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PubMedDate not supplied

[Qualitative techniques for public health research and the development of health care services: more than just another technique].

Regarding the debate on the existence of two current focuses on health science research (qualitative and quantitative), the paper states the need for complementing the techniques which contribute to a better knowledge of populations and communities, and the need for offering effective solutions to different problems. The article analyses the usefulness of qualitative methods, describes the techniques and procedures more frequently used to guarantee the validity and reliability of research findings and ends bringing up the need for using qualitative and quantitative approaches. This way of working together or learning from each other will enrich research and interventions on public heath and health management fields. Qualitative methods are useful for sound understanding of a given issue that is being investigated or evaluated taking into account the point of view of the participants under research. Key techniques, listed from the most structured to the less structured are among others: structured interview, Delphi, nominal group, case study, semistructured interview, focal group, brainstorming, discussion group, in depth interview, life story and participant observation.

Health Services Administration↗
PubMed1973

Measurement of the third component of complement bound to red blood cells in patients with the cold agglutinin syndrome.

The amount of the third component of complement (C3) bound to red cells of patients with the cold agglutinin syndrome was determined by a quantitative assay, measuring the fixation of the first component of complement by anti-C3. Abrupt reduction in the serum concentration of cold agglutinin by plasmapheresis markedly decreased the hemolytic rate, but the amount of C3 bound to circulating cells did not change appreciably. When this patient was transfused with normal cells. C3 accumulated on the transfused cells within 48 h to the level present on his own cells, but selective destruction of the transfused cells did not occur. When patients were subjected to acute cold stress, cell-bound C3 rose abruptly and intravascular hemolysis occurred. These studies suggest that most of the C3 detected on the circulating red cells of cold agglutinin patients is in an inactive form, and that the rate of attachment of C3 to the membrane is important in determining hemolysis.

Aged↗
PubMed1987

Guillain-Barré syndrome: activated complement components C3a and C5a in CSF.

Plasma and spinal fluid levels of complement activation products C3a and C5a were quantitated by radioimmunoassay in a group of 16 patients suffering from acute monophasic Guillain-Barré syndrome (GBS). Median CSF levels of C3a (118 ng/ml) and of C5a (9.6 ng/ml) were significantly elevated when compared with samples from a control group of patients with noninflammatory neurologic diseases. Plasma concentrations of these anaphylotoxic peptides were not significantly different between the two populations. Our findings indicate that the complement system is activated in the CSF of patients with acute GBS. Complement activation products may contribute to the inflammatory changes observed in this disorder.

Complement C3↗
PubMed1984

Biology of Langerhans cells: analysis by experiments to deplete Langerhans cells from human skin.

In vivo studies have demonstrated that various treatments of skin, e.g., UV irradiation, topical corticosteroids, and tape-stripping, will temporarily deplete the epidermis of Langerhans cells (LC). Whether this loss represents simply a loss of cell surface markers unique to LC, or actual depletion of cells, is unknown. By design, normal human skin transplanted to the congenitally athymic (nude) mouse is a system devoid of circulating precursors for human LC. Because LC have been shown to be of bone marrow origin, any depletion of these cells in this system should be permanent. Treatments to deplete LC from human skin grafts on nude mice after grafting included: (a) large doses of UV radiation (400 mJ/cm2 every 48 h, X 3), (b) potent high-dose topical corticosteroids (2.5 mg betamethasone valerate/cm2 every day, X 5), (c) tape-stripping (X 20). Treatments before grafting included: (a) treating donor skin with 900 R of gamma irradiation, (b) complement fixing monoclonal antibody to Ia-like antigens of LC, followed by fresh complement, (c) monoclonal antibody conjugated to toxins. Quantitation of the number of LC was analyzed on control and treated epidermal sheets using immunodiagnostic reagents, anti-HLA-DR, and surface ectoenzymes , ATPase. Results show that both UV irradiation and topical corticosteroids reduce the number of LC by these analyses. However, within 3 weeks, recovery to pretreatment levels has occurred. X-irradiation and tape-stripping were without effect. Despite evidence that the monoclonal antibody, complement, and toxic systems were delivered to the LC within the epidermis, there is no evidence that these treatments resulted in a decrease in LC. It appears that LC are currently either long-lived or replaced locally from a proliferative pool and that certain cell membrane determinants of human LC are somewhat differentially sensitive to UV radiation and corticosteroids.

Adrenal Cortex Hormones↗
PubMed1986

The interaction of complement components with Aeromonas species.

The interaction of seven serum-sensitive Aeromonas strains with the complement system was investigated using a 2-h quantitative assay. Of the strains tested, four isolates activated both the alternative and classical pathways, two activated only the alternative pathway, and one strain was sensitive to the bactericidal action of complement through the classical pathway only. Two of the four Aeromonas caviae strains were such efficient activators of the complement system that when challenged with human sera deficient in normal concentrations of C3 and C4, they were still subject to complement-mediated bacterial lysis. This phenomenon, in conjunction with previous studies on complement activation by Aeromonas spp., may help account for the decreased incidence observed of systemic disease caused by Aeromonas caviae.

Aeromonas↗
PubMed2004

Antibodies directed against O-acetylated sialoglycoconjugates accelerate complement activation in Leishmania donovani promastigotes.

BACKGROUND: An enhanced presence of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these epitopes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated. METHODS: Complement activators were affinity purified, complement activation via the CP, AP, and lectin-mediated complement pathway was measured by use of an anti-C3 radio-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of propidium iodide (PI) was measured by flow cytometry. RESULTS: Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame. CONCLUSIONS: Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.

Animals↗
PubMed1992

Antibacterial and protective properties of monoclonal antibodies reactive with Escherichia coli O111:B4 lipopolysaccharide: relation to antibody isotype and complement-fixing activity.

In vitro and in vivo antibacterial and protective properties of murine monoclonal antibodies (MAbs) to Escherichia coli O111:B4 lipopolysaccharide (LPS) were evaluated in relation to antibody isotype and complement-fixing activity. Six O side chain-specific MAbs, including two IgMs and one of each IgG subclass, were analyzed for quantitative binding and C3 deposition on intact bacteria, complement-mediated bactericidal and opsonophagocytic activity, and protection against intraperitoneal infections in mice. Although C3 was deposited on bacteria in the presence of normal human serum (NHS) alone, LPS-specific MAbs increased C3 attachment in a dose-dependent manner. Bacterial killing occurred only in the presence of both antibody and complement NHS and required an intact alternative pathway. The efficiency of bacterial killing varied by antibody isotype (IgM greater than IgG2a greater than other IgG subclasses) and correlated with C3-fixing capacity. Opsonophagocytic activity of MAbs exhibited a similar isotype-related rank order. Likewise, IgM was more active than IgG, and IgG2a was superior to other IgG subclasses, in MAb-mediated protection against intraperitoneal infection. These data document the interdependent antibacterial and complement-fixing properties of LPS-reactive MAbs and the degree to which both activities are determined by antibody class and isotype.

Animals↗
PubMed2006

Role of swarming migration in the pathogenesis of bacillus endophthalmitis.

PURPOSE: Bacillus cereus causes one of the most rapidly blinding forms of bacterial endophthalmitis. Migration of B. cereus throughout the eye during endophthalmitis is a unique aspect of this disease that may contribute to intraocular virulence. This study was conducted to analyze the contribution of swarming and intraocular migration to the pathogenesis of experimental endophthalmitis. METHODS: Eyes were injected intravitreally with 100 colony-forming units (CFU) of either wild-type, nonswarming, or swarming-complemented strains of B. cereus. Pathogenicity was compared throughout the course of infection by biomicroscopy, histology, electroretinography, and bacterial and inflammatory cell quantitation. RESULTS: Wild-type, nonswarming, and swarming-complemented B. cereus strains grew to a similar number in the vitreous throughout the course of infection. Unlike the wild-type and swarming-complemented strains, the nonswarming mutant did not migrate to the anterior segment during infection. The rate of decrease in retinal responses of eyes infected with the all strains was similar, resulting in near complete elimination of retinal function by 12 hours. All Bacillus strains caused similar degrees of posterior segment inflammation and retinal destruction. However, the accumulation of inflammatory cells in the anterior chamber, hyphemae, and corneal ring abscesses did not occur in eyes infected with the nonswarming mutant. CONCLUSIONS: The deficiency in swarming had little effect on retinal function loss or the overall course or severity of experimental B. cereus endophthalmitis. However, a deficiency in swarming prevented Bacillus from migrating to the anterior segment, leading to less severe anterior segment disease.

Animals↗
PubMed1989

[The importance of the microprecipitation reaction in agar in the diagnosis of toxoplasmosis].

The authors examined 56 pigs by an isolation experiment for Toxoplasma gondii and simultaneously by the Sabin-Feldman reaction, microprecipitation reaction in agar (MPA), quantitative microprecipitation reaction in agar (MPAK), reaction with complement-fixing and indirect haemagglutination reaction with toxoplasma antigen. In nine pigs with positive isolation finding, the Sabin-Feldman reaction and quantitative microprecipitation reaction in agar was positive in eight cases. In cases of negative Sabin-Feldman reaction, the quantitative microprecipitation reaction was always negative. The quantitative microprecipitation reaction in agar is therefore a suitable indicator of the success of isolation experiments. For a simplicity of the test it is suitable for immunological diagnostics of toxoplasmosis in pigs in common practice.

Animals↗
PubMed1977

Circulating complement breakdown products in patients with rheumatoid arthritis. Correlation between plasma C3d, circulating immune complexes, and clinical activity.

Quantitative determination of the small C3 breakdown product, C3d, was used to investigate complement activation in 45 plasma samples from 30 patients with rheumatoid arthritis (RA). The mean plasma C3e level in these samples (3.0 +/- 1.3 mg/100 ml) was significantly increased (P less than 0.001) as compared to patients with degenerative joint disease (0.9 +/- 0.4 mg/100 ml) and healthy blood donors (0.8 +/- 0.5 mg/100 ml). C3d levels were increased by more than s SD in 79% of RA samples. Plasma C3d levels were compared with C3d concentrations in synovial fluid. In most RA patients, the C3d levels were higher in synovial fluid than in plasma. A very significant correlation between plasma C3d levels and circulating immune complexes, as measured by determination of Clq binding activity (Clq BA), was observed (P less than 0.001). C3d levels were more elevated in RA patients with extra-articular disease manifestations (3.8 +/- 1.2 mg/100 ml) as compared to patients with joint disease alone (2.2 +/- 1.0 mg/100 ml). C3d levels and Clq BA were also significantly correlated (P less than 0.001) with the RA disease activity expressed by an index derived from sedimentation rate, joint score, and duration of morning stiffness. A close relationship between C3d levels, Clq BA, and the clinical activity further appeared during follow-up studies. The present observations suggest that a parallel but rather independent activation of the complement system may be induced by immune complexes in circulating blood and in the joint spaces during the course of rheumatoid arthritis.

Adult↗
PubMed1984

Quantitation of enterovirus 70 antibody by microneutralization test and comparison with standard neutralization, hemagglutination inhibition, and complement fixation tests with different virus strains.

We describe here a microneutralization procedure for conveniently testing large numbers of specimens for antibodies to enterovirus 70. The test utilized human rhabdomyosarcoma cells and was read by staining with crystal violet after 4 days of incubation. The test compares well with other serological assays, being more sensitive than the standard tube neutralization test and the complement fixation test, but less sensitive than the hemagglutination inhibition test. However, the hemagglutination inhibition test required concentrated, partially purified virus as antigen, as did the complement fixation test, and was difficult to read, so that its greater sensitivity may not be of practical significance. By all four test procedures, a recent isolate of enterovirus 70 was a more sensitive antigen than the prototype strain, as shown by greater geometric mean titers in sera of patients from various epidemics.

Antibodies, Viral↗
PubMed1990

[The quantitative analysis of lipopolysaccharide antigen in the blood serum of patients with salmonellosis by using complement-bound liposome lysis].

Titration of group B Salmonella O-antigen in the blood sera of patients and donors was carried out by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium LPS. Good correlation (r = 0.95) of the levels of S. typhimurium somatic O-antigen in the patients' sera determined by liposomal immunoassay and aggregate hemagglutination test was established. The concentration of the antigens in the tested samples was within 0.5-50 micrograms/ml. Statistical analysis of the results obtained by liposomal immunoassay techniques demonstrated differences in the distribution functions for the blood sera of patients with different diseases and of donors.

Adolescent↗
PubMed1991

Neutralizing antibody to herpes simplex virus by a novel neutralization method with peroxidase-labelled complement Clq.

A theoretically new neutralization method was developed for rapid and quantitative detection of virus neutralizing antibodies. The method involves the specific measurement of viral antigens produced by unneutralized virus by antiviral immune serum and peroxidase-labelled complement Clq (P*-Clq). After incubation of 37 degrees C for 18 h for amplification of herpes simplex virus (HSV) as a model, to detect unneutralized HSV, the amounts of HSV antigens produced in cells are measured by OD reading of enzymatic activities of P*-Clq bound to the HSV antigen-antibody complex formed after addition of anti-HSV probe serum and P*-Clq. Results showed that the OD reading of bound P*-Clq was proportional to the input m.o.i. of HSV and that neutralization of HSV with immune serum or human sera resulted in significant reduction of HSV-specific OD readings, depending on the antibody titers of serum samples used. The neutralizing antibody activity can be expressed as a percentage (NT%) of the quantity of neutralized virus, and the entire assay procedure can be completed within 24 h. This new method can replace the conventional neutralization test.

Animals↗
PubMed1990

Flow cytometry: its applications to immunohaematology.

Flow cytometry, using fluorescent probes, has only recently been applied to practical aspects of immunohaematology. It has been used for the detection and quantitation of cell-bound immunoglobulins and complement, minor cell populations, cellular antigens, and platelet and granulocyte antibodies. Practical applications of detecting minor cell populations have been successful in determining the extent of fetal maternal haemorrhage; determining the survival of transfused RBCs (small and large volumes); demonstrating RBC chimaerism (e.g. following bone marrow transplantation) and RBC mosaicism (e.g. demonstration of the McLeod phenotype carrier status). A large variety of cellular antigens have been demonstrated on RBCs, platelets and white cells using flow cytometry. Quantitation of RBC antigens by flow cytometry has been found to be much more objective and accurate than the routinely used serological approaches for establishing zygosity in paternity studies. Flow cytometry has been found to be a sensitive, objective and reproducible method for detecting platelet auto-and alloantibodies, and has also been used to detect granulocyte antibodies. More recently, it has been used to detect antibodies to monocytes and lymphocytes. As flow cytometers become more commonly available, many of these applications may become routine in clinical pathology laboratories.

Antigens, Surface↗
PubMed1958

Immunoreactions involving platelets. III. Quantitative aspects of platelet agglutination, inhibition of clot retraction, and other reactions caused by the antibody of quinidine purpura.

Quantitative aspects of platelet agglutination and inhibition of clot retraction by the antibody of quinidine purpura were described. The reactions appeared to depend on formation of types of antibody-quinidine-platelet complexes which could fix complement but complement was not necessary for these reactions. Complement fixation was at least 10 times more sensitive than platelet agglutination or inhibition of clot retraction for measurement and detection of antibody activity. Although it has been considered that antibodies of drug purpura act as platelet lysins in the presence of complement and that direct lysis of platelets accounts for development of thrombocytopenia in drug purpura, the present study suggests that attachment of antibody produces a change in platelets which is manifested in vitro only by increased susceptibility to non-specific factors which can alter the stability of platelets in the absence of antibody. The attachment of antibody to platelets in vivo may only indirectly affect platelet survival. In contrast to human platelets, dog, rabbit, and guinea pig platelets, and normal or trypsin-treated human red cells did not agglutinate, fix complement, or adsorb antibody; and intact human endothelial cells did not fix complement or adsorb antibody. Rhesus monkey platelets were not agglutinated by the antibody but did adsorb antibody and fix complement although their activity in these reactions differed quantitatively from that of human platelets. Cinchonine could be substituted for quinidine in agglutination and inhibition of clot retraction reactions but quinine and cinchonidine could not. Attempts to cause passive anaphylaxis in guinea pigs with the antibody of quinidine purpura were not successful.

Agglutination↗
PubMedDate not supplied

[The role of the complement system in the pathogenesis of yersinioses].

In 95 patients with Yersinia infections (yersiniosis and pseudotuberculosis) the character of changes in the complement system was studied, taking into account the functional activity of C1-C5, Co. The study demonstrated a decrease in the activity of the complement system in the dynamics of Yersinia infections and revealed quantitative changes of characteristics in yersiniosis and pseudotuberculosis. The results of the study suggest the mobilization of the complement system in Yersinia infections by the classical path of activation. Regularities of changes in the activity of C1-C5, Co, corresponding to the severity and clinical variants of Yersinia infections, make it possible to recommend the use of the above-mentioned parameters in the clinic of infectious diseases for evaluating the effectiveness of treatment and the course of the disease.

Complement System Proteins↗
PubMed2001

Deposition of C3b/iC3b leads to the concealment of antigens, immunoglobulins and bound C1q in complement-activating immune complexes.

Complement activation by bound IgG in serum at physiological concentrations is reflected in the deposition of C3b/iC3b in the absence of antigenic expression of the IgG or of any bound C1q on the target. The aim of this study was to investigate the functional requirements for this phenomenon and to establish its relationship to a release or concealment of the antigens. Microtiter wells coated with IgG by direct adsorption or by binding of IgG antibodies to pre-adsorbed homologous antigen were incubated with serum or serum reagents at 37 degrees C. The complement reaction was analyzed by ELISA to quantitate bound or released reaction products, and the release of IgG from the coated microtiter wells was gauged radiometrically. In the presence of serum, rapid binding of C1q and C3b occurred and was soon followed by a rapid loss of C1q expression; C3b binding remained high. Loss of IgG paralleled that of C1q. The functional requirement for the reaction was restricted to the activation and deposition of C3b/iC3b but was dependent of the combined function of the classical and alternative complement pathways. The loss of the IgG antigen was solely the result of antigen concealment, whereas the loss of C1q was only partly so. In biological terms, the concealment of bound IgG and C1q may reflect mechanisms by which complement down-regulates leukocyte responses stimulated by ligand-cell membrane receptor interactions.

Antigen-Antibody Complex↗
PubMed2006

Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites.

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized 15N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic 15N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.

Journal Article↗