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PubMed1982

Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory.

Complement C3↗
PubMed1989

Optimal conditions for in vitro T cell depletion of human bone marrow by Campath-1a plus complement as demonstrated by limiting dilution analysis.

Quantitations of residual T cells by limiting dilution analysis (LDA), immunofluorescence analysis, sheep red cell rosetting, and proliferative responses to phytohemagglutinin were done to identify treatment conditions that maximized the ex vivo T cell depletion (TCD) of human bone marrow (BM) with the rat monoclonal antibody Campath-1 (CP1) and complement (C'). Different treatment approaches achieved levels of TCD varying from 0.4 to 2.6 log10. However, under optimal treatment conditions, a mean (+/- SEM) log10 TCD of 2.60 +/- 0.12 was demonstrated by LDA. Concentrations of CP1 ranging from 5 micrograms to 300 micrograms/10(7) cells/ml achieved equally effective TCD as determined by LDA. An inverse relationship between the concentration of BM cells/ml and the extent of TCD was observed. Additional C' treatment did not increase TCD as detectable by LDA. Mean recoveries of CFU-GM (day 7), CFU-GM (day 14), CFU-GEMM, and BFU-E growth following CP1 + C' were 51, 43, 42, and 45% respectively. These results demonstrate the importance of cell concentration and treatment conditions for maximizing the depletion of BM T cells with CP1 + C'.

Animals↗
PubMed1976

Human deficiency of the eighth component of complement. The requirement of C8 for serum Neisseria gonorrhoeae bactericidal activity.

The serum of a 23-yr-old woman with prolonged disseminated gonococcal infection syndrome failed to normally promote hemolysis of sensitized sheep red blood cells (RBC). The patient's serum was deficient in the eight component of complement (C8) as determined by functional assays, immunoelectrophoresis, and quantitative immunoprecipitation. Functional titers of each of her other complement components were normal. No serum inhibitors of C8 were detected. The patient's serum supported activation of both the classical and alternate complement pathways. Her fresh serum lacked any bactericidal activity against Neisseria gonorrhoeae, but addition of purified C8 or complement donor serum restored bactericidal activity as well as RBC hemolytic activity. Her serum gave normal opsonization of yeast particles and staphylococci and had normal capacity to coat sensitized RBC with C8 and C4 and to generate chemotactic activity. No defects were observed in the patient's blood coagulation mechanisms. Complement-mediated bacterial lysis may be important in human defense against bacteremic Neisseria infections.

Adult↗
PubMed1991

[Immunological implications of surgical intervention in critical and noncritical patients].

Immunological implications are important in every surgical operation, specially when it is necessary to remove the spleen. She plays an important role in immunological aspecific (filter, phagocytosis) and specific processes (production of IgM and regulation of T- and B-lymphocytic system). Splenectomy causes an immunodeficiency with frequent post-operative complications (the most important is OPSI). Each operated patient is considered generically immunodeficient because surgical trauma and anesthesiologic practice are at the base of immunological alterations (biological barriers, aspecific immunity, A.P.P., complement, specific immunity, NK cells). It's indispensable to know pathological situations that make "critical" the immunological state: caloric-proteic malnutrition, elderly (greater than 70 years old), immunosuppressive therapy, sepsis, shock, neoplasms. I. e.: a patient about seventy years old presents a reduced endocrine secretion of thymic hormone and, probably, a low synthesis of immunoglobulins. Besides the corticosteroids modify the answer of T-lymphocytes and NK cells. Sepsis induces metabolic and immunological alterations after early activation of humoral mediators, modified quantity and life of A.P.P., activation of complement, inhibition of cell-mediate immunity, modification of number and activity of haematic lymphocytes. Trauma induces a hypersecretion of corticosteroid, adrenalin, noradrenaline, glucagon with consequent hypercatabolism that causes malnutrition. The hormonal hypersecretion is a determining factor of reduced phagocytic activity (inhibited migration of neutrophils and monocytes), quantitative and qualitative alterations of complement, deficit of T-cells, hyporeactivity to skin test, depressed answer of antibodies to bacterial and viral antigens. Progressive neoplasms are characterized by modification of T-lymphocytes number, depressed macrophagic activity, hyporeactivity to skin tests.

Adult↗
PubMed1983

Deposition of the terminal C5b-9 complement complex on erythrocytes by human red cell autoantibodies.

A radioassay for the detection of complement activating autoantibodies (autohaemolysins) in patients with autoimmune haemolytic anaemia (AIHA) is described. The method is based on immunoradiometric quantitation of the cytolytic C5b-9 complement complex following its antibody-dependent deposition on red blood cells (RBC). The use of affinity-purified, radiolabelled antibodies directed against the neoantigens of the C5b-9 complex ensured specificity of the test which proved more sensitive than conventional haemolytic assays and was not subject to disturbances by haemolytic sera. The results obtained in 70 patients with various forms of AIHA (warm type N = 45); cold type (N = 22); Donath-Landsteiner type (N = 3] support the prevailing assumption that autohaemolysins can trigger the complement cascade to completion. Since intact RBC from patients with detectable autohaemolysins carried C3/C4 components but never C5b-9, it is inferred that regulatory mechanisms operate in vivo at the level of C4b/C3b inactivation to arrest the cascade and rescue the autologous cells.

Adolescent↗
PubMed1985

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12.

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO3. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA-chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by 'rocket immunoelectrophoresis' using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants.

Bacterial Proteins↗
PubMed1998

Flow cytometry in diagnostic cytology.

Flow cytometry (FCM) is a useful adjunct to cytologic examination, because the quantitative biochemical information it provides complements the morphologic information gained during visual examination. It aids in the interpretation of bladder washings, and is particularly useful for the assessment of lymphoid lesions, whether they originate from fine-needle aspiration, cerebrospinal fluid, or effusions. Optimal use of FCM frequently requires assessment of more than one parameter; simultaneous use of cell differentiation markers and nuclear DNA quantitation is often significantly more useful than either alone. Despite the utility of FCM, however, the potential for future development appears to be limited. Improvements in image cytometry allow reasonable assessment of ploidy and S-fraction to be made from specimens prepared on glass slides. Multiparameter measurements may also be accomplished with imaging techniques, which allow the further advantage of visual identification of cells with equivocal morphologic changes. The development of artificial intelligence methods for use with imaging technology has also significantly exceeded that of FCM. Finally, image cytometry is often more useful for samples with few cells. Other challenges are posed by immunocytochemical methods which compete with flow cytometry as tools for assessment of proliferation. Given the relatively high cost of FCM instrumentation, survival of FCM as an ancillary technique in cytopathology will require further technical refinements to offset the advantages currently associated with image cytometry and immunocytochemistry.

Biopsy, Needle↗
PubMed1983

Complement-activating immune deposits in systemic lupus erythematosus skin.

Immune deposits at the cutaneous basement membrane zone are a characteristic feature of systemic lupus erythematosus. Previous studies using immunofluorescent methods to detect complement components have provided evidence that some deposits contain immune complexes capable of activating complement. However, this important biologic property of complexes has not been detected or measured using functional assays, and it has not been determined whether immune deposits can activate complement at the basement membrane zone. In this study immune deposits in biopsies of lupus skin have been examined using direct immunofluorescence for the third component of complement (C3) to detect complement deposited in vivo. In addition, the deposits have been studied using the leukocyte attachment assay and indirect C3 binding immunofluorescence to detect and measure complement activation at the basement membrane zone in vitro. The results show that complement activation occurs at the basement membrane in some but not all lupus skin containing immunoglobulin deposits, that deposits differ quantitatively in their ability to activate complement, and that direct C3 immunofluorescence is a relatively insensitive method for detecting complement-activating complexes. The results provide functional evidence suggesting that immune deposits in some lupus skin are complement-activating complexes and potentially capable of activating complement at the basement membrane in vivo. Furthermore, the results suggest functional assays for evaluating complement-activating complexes may be valuable supplements to immunofluorescence in exploring the relationship between immune deposits and systemic and cutaneous disease.

Basement Membrane↗
PubMed1992

An improved europium release assay for complement-mediated cytolysis.

An improved assay for complement-mediated cytolysis is described. The target cells are labeled with europium complexed to diethylenetriaminopentaacetate (Eu-DTPA). Cytolysis caused by antibody plus complement leads to the release of the Eu-DTPA complex which is then formed into a highly fluorescent chelate by the addition of 2-naphthoyltrifluoroacetone (2-NTA). The amount of europium chelate formed--a measurement of cell death--is then quantified with a time-resolved fluorometer. The results of the assay are reproducible. Complement-mediated cytolysis when measured by europium release was five times more sensitive than when measured by conventional 51Cr release and three times than when measured by trypan blue exclusion. Because europium does not decay, target cells can be labelled in batches and stored frozen until use, which speeds and simplifies the assay. Thus, europium release assay is a simple and quantitative method to measure complement-mediated cytolysis which is sensitive and more rapid than conventional assays.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1985

The prognostic value of preoperative serum immunoglobulin and complement component concentrations in patients with gastric carcinoma.

Patients with and without recurrence of gastric carcinoma after potentially curative resection (stages I, II and III--pTNM) were studied. The preoperative serum concentrations of IgG and C1-INH were significantly different in a group of 22 patients with recurrence and a group of 33 survivors who were free from recurrence. The patients with recurrence had lower concentrations of IgG (8.3 +/- 2.6 g/l) and higher concentrations of C1-INH (0.4 +/- 0.07 g/l) than the recurrence-free survivors (10.8 +/- 3.5 and 0.35 +/- 0.08 g/l, respectively). The results indicate that preoperative quantitation of serum immunoglobulins and complement components may be helpful in predicting the prognosis in gastric carcinoma.

Aged↗
PubMed1981

Complement-mediated solubilization in patients with systemic lupus erythematosus, nephritis or vasculitis.

Solubilization of an immune precipitate by serum is a complement function mediated by the alternative pathway and enhanced by the classical pathway--it therefore provides the basis of a simple quantitative assessment of the integrity of complement function. Using a preformed radiolabelled precipitate of BSA-alpha BSA Ab, the solubilization capacity of serial sera from 75 patients with various immune complex diseases or diseases associated with hypocomplementaemia was investigated to correlate this assay of complement function prospectively with disease activity and with measurements of circulating immune complexes (CIC). Reduction in solubilization, defined by more than 25% of values in a given patient being below the normal range, was found in 11 of 12 patients with active SLE, two of 19 patients with active systemic vasculitis, three of three patients with post-streptococcal glomerulonephritis and in three of six patients with nephrotic syndrome due to other types of nephritis. In serial studies, solubilization correlated with disease activity in patients with SLE (P less than 0.005), systemic vasculitis (P less than 0.05) and post-streptococcal glomerulonephritis (P less than 0.05). CIC were found more frequently than abnormalities in solubilization; however, the solubilization assay identified a population of patients with CIC more likely to have active disease. This simple assay of complement function provides data on an aspect of immune complex disease not readily apparent from standard estimations of circulating immune complexes, and appears to be a better measure of their potential phlogistic effects.

Adult↗
PubMed2006

Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data.

As one of the most recent members of the omics family, large-scale quantitative metabolomics data are currently complementing our systems biology data pool and offer the chance to integrate the metabolite level into the functional analysis of cellular networks. Network-embedded thermodynamic analysis (NET analysis) is presented as a framework for mechanistic and model-based analysis of these data. By coupling the data to an operating metabolic network via the second law of thermodynamics and the metabolites' Gibbs energies of formation, NET analysis allows inferring functional principles from quantitative metabolite data; for example it identifies reactions that are subject to active allosteric or genetic regulation as exemplified with quantitative metabolite data from Escherichia coli and Saccharomyces cerevisiae. Moreover, the optimization framework of NET analysis was demonstrated to be a valuable tool to systematically investigate data sets for consistency, for the extension of sub-omic metabolome data sets and for resolving intracompartmental concentrations from cell-averaged metabolome data. Without requiring any kind of kinetic modeling, NET analysis represents a perfectly scalable and unbiased approach to uncover insights from quantitative metabolome data.

Computational Biology↗
PubMed1988

Complementary oligodeoxynucleotide probes of RNA conformation within the Escherichia coli small ribosomal subunit.

The large RNA molecule within each ribosomal subunit is folded in a specific and compact form. The availability of specific 16S RNA sequences on the surface of the small ribosomal subunit has been probed by using complementary oligodeoxynucleotides. The hybridization of 8-15-nucleotide-long oligomers to their RNA complements within the subunit was quantitated by using a nitrocellulose membrane filter binding assay. The probes have been grouped into classes on the basis of sequence-specific binding ability under different conditions of ionic environment, incubation temperature, and subunit activation state [as defined by the ability to bind phenylalanyl-tRNA in response to a poly(uridylic acid) message]. Oligodeoxynucleotides complementary to nucleotides flanking 7-methylguanosine residue 527 and to the 3'-terminal sequence bound 30S subunits regardless of the activation state. Oligodeoxynucleotides that complement 16S ribosomal RNA residues 1-16, 60-70, 685-696, and 1330-1339 and the sequence adjacent to the colicin E3 cleavage site at residue 1502 all bound efficiently only to subunits in an inactivated conformation. Probes complementary to residues 1-11 and 446-455 bound only inactivated subunits, and then with low efficiency. Sequences complementary to nucleotides 6-16, 99-109, 1273-1281, and 1373-1383 bound 30S subunits poorly regardless of the activation state. With one exception, each probe was bound by native or heat-denatured 16S ribosomal RNA (as determined by size-exclusion chromatography). We conclude that complementary oligodeoxynucleotide binding efficiency is a sensitive measure of the availability of specific RNA sequences under easily definable conditions.

Base Sequence↗
PubMed1999

Complement deposition on immune complexes reduces the frequencies of metabolic, proteolytic, and superoxide oscillations of migrating neutrophils.

Neutrophils exhibit intrinsic sinusoidal metabolite concentration oscillations of 3 min in resting cells and an additional approximately 10- or 20-s oscillation in migrating/adhering cells. To better understand immune complex (IC)-mediated leukocyte activation, we have studied neutrophil metabolic oscillations in the presence of ICs either with or without fixed complement. Using a microscope photometer we quantitated NAD(P)H autofluorescence oscillations. Cells exposed to ICs exhibited metabolic oscillation periods of approximately 12 s in the absence of complement and approximately 22 s in the presence of complement opsonization. To determine if the effects could be associated with C3 deposition, we used ICs opsonized with only C3 or only C1 and C4. Untreated ICs, heat-inactivated complement-treated ICs, and C1,C4-treated ICs trigger rapid metabolic oscillations, as do fMLP and yeast; in contrast, ICs treated with full complement or C3 alone did not affect NAD(P)H oscillations in comparison to controls. The induction of higher frequency (approximately 10 s) NAD(P)H oscillations by ICs could be blocked by addition of anti-FcgammaRII, but not FcgammaRIII mAb fragments, suggesting the participation of FcgammaRII in cellular metabolic responses to ICs. Parallel changes in the frequencies of oxidant release and pericellular proteolysis were found for all of these stimuli. Thus, immune complex composition affects both intracellular metabolic signals and extracellular functional oscillations. We suggest that complement attenuates the phlogistic potential of ICs by reducing the frequency of cytoplasmic NAD(P)H oscillations.

Adult↗
PubMed2006

A proteomic study of serum from children with autism showing differential expression of apolipoproteins and complement proteins.

Modern methods that use systematic, quantitative and unbiased approaches are making it possible to discover proteins altered by a disease. To identify proteins that might be differentially expressed in autism, serum proteins from blood were subjected to trypsin digestion followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) on time-of-flight (TOF) instruments to identify differentially expressed peptides. Children with autism 4-6 years of age (n=69) were compared to typically developing children (n=35) with similar age and gender distributions. A total of 6348 peptide components were quantified. Of these, five peptide components corresponding to four known proteins had an effect size >0.99 with a P<0.05 and a Mascot identification score of 30 or greater for autism compared to controls. The four proteins were: Apolipoprotein (apo) B-100, Complement Factor H Related Protein (FHR1), Complement C1q and Fibronectin 1 (FN1). In addition, apo B-100 and apo A-IV were higher in children with high compared to low functioning autism. Apos are involved in the transport of lipids, cholesterol and vitamin E. The complement system is involved in the lysis and removal of infectious organisms in blood, and may be involved in cellular apoptosis in brain. Despite limitations of the study, including the low fold changes and variable detection rates for the peptide components, the data support possible differences of circulating proteins in autism, and should help stimulate the continued search for causes and treatments of autism by examining peripheral blood.

Apolipoproteins↗
PubMed2005

Quantitative methods for developing Fc mutants with extended half-lives.

Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half-lives in vivo, and represent an attractive means for extending the half-lives of therapeutic antibodies. The half-lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half-life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (DeltaDeltaGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental DeltaDeltaGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell-level pulse-chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments.

Animals↗
PubMed2001

Continual low-level activation of the classical complement pathway.

There is evidence that the classical complement pathway may be activated via a "C1-tickover" mechanism, analogous to the C3-tickover of the alternative pathway. We have quantitated and characterized this pathway of complement activation. Analysis of freshly collected mouse and human plasma revealed that spontaneous C3 activation rapidly occurred with the generation of C3 fragments in the plasma. By the use of complement- and Ig-deficient mice it was found that C1q, C4, C2, and plasma Ig were all required for this spontaneous C3 activation, with the alternative complement pathway further amplifying C3 fragment generation. Study of plasma from a human with C1q deficiency before and after therapeutic C1q infusion confirmed the existence of a similar pathway for complement activation in humans. Elevated levels of plasma C3 were detected in mice deficient in complement components required for activation of either the classical or alternative complement pathways, supporting the hypothesis that there is continuous complement activation and C3 consumption through both these pathways in vivo. Blood stasis was found to stimulate C3 activation by classical pathway tick-over. This antigen-independent mechanism for classical pathway activation may augment activation of the complement system at sites of inflammation and infarction.

Animals↗