Inclusion of a non-immunoglobulin binding protein in two-site ELISA for quantification of human serum proteins without interference by heterophilic serum antibodies

Measurement of human serum molecules with two-site ELISA can be biased by the presence of human heterophilic anti-animal immunoglobulin antibodies (HAIA) that cause false-positive signals by cross-linking the monoclonal (mAb) and/or polyclonal antibodies (pAb) used for the pre- (capture) and post-analyte steps (detection). To evaluate a novel ELISA format designed to avoid interference by HAIA, a target-specific non-immunoglobulin (Ig) affinity protein (affibody) was used to replace one of the antibodies. First, a human IgA-binding affibody (Z(IgA)) selected by phage display technology from a combinatorial library of a single Staphylococcus aureus protein A domain was used. The detection range of IgA standard using an ELISA based on Z(IgA) for capture and goat pAb against IgA (pAb(IgA)) for detection was comparable with that of using pAb(IgA) for both capture and detection. Secondly, another affibody (Z(Apo)) was combined with mAb and used to detect recombinant human apolipoprotein A-1. The affibody/antibody ELISAs were also used to quantify human serum levels of IgA and apolipoprotein A1. To verify that human serum did not cause false-positive signals in the affibody/antibody ELISA format, the ability of human serum to cross-link affibodies, mAb (mouse or rat) and/or pAb (goat) displaying non-matched specificities was assessed; affibodies and antibodies were not cross-linked whereas all combinations of mAb and/or pAb were cross-linked. The combination of affibodies and antibodies for analysis of human serum molecules represents a novel two-site ELISA format which precludes false-positive signals caused by HAIA.     

Interleukin 2 and gamma interferon production, interleukin 2 receptor expression, and DNA synthesis induced by tularemia antigen in vitro after natural infection or vaccination.

The T-cell response induced by Francisella tularensis antigen in sensitized subjects was characterized in vitro by measuring DNA synthesis in whole-blood and mononuclear cell cultures, interleukin 2 (IL-2) and gamma interferon (IFN-gamma) production, and IL-2 receptor expression. Correlations between these variables were estimated. The strengths of the responses were compared in 21 subjects naturally infected 2 years ago, 6 subjects vaccinated 5 to 6 years ago, and 13 control subjects with no history of infection or vaccination. Subjects with a history of natural infection synthesized more DNA in both whole-blood and mononuclear cell cultures, secreted more IL-2 and IFN-gamma, and expressed more IL-2 receptors than control subjects did. All these responses differed highly significantly (P less than 0.001) from those of the control subjects. The vaccinees exhibited somewhat lower responses than the naturally immunized subjects did, but the vaccinees could be distinguished from the control subjects by their DNA synthesis, receptor expression, and IFN-gamma production (P less than 0.01 to 0.001). The vaccinees showed a lower response, in terms of DNA synthesis and IL-2 secretion (P less than 0.05), than the infected group did but responded in a manner similar to that of this group, with respect to receptor positivity and IFN-gamma secretion (P greater than 0.10). The correlations between all the T-cell functions were good, with highly significant correlations (P less than 0.001) between whole-blood DNA synthesis and IL-2 and IFN-gamma secretion and between the two lymphokines (P less than 0.001). The results not only increase our knowledge of the T-cell response to tularemia antigen but also give an alternative approach to DNA synthesis measurement for the quantitation of T-cell responses. The results for the low-responding sensitized subjects seem to indicate that the parameters were comparable in sensitivity.     

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20–30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di- and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ~20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.     

Evaluation of the image quality of ink-jet printed paper copies of digital chest radiographs as compared with film: A receiver operating characteristic study

Paper copies of digital radiographs printed with the continuous ink-jet technique have proved to be of a high enough quality for demonstration purposes. We present a study on the image quality of ink-jet printed paper copies of digital chest radiographs, based on receiver operating characteristic (ROC) analysis. Eighty-three digital radiographs of a chest phatom with simulated tumors in the mediastinum and right lund, derived from a computed radiography (CR) system were presented in two series of hard copies as ink-jet printed paper copies and as laser recorded film. The images, with a matrix of 1,760×2,140 pixels, were printed with a spatial resolution of 10 pixels/mm in the CR film recorder as well as in the ink-jet printer. On film, every image was recorded in two versions, one optimized for the mediastinum and one for the lungs. On paper, only one image was printed; this constituted an effort to optimize both the mediastinum and the lungs. The ink-jet printed images, printed on a matt coated paper, were viewed as on-sight images with reflected light. The exdaminations were reviewed by six radiologists, and ROC curves were constructed. No significant difference was found between the performance of film and that of ink-jet paper prints. Because the cost for a paper copy is only a tenth of that of film, remarkable cost reductions can be achieved by using the ink jet technique instead. Our results show that further quality studies of ink-jet printed images are worthwhile.     

Suppression of macrophage activation with CNI-1493 increases survival in infant rats with systemic Haemophilus influenzae infection

CNI-1493, a potent macrophage deactivator, was used to treat infant rats systemically infected with Haemophilus influenzae type b (Hib). CNI-1493 was injected 1 h prior to bacterial inoculation and 24 h later and resulted in a 75 percent increased rate of survival compared to that for untreated controls. The effect of CNI-1493 on the inflammatory response was studied by immunohistochemical detection of individual tumor necrosis factor alpha (TNF-alpha)-, interleukin 1 beta (IL-1beta)-, and gamma interferon (IFN-gamma)-producing cells in the spleen. A significant reduction of the incidence of TNF-alpha- and IL-1beta-expressing cells was found for CNI-1493-treated animals. IFN-gamma expression was not suppressed by CNI-1493, indicating that cytokine inhibition was specific in macrophages. CNI-1493 significantly reduced the number of infiltrating granulocytes in the brain from that for controls. This study provides evidence that CNI-1493 protects against lethal Hib infection by deactivating the inflammatory cascade in infant rats.     

Inhibition of nitric oxide synthase reduces Sephadex-induced oedema formation in the rat lung: Dependence on intact adrenal function

In the present study we have investigated the effect of L-nitro arginine mono methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase on Sephadex induced inflammation in the rat lung. Instillation of Sephadex into the airways induced an inflammatory reaction characterized by a long-lasting interstitial oedema, measured as an increase in lung weight, and an influx of inflammatory cells into the airways. L-NAME given s.c. prevented the increase in lung weight following Sephadex instillation. The inactive enantiomer D-NAME had no effect, nor did aminoguanidine which indicates that this effect of L-NAME was mediated by inhibition of the constitutive form of NOS. Treatment with L-NAME did not reduce an established oedema. In contrast, L-NAME tended to enhance the influx of oesinophils into the airways of Sephadex-instilled animals.L-NAME did not have any effect on the development of oedema in adrenalectomized rats or in animals where formation of glucocorticosteroids (GCS) was inhibited with metyrapone. L-NAME did not however, increase plasma levels of corticosterone. The present results indicate that, in this model, inhibition of NO-synthesis has marked anti-inflammatory effects. The underlying mechanism is complex but seems not to involve prevention of overproduction of NO.     

Insulin production by pancreatic islets of obese-hyperglycemic mice cultured for one week in different glucose concentrations.

Culture of pancreatic islets isolated from obese-hyperglycemic mice (gene symbol ob) for one week in media containing widely different concentrations of glucose (3.3, 5.6 or 16.7 mM) was found to markedly influence the functional behaviour of the islet B-cells. Thus, the insulin content of islets cultured at 3.3 or 16.7 mM glucose (subphysiological or supraphysiological glucose concentrations respectively) was markedly reduced. Islets cultured in 5.6 or 16.7 mM glucose displayed a normal insulin secretory response when stimulated with glucose, whereas islets cultured in a subnormal glucose concentration (3.3 mM) showed a reduced insulin response to glucose stimulation in batch type incubations and also lacked a second phase of insulin secretion in islet perifusion experiments. The rate of insulin biosynthesis of non-cultured ob/ob islets was higher than that of islets from their lean siblings but culture for one week in 3.3 mM glucose induced a pronounced impairment of the insulin biosynthesis in islets of obese as well as lean mice. The present data indicate that the hyperfunction of the islets of the ob/ob mouse at least in part is a reversible phenomenon, suggesting that inherent properties of islet B-cells do not act as "primary" factors in the development of the obese-hyperglycemic syndrome.     

Chemoattractant-induced NADPH oxidase activity in human monocytes is terminated without any association of receptor-ligand complex to cytoskeleton

When the chemotactic peptide formylmethionyl-leucyl-phenylalanine binds to its cell surface receptor, a transmembrane signal is generated that activates the superoxide-producing NADPH oxidase of human phagocytes. Comparing monocytes and neutrophils with regard to the production of superoxide anion induced by the peptide, we found a similar time-course for both types of cells. In neutrophils, ligand binding induced a conversion of the receptor to a high-affinity form, a change suggested to be due to an association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton. This event has been hypothesized to terminate the signal that activates the NADPH oxidase and thereby results in cessation of the cellular production of superoxide anion. Neutrophils preincubated with the cytoskeleton-disrupting drug cytochalasin B showed an increased and prolonged superoxide anion production after activation with the peptide, thus indicating that the cytoskeleton is involved in terminating this response. Formylmethionyl-leucyl-phenylalanine was also found to induce polymerization of actin in monocytes; however, cytochalasin B had no effect on the peptide-induced generation of superoxide anion in these cells. Furthermore, also in monocytes, ligand binding induced a conversion of the receptor to a high-affinity form; however, the receptor-ligand complex did not coisolate with the Triton X-100-insoluble cytoskeleton. These results indicate that, in monocytes, the NADPH oxidase activating pathway is terminated without any association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton.     

Expression of interleukin-1 alpha, interleukin-1 beta and interleukin-6 in chronic B lymphocytic leukaemia (B-CLL) cells from patients at different stages of disease progression.

We have previously found that isolated B-CLL cells from progressive disease produce less interleukin-1 beta (IL-1 beta) as compared with cells from patients with indolent disease. Here we extend that finding to include measurements of IL-1 beta mRNA and secretion of IL-1 alpha and interleukin-6 (IL-6). As before, a lower production of IL-1 beta was found in cells from progressive disease. IL-6 was produced by cells from patients at all stages, with a tendency to follow the IL-1 beta production. Low secretion of IL-1 alpha was noted. When viable cells were permeabilized and analysed at the single cell level with monoclonal antibodies, most B-CLL cells were found to contain IL-1 alpha. A minor fraction of non-permeabilized cells expressed IL-1 alpha at the cell membrane. However, only small fractions of cells were positive for intracellular IL-1 beta (less than 1%) and almost no IL-6-positive cells were found. We conclude that either IL-1 beta and IL-6 are produced by a minor population of undefined cells, or that a more sensitive in situ method is needed to detect production of these cytokines in B-CLL cells. The possible biological significance of secreted, and membrane-expressed helper factors in B-CLL is discussed.