Detachment of L cells in the presence of normal mouse spleen cells in vitro; a quantitative study.
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Animals↗
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Application of a solid-phase immunofluorometric assay to the selection of monoclonal antibody specific for the adenovirus group-reactive hexon antigen.
An immunofluorometric assay (IFMA) has been evaluated as a screening assay to detect monoclonal antibodies to the group-specific antigen of the adenovirus hexon component. The antibodies were produced as mouse ascitic fluids from hybridoma cells generated from Balb/C mice immunized with purified adenovirus type 2 hexon component and crude adenovirus type 3 culture supernatants. The purified IgG fractions from all monoclonal ascitic fluids tested were identified as the IgG1 K mouse isotype. Antibody titers ranged from 102,400 to 204,800 by the IFMA, from 200 to 12,800 by indirect FA, and were generally nonreactive in counterelectrophoresis, complement fixation, hemagglutination-inhibition, serum neutralization, and immune electron microscopy titrations. The IFMA is a reliable method for quantitating low levels of specific antibody in large numbers of test samples, and is therefore ideal as a screening assay for monoclonal antibody in tissue culture fluids and in mouse ascitic fluids.
Adenoviruses, Human↗
New approaches to the genetic analysis of neuroticism and anxiety.
The completion of the human genome project and the complementary genome projects for other species has broadened the scope for novel bioinformatic approaches to quantitative trait locus (QTL) identification. A key issue for quantitative trait nucleotide (QTN) identification is progressing from a large QTL peak, spanning perhaps 50 cM and many hundreds of genes, to a gene or nucleotide variant which is responsible for that QTL effect. The complementary use of mouse models to dissect large syntenic loci in humans is a powerful method for reducing QTL intervals to the order of 1 Mb. This paper presents an overview of the approaches used in our laboratory to ultra-fine map QTLs for anxiety-related traits, and to identify quantitative trait genes (QTG). As new genetic techniques and statistical approaches arise, we are getting closer to identifying those long sought after QTNs.
Animals↗
Rapid quantitation of gene therapy specific CFTR expression using the amplification refractory mutation system.
Gene therapy offers the potential of correcting genetic disorders such as cystic fibrosis (CF). By complementing the non-functional endogenous cystic fibrosis transmembrane conductance regulator (CFTR) gene with a functional transgene, we anticipate it may alleviate the disease phenotype. All approaches to CF gene therapy rely upon sensitive assays to monitor delivery, expression and maintenance of CFTR vectors. Here, we describe the adaptation of the amplification refractory mutation system (ARMS) to discriminate between different forms of CFTR. A LightCycler PCR machine allows realtime continuous fluorescence monitoring of rapid-cycle PCR. We show quantitation of and discrimination between expression of endogenous (wild-type or mutant) CFTR and the introduced transgene.
Cystic Fibrosis Transmembrane Conductance Regulato↗
A microtiter assay for cell-mediated cytotoxicity to cytomegalovirus antigens.
The cell-mediated immune response is important to host defense against viruses, yet techniques with which to measure the contribution of lymphocytes (particularly K cells) to anti-viral cytotoxic reactions are not well-established. We describe a new procedure for measuring lymphocyte-mediated antibody-dependent immunity to cytomegalovirus. Central to the assay is the use of 51Cr-containing red blood cells labeled with purified viral antigen, which can be easily quantitated and readily stored for at least 72 hours. High sensitivity was observed, as sera with a 1: 512 complement fixation titer could be diluted 10,000-fold without extinguishing the detectable cytotoxic response. A set of conditions (target/effector ratio; dilution of serum; source and age of target cells; and incubation medium and time) was defined that optimized sensitivity, maintained a low level of background release, yet utilized small amounts of reagents. A kinetics study revealed that 80% of specific release of radioisotope occurred in the first 4 hours. Investigation of the epidemiology, diagnosis, and management of disease caused by CMV may be greatly aided by this new method for quantitating specific cell-mediated anti-viral immune reactivity.
Animals↗
Interference with total hemolytic complement assay in rheumatoid arthritis: correlation with disease activity.
A subgroup of patients with rheumatoid arthritis (RA) whose sera agglutinated sensitized sheep erythrocytes used for total hemolytic complement determination has been identified. These patients have higher titers of rheumatoid factor (RF) and more severe rheumatoid disease compared to controls. The agglutination of sensitized sheep erythrocytes by sera of some patients with RA may be related to qualitative or quantitative variations in their RF.
Adult↗
Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling.
Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and <9%, respectively.
Dose-Response Relationship, Drug↗
[Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent].
An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.
Animals↗
Overview of host defenses in fungal infections.
Humans have a broad array of host defense mechanisms against fungal invasion. Most serious mycoses occur in patients who have defects in one or more of these defenses. Defense mechanisms not related to the immune system include barriers created by the skin and mucous membrane, competition from the normal indigenous bacterial flora, mucociliary clearance mechanisms, and fungistatic products in serum such as metal chelators. Among the immune mechanisms, activation of the complement system and generation of an antibody response facilitates recognition of fungi by specific receptors on immune cells. Quantitative and qualitative disorders of phagocytic function predispose patients to the development of invasive aspergillosis and candidiasis. Impairment of cell-mediated immunity predisposes patients to cryptococcosis, histoplasmosis, coccidioidomycosis, and mucocutaneous candidiasis. An overexuberant inflammatory response to fungal antigens can result in morbidity and mortality because of damage to host tissues. Knowledge of the type of immunocompromise that is afflicting a patient enables the clinician to predict which mycoses the patient is likely to have and to which the patient is predisposed.
Antibodies, Fungal↗
Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape.
Members of the genus Rickettsia possess the ability to invade host cells and promptly escape from phagosomal vacuoles into the host cell cytosol, thereby avoiding destruction within the endosomal pathway. The mechanism underlying rickettsial phagosomal escape remains unknown, although the genomic sequences of several rickettsial species have allowed for the identification of four genes with potential membranolytic activities (tlyA, tlyC, pat1, and pld). This study was undertaken to determine which of the selected genes of Rickettsia prowazekii mediate the escape process. Quantitative ultrastructural analyses indicated that the period of active phagosomal escape was between 30 and 50 min postinfection. Reverse transcriptase PCR analyses determined that tlyC and pld were transcribed during the period of active phagosomal escape but that tlyA and pat1 were not. The functionality of both tlyC and pld was determined by complementation studies of Salmonella, which replicates within endosomes. Complementation of Salmonella organisms with either tlyC or pld resulted in the escape of transformants from endosomal vacuoles into the host cell cytosol demonstrated by quantitative ultrastructural analyses. These data suggest a role for tlyC and pld in the process of phagosomal escape by R. prowazekii.
Animals↗
Lymphoma-associated paraneoplastic angioedema with normal C1-inhibitor activity: does danazol work?
A patient with splenic marginal zone lymphoma presented with severe, symptomatic acquired angioedema. Unlike previously reported cases, his serum levels of complement and C1 inhibitor (C1-INH) were not decreased. Nonetheless, he responded clinically to treatment with an attenuated androgen and, after therapeutic splenectomy, has been maintained asymptomatic without androgen therapy for 5 years. Thus stimulation of C1-INH synthesis may overcome paraneoplastic angioedema in patients with lymphoproliferative disorders despite the absence of typical evidence for a quantitative or qualitative defect in C1-INH. Androgens should not be withheld despite a normal level of C1-INH and complement in symptomatic patients.
Adult↗
Inhibition of growth of ASL-1w murine leukemia cells by anti-thymus leukemia antigen (TL) serum in the absence of complement.
Previous studies have shown that the cell surface expression of thymus leukemia antigen (TL) on ASL-1w leukemia cells varies with the progression of the cells through the growth cycle. Expression of TL is maximal in S phase, and its quantitative expression varies directly with DNA synthesis. In the present study, the effect of anti-TL serum on the growth of ASL-1w cells was examined. The antiserum, tested in the absence of complement, affected the growth of these cells in biphasic manner. When the antiserum concentration was 0.1% or greater, there was a rapid decline in the rate of DNA synthesis, and after 5 to 7 hr, cell death. When the antiserum concentration was 0.067% or less, the decline in the rate of synthesis of DNA did not become apparent until 5 to 6 hr after treatment. Under these conditions, there was approximately a 20% increase in cell number after 24 hr of culture. The hypothesis that treatment of ASL-1w cells with the lesser concentration of anti-TL serum blocks the cells in G2 phase of the cell cycle is discussed.
Animals↗
Complement deficiency states and meningococcal disease.
Analysis of complement deficiency states has supported the role of complement in host defense and elucidated diseases associated with defective complement function. Although neisserial infection plays a prominent role in these deficiency states, examination of individuals with late complement component deficiency (LCCD) reveals a particular propensity for recurrent meningococcal disease and provides important clues to the role of complement in neisserial infections. In response to meningococcal disease, LCCD individuals produce significantly greater amounts of antilipooligosaccharide (LOS) antibody which can kill group B meningococcus in a complement-sufficient in vitro system. Further studies of antibody cross-reactivity to other meningococci has led to a clearer understanding of its epitopic specificity. Nevertheless, epidemiologic evidence is consistent with the relative absence of protective immunity in LCCD persons following an episode of infection and supported by quantitation of antibody to capsular polysaccharide. However, compared to anti-LOS antibodies, anticapsular antibodies can offer immune protection to LCCD individuals via complement-dependent opsonophagocytosis--the only form of complement-mediated killing available to these persons. Thus vaccination of LCCD persons with capsular antigens is considered an important means of protecting these high-risk individuals against meningococcal disease.
Complement System Proteins↗
Disorders of neutrophil function.
Major bacterial infections are most commonly associated with agranulocytosis or an abnormality of immunoglobulins or complement. Occasionally, repeated infections cannot be attributed to these relatively common causes. In such cases, a quantitative abnormality in neutrophil function should be sought. Complete evaluation of neutrophil function, including: chemotaxis, adhesion, aggregation, phagocytosis, granule content and degranulation, respiratory burst activity and bacterial killing is expensive and requires the services of a specialized laboratory. However, preliminary screening of a patient with a predisposition towards infection can be carried out using simple and inexpensive methods. These include examination of blood films, chemotaxis assay, NBT test and peroxidase staining. For final diagnosis and determination of genetic transmission and treatment, specific tests are indicated. Investigation of neutrophil functions may be useful for the diagnosis of congenital and acquired neutrophil disorders. These assays may also be useful in research, diagnosis and follow up of non-infectious diseases with active inflammatory component.
Bacterial Infections↗
Capsule does not block antibody binding to PspA, a surface virulence protein of Streptococcus pneumoniae.
Of the proteins on the surface of Streptococcus pneumoniae, one of those best able to elicit protection against pneumococcal infection is pneumococcal surface protein A (PspA). Although this protein is attached to the membrane molecule, lipoteichoic acid, which is well beneath the capsule, PspA's ability to inhibit complement deposition and killing by apolactoferrin, suggests that it must have surface exposure. This study provides quantitative data showing that the capsular polysaccharide on types 2 and 3 pneumococci provides little or no masking ability of antibodies to bind PspA. Capsule was even observed to enhance, rather than inhibit the binding of two protective monoclonal antibodies to their epitopes on cell surface PspA. These results with antibodies to PspA are in contrast to binding by antibodies to the phosphocholine (PC) epitope of the lipoteichoic and teichoic acids. The binding of antibody to PC was largely, but not completely, blocked by capsular polysaccharide.
Antibodies, Bacterial↗
Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5' nuclease assay (TaqMan).
The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5' nuclease PCR (TaqMan) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7kb beta-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using 32P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas 32P-postlabeling with nuclease P(1) enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B(1). Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing 32P-postlabeling assay with nuclease P(1) enrichment.
Animals↗
Gene profiling reveals unknown enhancing and suppressive actions of glucocorticoids on immune cells.
Glucocorticoids continue to be the major immunomodulatory agents used in clinical medicine today. However, their actions as anti-inflammatory and immunosuppressive drugs are both beneficial and deleterious. We analyzed the effect of glucocorticoids on the gene expression profile of peripheral blood mononuclear cells from healthy donors. DNA microarray analysis combined with quantitative TaqMan PCR and flow cytometry revealed that glucocorticoids induced the expression of chemokine, cytokine, and complement family members as well as of newly discovered innate immune-related genes, including scavenger and Toll-like receptors. In contrast, glucocorticoids repressed the expression of adaptive immune-related genes. Simultaneous inhibitory and stimulatory effects of glucocorticoids were found on inflammatory T helper subsets and apoptosis-related gene clusters. In cells activated by T cell receptor cross-linking, glucocorticoids down-regulated the expression of specific genes that were previously up-regulated in resting cells, suggesting a potential new mechanism by which they exert positive and negative effects. Considering the broad and continuously renewed interest in glucocorticoid therapy, the profiles we describe here will be useful in designing more specific and efficient treatment strategies.
Anti-Inflammatory Agents↗
Radionuclide imaging in hip abnormalities.
The uptake of Tc-99m-diphosphonate was studied topographically and quantitatively on 258 diseased hips. For many diseased hips, bone scintigraphy provides items of information that complement the clinical, radiological, and biological data, with which it should always be compared. The localization, extent, and intensity of the uptake differ greatly according to the hip disorder studied, and depend not only on the cause of the lesion but also on its stage of evolution. Bone scintigraphy can shed light on the etiologic diagnosis of a painful hip at the beginning of its evolution, before definite roentgenographic signs appear, thanks to some very precise characteristics of the uptake, this is the case for transient osteoporosis of the hip, aseptic osteonecrosis, Legg-Perthes disease, early arthritis of the hip joint, and stress fractures of the femoral neck or of the pubic rami.
Adult↗