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Results for “quantitative complementation”

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PubMed1998

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products.

Quantification of microorganisms is an important part of the normal diagnostic work of a clinical microbiology laboratory. Traditionally the diagnosis of pertussis is subject to a yes or no approach with no quantitative dimension. This can, however, be of interest as a factor when judging the risk of a patient spreading the bacterium and as a research tool. The aim of the present study was to develop a PCR-based quantitative assay for Bordetella pertussis DNA in clinical nasopharyngeal aspirates by combining a quantitative PCR with a colorimetric detection principle, DIANA (detection of immobilised amplified nucleic acid). A competitor to the PCR target sequence in IS-481, containing a lac-operator, was constructed and calibrated, and a test protocol prepared. A total of 46 clinical nasopharyngeal aspirates, previously diagnosed using a standard nested PCR assay and quantified by culture, were analysed by the quantitative PCR. The method showed acceptable precision and accuracy considering that it estimates the total number of bacterial genomes while culture detects viable bacteria. Recognised advantages were the simple colorimetric detection, the inborn indication of a working PCR assay, and the possibility of obtaining results even when partial inhibition of the PCR assay was seen. In addition, the quantitative PCR result can be obtained within one day compared to 3-10 days for culture. The present results and the qualities of the quantitative PCR suggest that this assay will be a useful complement in routine diagnostics and in research.

Bacterial Typing Techniques↗
PubMed1972

Simplified radioimmunoassay for diagnostic serology.

A simplified, indirect radioimmunoassay is described for Escherichia coli, vaccinia virus, and herpesvirus. The antigens were affixed to glass cover slips; thus both the primary and secondary reactions take place on the cover slips, and the unbound antiserum is easily separated from the bound antiserum by rinsing. Rabbit or human immune sera were reacted with the antigens, and the primary immune complex was quantitated by a secondary reaction with (125)I-indicator globulin (anti-rabbit or anti-human). A direct relationship between the antiserum concentration and the (125)I absorption was established. Variations in titers were detectable, and the titers were comparable to complement fixation titers. Homologous and heterologous reactions were distinguishable. The method affords an objective, quantitative, and qualitative evaluation of antibody, and results are reproducible.

Absorption↗
PubMed2000

Immunostaining for cell picking and real-time mRNA quantitation.

Microdissection techniques allow a cell-type or even cell-specific mRNA analysis within complex tissues. Furthermore, valid mRNA quantitation can be performed by real-time reverse transcriptase-polymerase chain reaction from a few isolated cells obtained from cryosections. For a more precise access to many cell types, this technique has to be complemented by a cell-type-specific immunostaining. To evaluate its effect on mRNA quantitation, we analyzed alveolar macrophages (AMs) from control rat lungs and those undergoing stimulation with lipopolysaccharide and interferon-gamma nebulization. Whereas AMs from the left lung were directly harvested for mRNA extraction by bronchoalveolar lavage, tissue sections of the right lung were stained with an optimized immunofluorescence protocol detecting AMs. Fifteen AM profiles per sample were picked by laser-assisted sampling technique. Normalizing to a standard gene, nitric oxide synthase II (NOSII) and tumor necrosis factor (TNF)-alpha mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. In stimulated lungs, the percentage of picked samples positive for NOSII or TNF-alpha mRNA increased significantly. Moreover, a marked increase in the ratio of target gene mRNA to standard gene mRNA was noted for both NOSII and TNF-alpha in picked AMs from stimulated lungs, which matched very well the increase detected in the lavaged AMs undergoing direct RNA extraction. Thus, when using an optimized protocol for immunofluorescence, this approach may be reliably combined with laser-assisted cell picking and real-time mRNA quantitation in a few immunohistochemically characterized cell profiles within complex tissues.

Animals↗
PubMed2006

Retinal synthesis and deposition of complement components induced by ocular hypertension.

Inappropriate activity of the complement cascade contributes to the pathophysiology of several neurodegenerative conditions. This study sought to determine if components of the complement cascade are synthesized in the retina following the development of ocular hypertension (OHT) and if complement accumulates in association with retinal ganglion cells. Toward this goal the gene expression levels of complement components 1qb (C1qb) and 3 (C3) were determined in the retina by quantitative polymerase chain reaction in human eyes with elevated intraocular pressure (IOP) and healthy retinal tissue as well as in a rat model of OHT induced by laser cauterization of the trabecular meshwork and episcleral veins. Immunohistochemical methods were employed to determine the sites of complement deposition in the retina and optic nerve head. Our data demonstrate that transcript levels for C1q and C3 are significantly elevated in retinae subjected to OHT, both in the animal model as well as in human eyes. Immunohistochemical analyses indicate that C1q and C3 accumulate specifically in the retinal ganglion cell layer and the nerve fiber layer. In addition, we demonstrate that the terminal complement complex, or membrane attack complex, is formed both in the human and rat model as a consequence of OHT. Complement activation, particularly formation of membrane attack complexes, has the potential to exacerbate ganglion cell death through bystander lysis or glial cell activation. The results show that complement activation occurs in the retina that has been subjected to elevated IOP, and may have implications in pathophysiology of glaucoma.

Animals↗
PubMed2005

Inflammatory response and re-stenosis after percutaneous coronary intervention in heart transplant recipients and patients with native atherosclerosis.

BACKGROUND: The clinical benefit of percutaneous coronary intervention (PCI) in heart transplant recipients (HTRs) with coronary allograft vasculopathy (CAV) has been questioned. We investigated the degree of inflammatory reaction during PCI in CAV compared to patients with native atherosclerosis, and the possible relationship between PCI-induced inflammation and the degree of re-stenosis in these 2 patient groups. METHODS: In 11 CAV patients and 10 patients with native atherosclerosis, blood samples were drawn before and 24 hours and 6 months after PCI, and analyzed with regard to hsCRP, MCP-1, components of complement activation, von Willebrand factor (vWf), soluble L-selectin and ICAM-1. Quantitative angiography was performed before and after PCI, and at 6-month follow-up. RESULTS: Baseline levels of hsCRP, vWf and MCP-1 were significantly elevated and levels of L-selectin and ICAM-1 and activation products of the alternative pathway of the complement system were decreased in CAV patients compared to those with native atherosclerosis. PCI induced significant increases of hsCRP in both groups as well as an increase in vWf in native atherosclerosis, whereas a decrease in L-selectin was observed in native atherosclerosis. Plasma levels of MCP-1 correlated with percent stenosis at follow-up in both groups, whereas a correlation between hsCRP and percent stenosis was evident only in patients with native atherosclerosis. There were no differences in rates of re-stenosis between the 2 groups. CONCLUSIONS: HTRs with CAV and patients with native atherosclerosis are characterized by different profiles of immune activation and respond differently to PCI. Nevertheless, an inappropriate inflammatory reactivity may predispose to re-stenosis after PCI in both groups of patients, with pre-procedural inflammation being of particular importance in CAV.

Aged↗
PubMed1983

Activation of the alternative pathway of complement by Acanthamoeba culbertsoni.

Normal human serum (NHS) contained an amoebicidal property for Acanthamoeba culbertsoni. Killing was quantitated by measuring the ability of the amoebae to undergo cell division subsequent to exposure to NHS, and also by microscopical examination. Plasma membrane disruption and extrusion of intracellular components occurred within 5-10 min following exposure to NHS. Adsorption of specific antibody did not remove the amoebicidal activity while heating serum at 56 degrees C/30 min or treatment with zymosan prevented the killing of A. culbertsoni. Haemolytic complement was consumed and C3 conversion occurred during the incubation of NHS with amoebae. Killing required the presence of the late complement components. The findings that (a) amoebae were killed in C2 deficient human serum and ethylene glycol tetra-acetic acid (EGTA), but not ethylenediamine tetra-acetic acid (EDTA) treated NHS; (b) haemolytic complement consumption, which occurred by incubating NHS with the amoebae, could be prevented by addition of EDTA, but not EGTA and (c) conversion of C3 occurred in the presence of EGTA, but not EDTA, indicated that activation of the alternative pathway of complement was involved. This may be of importance as a natural defence mechanism in humans against A. culbertsoni infections.

Amoeba↗
PubMed1983

Effect of intraocular lenses on complement levels in human serum.

Polymethylmethacrylate intraocular lenses with polypropylene loops were incubated in fresh human serum to determine and quantitate the effect of these lenses on haemolytic levels of C1, C4, C2, C3 and C5 and levels of Factor B and C3c measured by radial immunodiffusion. We were unable to demonstrate any statistically significant differences in complement levels in serum after incubation for 1 h with and without an intraocular lens. After incubation of serum for 3 h with and without an intraocular lens, we were able to demonstrate a statistically significant reduction in C3 by haemolytic assay but not in C3c by radial immunodiffusion. There were no statistically significant differences at 3 h in C1, C4, C2, C5 and Factor B. The results of this study suggest that intraocular lenses do not have a substantial effect on complement levels.

Complement C1↗
PubMed1998

The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence.

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene, alb1, which is required for conidial pigmentation. The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype. Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface. Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model. These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence.

Amino Acid Sequence↗
PubMed2001

Serologic studies in coccidioidomycosis.

Serologic tests are valuable in the management of patients with suspected or confirmed coccidioidomycosis. Qualitative tests (immunodiffusion, enzyme immunoassay, or latex particle agglutination) permit detection in the serum of the major antibody responses-coccidioidal IgM in early coccidioidomycosis, and complement fixing (CF) IgG, which appears later and is more persistent. Quantitation of the level (titer) of coccidioidal IgG is useful in prognosis and diagnosis. The preferable antigen for these tests is coccidioidin from the hyphal phase of Coccidioides immitis. When coccidioidal disease has disseminated to an extrapulmonary site, the corresponding fluid-cerebrospinal, synovial, peritoneal (as well as pleural)-can be tested for coccidioidal antibody provided that the serum is known to be positive. Some immunocompromised patients may have a limited humoral responsiveness; therefore, it will be important to develop a practical method for the detection of coccidioidal antigen.

Coccidioidomycosis↗
PubMedDate not supplied

[Autoantigen T in guinea pig germinal cells: fine localization and ultrastructural lesions induced in vitro by specific anti-T autoantibody and complement].

Four autoantigens (S, P, T, Z), are known to be present in guinea pig spermatozoa. The only anti-T antibody is able to fix complement and is spermotoxic. Using immunoenzymatic technics, autoantigen T has been localized on the plasma membrane of spermatozoa and spermatids. A quantitative ultrastructural study has shown the anti-T induced irreversible, specific lesions on the germinal cells in presence of complement. A few minutes after addition of complement, almost all the cells are injured. Control sere (normal serum, anti-ova or anti-S or anti-P sera are inefficient. These findings are related to the mechanisms of autoimmune aspermatogenetic orchitis.

Animals↗
PubMed1999

Immune adherence-mediated opsonophagocytosis: the mechanism of Leishmania infection.

To mimic the sandfly pool feeding process and characterize the cellular and biochemical events that occur during the early stages of promastigote-host interaction, we developed an ex vivo model of human blood infection with Leishmania promastigotes. Within 30 s of blood contact, Leishmania promastigotes bind natural anti-Leishmania antibodies, which then activate the classical complement pathway and opsonization by the third component of complement. The opsonized promastigotes undergo an immune adherence reaction and bind quantitatively to erythrocyte CR1 receptors; opsonized Leishmania amastigotes also bind to erythrocytes. Progression of infection implies promastigote transfer from erythrocytes to acceptor blood leukocytes. After 10 min of ex vivo infection, 25% of all leukocytes contain intracellular parasites, indicating that blood cells are the early targets for the invading promastigotes. We propose that adaptation to the immune adherence mechanism aids Leishmania survival, promoting rapid promastigote phagocytosis by leukocytes. This facilitates host colonization and may represent the parasite's earliest survival strategy. In light of this mechanism, it is unlikely that infection-blocking vaccines can be developed.

Animals↗
PubMed1993

A quantitative analysis of the microglial cell reaction in central primary sensory projection territories following peripheral nerve injury in the adult rat.

The time course of the microglial cell reaction in central nervous system primary sensory projection territories has been examined following peripheral nerve injury in the adult rat using qualitative and quantitative analysis of immunoreactivity with the monoclonal antibody OX-42, which recognises the complement receptor CR3. The regions examined included the gracile nucleus, the column of Clarke and the spinal cord dorsal horn (superficial and deep laminae separately) after unilateral sciatic nerve transection, and the spinal trigeminal nucleus following unilateral infraorbital nerve transection. In all territories examined a qualitative increase in OX-42 immunoreactivity was observed 24 h postlesion. Further, quantitative analysis revealed an exponential development of the OX-42 immunoreactivity, with a peak at one week postlesion, thereafter showing a slow exponential decline. Our results show that the signal (or signals) that induces the microglial cell response in primary sensory projection territories is rapid in comparison to previously described central degenerative changes following peripheral nerve lesions (transganglionic degeneration). These findings are compatible with the hypothesis that activated microglia play a pathogenetic role in the development of transganglionic degeneration.

Afferent Pathways↗
PubMed1981

Rapid in vivo clearance of C5ades arg: a possible protective mechanism against complement-mediated tissue injury.

C5ades arg is an important mediator of the tissue injury associated with intravascular complement activation and exerts its effects by causing granulocyte aggregation, leukoembolism, and oxidative damage to endothelial cells. We have now investigated some of the mechanisms responsible for deactivation of this potent complement fragment by studying the clearance of C5ades arg from the plasma of New Zealand white rabbits in vivo. Quantitative aggregation of human granulocytes was used as a bioassay for rabbit C5ades arg. After a bolus intravenous infusion of activated autologous plasma complement, athe plasma C5ades arg content peaked within 60 sec and was cleared rapidly and exponentially, with a mean half-life of 3.0 +/- 0.6 (S.E.M.) min. In neutropenic animals the half-life of C5ades arg was prolonged by 57% +/- 22 and splenectomy, but not hepatectomy, also significantly lengthened the half-life by 48% +/- 18. The most striking delay in C5ades arg clearance was induced by inhibition of endogenous proteases with EACA and aprotinin, which prolonged the half-life by 87% +/- 4 and 94% +/- 12, respectively. These data demonstrate that like other complement fragments such as C3a, C5ades arg has a very brief functional half-life in experimental animals and that although other mechanisms may be operative, endogenous proteases, circulating granulocytes, and the spleen may be important in deactivating this biologically important complement fragment. Impairment of these and other clearance mechanisms could lead to prolonged and excessive complement activation and subsequent exaggerated tissue injury.

Agranulocytosis↗
PubMed1994

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibroblasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras proto-oncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 microM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.

Animals↗
PubMed2003

Dancing with complement C4 and the RP-C4-CYP21-TNX (RCCX) modules of the major histocompatibility complex.

The number of the complement component C4 genes varies from 2 to 8 in a diploid genome among different human individuals. Three quarters of the C4 genes in Caucasian populations have the endogenous retrovirus, HERV-K(C4), in the ninth intron. The remainder does not. The C4 serum proteins are highly polymorphic and their concentrations vary from 100 to approximately 1000 microg/ml. There are two distinct classes of C4 protein, C4A and C4B, which have diversified to fulfill (a) the opsonization/immunoclearance purposes and (b) the well-known complement function in the killing of microbes by lysis and neutralization, respectively. Many infectious and autoimmune diseases are associated with complete or partial deficiency of C4A and/or C4B. The adverse effects of high C4 gene dosages, however, are just emerging, as the concepts of human C4 genetics are revised and accurate techniques are applied to distinguish partial deficiencies from differential expression caused by unequal C4A and C4B gene dosages and gene sizes. This review attempts to dissect the sophisticated genetics of complement C4A and C4B. The emphases are on the qualitative and quantitative diversities of C4 genotypes and phenotypes. The many allotypic variants and the processed products of human and mouse C4 proteins are described. The modular variation of C4 genes together with the serine/threonine nuclear kinase gene RP, the steroid 21-hydroxylase CYP21, and extracellular matrix protein TNX (RCCX modules) are investigated for the effects on homogenization of C4 protein polymorphisms, and on the unequal genetic crossovers that knocked out the functions of CYP21 and/or TNX. Furthermore, the influence of the endogenous retrovirus HERV-K(C4) on C4 gene expression and the dispersal of HERV-K(C4) family members in the human genome are discussed.

Animals↗
PubMed1976

Immune complexes and complement hypercatabolism in patients with leprosy.

The occurrence of immune complexes in the serum and the level of the C3 breakdown product C3d in the plasma from patients with leprosy were studied by quantitative methods and the results were compared in various forms of the disease. These studies were performed on sixty-two samples from twenty-six patients. The serum 125I-C1q binding activity was found to be increased by more than 2 s.d., as compared to the normal values, in most of the sera from patients with erythema nodosum leprosum (ENL) (80%) and uncomplicated lepromatous leprosy (82%), but also in the sera from patients with tuberculoid leprosy (58%). In vitro studies suggested that immune complexes involving mycobacterial antigens were present in leprosy sera. An increased C3d level (greater than 2s.d.) was also found in most of the plasma from patients with ENL (70%), but rarely in the plasma from patients with uncomplicated lepromatous leprosy (18%) and never in tuberculoid leprosy patients' plasma. The absence of a significant correlation between the 125I-C1q binding activity and the C3d level in leprosy patients may suggest that extravascular immune complexes are involved in the complement activation occurring in ENL. The quantitation of C3d in plasma may be of some practical interest in the early diagnosis of ENL complications of leprosy.

Adult↗
PubMed1999

Nondestructive NMR determination of oil composition in transformed canola seeds.

Magic-angle spinning (MAS) 13C nuclear magnetic resonance (NMR) spectroscopy is a convenient method for nondestructive, quantitative characterization of seed oil composition. We describe results for intact hybrid and transformed canola seeds. The MAS 13C NMR technique complements and agrees with gas chromatography results. The spectral resolution approaches that of neat, liquid oils. MAS 13C NMR data allow quantitative analysis of major oil components, including saturates and oleic, linoleic, and linolenic acyl chains. 13C NMR directly and quantitatively elucidates, triglyceride regiochemistry and acyl chain cis-trans isomers that cannot be quickly detected by other methods. MAS 13C NMR can serve as the primary method for development of near-infrared seed oil calibrations. These NMR methods are nondestructive and attractive for plant-breeding programs or other studies (e.g., functional genomics) where loss of seed viability is inconvenient.

Carbon Isotopes↗
PubMed1994

Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1).

The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b2) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR-A required for C4b2 binding and the potential of C3b binding sites in CR1 to bind C4b2. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b2 with similar affinities (Kd, approximately 4 x 10(-7) M). Construct SCR(1-2)D bound C4b2 with lower affinity (Kd, 1.4 x 10(-6) M) indicating that SCR(1-4) are required for a fully functional C4b2 binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b2 with lower affinity (Kd 1.2 x 10(-6) M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b2. Each CR1 construct that bound C4b2 functioned as a cofactor for factor I-mediated cleavage to C4d.

Animals↗