1600 U/ml, which corresponds to about 0.8 μg homogeneous IL-2/ml. The patient was treated over a 50-day period with a total dose of 20,000 U IL-2, which was injected subcutaneously. IL-2 was well tolerated. Within 3 weeks, the treatment led to a normalization of a lymphocytosis which had prevailed for the previous 3 months. A pronounced eosinophilia also improved but did not reach normal levels. The most striking effect was a normalization of the OKT4⁺/OKT8⁺ ratio with a concomitant relative increase in OKT3⁺ cells in the peripheral blood. No effects were seen on E rosette formation, B cell counts or serum Ig levels. Also NK or ADCC activity remained high, as before the treatment. Infectious episodes and requirement for antibiotic treatment were less frequent during IL-2 therapy. Some effects of IL-2 were transient, e.g., the counts of OKT4⁺ and OKT3⁺ cells which returned to pathological values a few weeks after the treatment was discontinued.     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.     

Specificity of anti-DNA antibodies in SLE-I. Definition and gross specificity of antibody populations in human SLE plasma

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations of anti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonucleotides (average length tetranucleotides). For one of the plasma tested (PS), pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.     

Enhancement of Fas-mediated apoptosis in ageing human keratinocytes

Cellular senescence and apoptosis are two metabolically related and seemingly synergistic processes that are involved in tissue maintenance and homeostasis, anti-tumor protection, and age-related diseases. Despite this apparent co-operativity, senescence can inhibit apoptosis in certain conditions. Here, we describe senescence-apoptosis relationships in human epidermal cells by comparing apoptosis-related effector concentrations in keratinocyte cultures and epidermal skin cells at various stages of ageing. Using western blots, flow cytometry, enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence, we determined the amounts of apoptotic effectors in aged cells compared to young ones, in parallel with beta-galactosidase activity at neutral pH (senescence-associated beta-galactosidase, SA beta-gal), found to be a good indicator of cellular ageing. We observed increased levels of several Fas-mediated apoptosis effectors (Fas, Fas ligand, FADD, FLICE), both in cell cultures at advanced passages and in skin cells of aged donors (above 45 years). Furthermore, we found that while the pro-apoptotic p53 increased, the anti-apoptotic Bcl-2 declined. In spite of this, the extent of spontaneous apoptosis did not change in senescent keratinocyte cultures. The cells, however, became notably more susceptible to apoptosis when kept in exhausted growth medium, or upon Fas receptor activation by anti-Fas antibody binding. Our results are consistent with recent findings in senescent fibroblasts, showing that the death-signaling pathway is enhanced at senescence.     

Purine nucleoside phosphorylase, a possible histochemical marker for T-cells in man.

A procedure is described for the histochemical detection of purine nucleoside phosphorylase (PNP) activity in circulating lymphocytes of man. The number of PNP-positive cells, as evaluated on smears of Ficoll--Hypaque purified cells, correlated well with the number of E-rosette-forming cells of the same blood samples of healthy and diseased people with normal or abnormal numbers of E-rosettes. In healthy people, the number of PNP-positive cells was within the range of 70-80% of the total lymphocyte population, whilst the corresponding E-rosette-forming cells were scored between 60-75%. Patients with unusually low or high E-rosettes had equally low or high numbers of PNP-reactive cells. More substantial evidence for the presence of PNP activity in T-cells and not in B cells was gathered from experiments in which PNP activity and surface membrane immunoglobulins (SMIg) were simultaneously demonstrated on the same preparation. These results showed, on the one hand, that the bulk of lymphocytes that are reactive for PNP do not reveal SMIg and, on the other hand, that most Ig-bearing cells were unreactive for PNP.     

Soy phytoestrogens are neuroprotective against stroke-like injury in vitro

Diets high in soy are neuroprotective in experimental stroke. This protective effect is hypothesized to be mediated by phytoestrogens contained in soy, because some of these compounds have neuroprotective effects in in vitro models of cell death. We tested the ability of the soy phytoestrogens genistein, daidzein, and the daidzein metabolite equol to protect embryonic rat primary cortical neurons from ischemic-like injury in vitro at doses typical of circulating concentrations in human populations (0.1-1 microM). All three phytoestrogens inhibited lactate dehydrogenase (LDH) release from cells exposed to glutamate toxicity or the calcium-ATPase inhibitor, thapsigargin. In cells exposed to hypoxia or oxygen-glucose deprivation (OGD), pretreatment with the phytoestrogens inhibited cell death in an estrogen receptor (ER) dependent manner. Although OGD results in multiple modes of cell death, examination of alpha-spectrin cleavage and caspase-3 activation revealed that the phytoestrogens were able to inhibit apoptotic cell death in this model. In addition, blockade of phosphoinositide 3-kinase prevented the protective effects of genistein and daidzein, and blockade of mitogen-activated protein kinase prevented genistein-dependent neuroprotection. These results suggest that pretreatment with dietary levels of soy phytoestrogens can mimic neuroprotective effects observed with estrogen and appear to use the same ER-kinase pathways to inhibit apoptotic cell death.     

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.     

Chloride transport in NCL-SG3 sweat gland cells: channels involved

The aim of the study was to assess whether NCL-SG3, the only immortalized sweat gland cell line available, can be used as an in vitro model to study chloride ion transport in cultured sweat gland cells. Cl(-) efflux was measured using the MQAE dye fluorescence technique after stimulating the cells with different agonists. A significant stimulation of chloride efflux was achieved with the calcium ionophore A23187 resulting in an efflux rate of 0.9 mM/s. Both ATP and UTP activated chloride efflux in these cells, with the ATP response being larger. IBMX and forskolin stimulation did not induce a rate of chloride efflux above the basal level. Immunocytochemistry showed no detectable CFTR in NCL-SG3 cells. This finding was confirmed with flow cytometry analysis. Niflumic acid (20 and 100 microM NFA) and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (100 ìM) decreased the rate of ATP-stimulated chloride efflux significantly (0.40 and 0.31 mM/s with NFA, 0.37 mM/s with H2DIDS). Gadolinium (20 ìM) had no effect on the chloride transport rate. In conclusion, the NCL-SG3 cells retain some of the aspects of human sweat gland epithelium, such as the ability to form cell-cell contacts. The CFTR protein is neither functional nor expressed in cultured NCL-SG3 sweat gland cells. Ca(2+)-activated chloride conductance is confirmed and the putative Ca(2+)-activated chloride channel (CaCC) is further characterized in term of its pharmacological sensitivity. The NCL-SG3 sweat gland cell line can be used to investigate the characteristics of the CaCC and to identify the channel.     

Use of monoclonal anti-rat IgE in a radioimmunoassay for antigen specific rat IgE

We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.