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A high-performance liquid chromatography method is reported for the quantitative determination of the intact C3, the third component of complement, in solutions containing inactivated C3. This method is simple and far more sensitive than a conventional method using 5,5'-dithiobis(2-nitrobenzoic acid).
The release of 51Cr from cells infected with herpes simplex virus type 1 or type 2 by antibody and complement was examined as a method of quantitating antibodies to the viruses. With decreasing concentration of antibody, a typical dose-response curve was observed; a region of antibody excess in which dilution did not affect percentage of specific 51Cr release followed by a region in which a linear relation existed between dilution and percentage of specific 51Cr release. Therefore, a quantitative expression of antibody titer was defined as that dilution of serum which yielded 50% specific 51Cr release. The slopes of the linear portion of the dose-response curves were characteristic of the type of virus used to infect the cells and not upon the source of antiserum, thus, the slopes could be used to estimate antibody titers. The multiplicity of infection influenced the antibody titers; reproducible results were obtained when cultures were infected with 3 to 5 plaque-forming units per cell; the antibody titers decreased when less virus was used. The antibody titers obtained by the 51Cr release test were similar to those obtained by a microneutralization test. The 51Cr release test was found to be reproducible and to be useful in estimating the percentages of antibody activity attributable to antibodies to cross-reacting and type-specific antigens.
Aberrations of cellular immune functions in pyoderma gangrenosum (PG) may lead to nonspecific activation of inflammatory cells or to an imbalance of suppression leading to autoaggression (chronic ulceration). A patient with severe unremitting PG had anergy to a battery of seven skin test antigens. Mixed lymphocyte reactions, autologous mixed lymphocyte reactions, lymphocyte proliferative responses to antigens, and the production of leukocyte inhibitory factor were substantially suppressed, while the lymphocyte responses to mitogens were unaffected. Quantitative immunoglobulin and complement levels were normal. The inhibition of cellular immune functions was mediated by a factor in the patient's serum. This factor also inhibited lymphocyte functions of normal unrelated control subjects. Preliminary studies demonstrated that the factor is nondialyzable, heat stable, and not adsorbed by Staphylococcus A protein. Pulse therapy with large doses of corticosteroids resulted in dramatic clinical improvement.
Allergic type I reactions to medicines range in their clinical presentation from rhinitis and urticaria to severe bronchoconstriction and anaphylactic shock. We examined all cases of suspected drug induced reactions classified as anaphylactic reactions or shock reported in Sweden between 1972 and 1995 with regard to patient characteristics and drug(s) suspected. Some comparisons with drug sales and prescription data were also made. During the study period of a total of 1338 reports concerned anaphylactic/oid shock or reactions with at least a possible causal relation to medicine giving an overall reporting rate of seven cases per million inhabitants per year of drug-induced anaphylaxis. Of these 1338 patients 51 (3.8%) died from their reactions. Among the non-fatal cases, 460 (34.4%) were diagnosed as shock and 827 (61.8%) as anaphylactic reactions. In total 46.3% of all reports concerned men but men were over-represented among the older patients and among the fatal cases (65%). There were 201 different drugs reported as 'suspected' them most common of which were dextrans (418 reports), X-ray contrast media (161 reports) and antibiotics (153 reports). For dextrans the rate of anaphylactic reactions, shock and fatal cases reported were 128,101 and 21 per million bottles respectively. This decreased to 10.3, 9.8, and 0.4 per million bottles after the introduction of preventive treatment with dextran 1 in 1983.The reporting rate for ionic contrast media were 0.14, 0.13 and 0.02 per 1000 l for reactions, shock and fatal cases respectively whilst for non-ionic contrast media they were 0.7/1000 l for reactions, 0.02/1000 l for shock, but there was no report of a fatal case. For phenoxymethylpenicillin the reported rate of anaphylaxis was 0.14 cases per million defined daily doses and for benzylpenicillin it was 3.7 cases per million defined daily doses. During the study period several drugs have been identified as important causes of anaphylaxis and measures have been taken to decrease the risk of anaphylaxis e.g. the introduction of preventive treatment with dextran 1, the shift from ionic to non-ionic contrast media and the abolition of polyethoxylated castor oil as a solvent. Spontaneous reporting of drug-induced anaphylaxis remains an important surveillance model but needs to be complemented by better quantitative methods.
The messenger RNAs for the outer membrane proteins in E. coli are more stable than the bulk of the messenger RNA s (Hirashima et al., 1973). Polysomes, enriched in those containing stable mRNAs have been isolated following rifampicin treatment and have been shown to contain quantitatively the same complement of ribosomal protein as normal polysomes. There is one exception: ribosomal protein S1 is present in larger amounts in the polysomes containing stable messengers. However, there are grounds for believing this finding to be an artifact. It is concluded that the differences between outer membrane protein synthesis and bulk protein synthesis are not due to a difference in the ribosomes.
Fourteen mammalian pancreatic ribonucleases of known amino acid sequence were compared by 1 or more of 3 different immunological methods: standard quantitative micro-complement fixation, spot-plate micro-complement fixation, and inhibition of phage inactivation. It was found that, while the results obtained by the 3 techniques were correlated with one another, the standard micro-complement fixation procedure was most versatile, economical of materials, and easiest to execute. The standard MC'F technique was more sensitive than the spot-plate technique to differences in amino acid sequence. The inhibition of phage inactivation method was more sensitive than the standard method for measuring differences among closely related RNases but proved impractical for amino acid differences over 15%; the MC'F method could be extended to at least 30% sequence differences. The standard method, moreover, readily detected the single amino acid difference between dromedary and camel RNases. A linear relationship was found between immunological distance (y) in the MC'F test and percent sequence difference (x) which fit the equation y = 7x. The strength of the correlation between immunological distance and percent sequence difference is consistent with the proposal that a large fraction of the evolutionary substitutions of amino acids in ribonuclease are immunologically detectable. This could be explained either by a multideterminant hypothesis or by a pauci-determinant hypothesis which says that substitutions occurring outside determinants produce small conformational changes influencing determinant reactivity.
Chromosome aberrations in two glioma cell lines were analyzed using biotinylated DNA library probes that specifically decorate chromosomes 1, 4, 7, 18 and 22 from pter to qter. Numerical changes, deletions and rearrangements of these chromosomes were readily visualized in metaphase spreads, as well as in early prophase and interphase nuclei. Complete chromosomes, deleted chromosomes and segments of translocated chromosomes were rapidly delineated in very complex karyotypes. Simultaneous hybridizations with additional subregional probes were used to further define aberrant chromosomes. Digital image analysis was used to quantitate the total complement of specific chromosomal DNAs in individual metaphase and interphase cells of each cell line. In spite of the fact that both glioma lines have been passaged in vitro for many years, an under-representation of chromosome 22 and an over-representation of chromosome 7 (specifically 7p) were observed. These observations agree with previous studies on gliomas. In addition, sequences of chromosome 4 were also found to be under-represented, especially in TC 593. These analyses indicate the power of these methods for pinpointing chromosome segments that are altered in specific types of tumors.
To investigate the relationship between serum ferritin and disease activity in systemic lupus erythematosus (SLE), we enrolled 128 patients with SLE (18 males and 110 females). Twenty-eight patients (2 males and 26 females) with rheumatoid arthritis (RA) served as controls. The SLE patients were subdivided into three groups according to Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores: groups A (0-5), B (6-9), and C (> or =10). We prospectively evaluated 48 SLE patients before and after treatment. Serum ferritin and anti-dsDNA antibody were measured by radioimmunometric assay. C-reactive protein (CRP) was measured quantitatively by immunonephelometry. Complements 3 and 4 (C3 and C4) were measured by nephelometry. Serum levels of ferritin during the more active stage of SLE (group C) exceeded those of RA patients and patients at less active stages of SLE (groups A and B). There were no significant differences between RA patients and groups A and B. Serum ferritin was elevated especially in serositis and hematologic manifestation. In this prospective study, changes in SLEDAI scores before and after treatment correlated significantly with serum ferritin levels and inversely to C3 and C4 levels. We confirm that serum ferritin levels can be a useful marker of disease activity in SLE patients.
OBJECTIVE: To determine the potential contribution of a new healthcare practice model, the specialist menopause pharmacist (SMP) role. METHOD: Post pilot, the SMP's remit was proposed as combining clinical practice (service delivery) and research studies, with emphasis on strengthening liaison between the secondary and primary care sectors. Action research, a qualitative research technique, was selected to document role development in the first year. Here the pharmacist-researcher's focus was a local situation where the effects of a particular change, involving people who were part of the situation, were assessed. The change factor was the introduction of the pharmacist to the multi-disciplinary specialist team. The pharmacist-researcher did not attempt to hold anything constant but observed the changes occurring in a systematic manner. Analysis of on-going collaborative professional activity generated the hypothesis that the role was of use in enhancing patient care. Using triangulation and focusing on the descriptive phrase 'of use', it was then possible to study SMP implemented 'actions' that would be accepted as being 'useful' SMP functions. The aim was to test for reliability and obtain data with greater range and accuracy. The three studies undertaken included a controlled, questionnaire study asking for patients' views on the pharmacist service, auditing health professionals usage of the pharmacist operated telephone help-line, and assessing the impact of structured on-site training on community pharmacists. MAIN OUTCOME MEASURE: Overall impact and achievements over 3 years, against a background where the SMP role continued to develop during the study. RESULTS: Action research methodology engendered reflective practice, enabling the SMP to be both the service delivery provider (the intervention) and the researcher. This pharmacist practice model is accepted both by patients and health professional colleagues. The remit combines clinical practice with on-going research studies. In the UK setting, the SMP can undertake numerous liaison activities between secondary and primary care sectors to facilitate enhanced delivery of menopause patient care. CONCLUSION: Using an action research approach, and combining qualitative and quantitative methods to complement data collection, it was possible to assess the specialist pharmacist role in depth.
Structural differences between various myosins were investigated by means of antibodies to heavy meromyosin, a tryptic subfragment of myosin. Heavy meromyosin was purified from rabbit white skeletal and from pig and human cardiac muscles by gel filtration, and antisera were produced in guinea pigs. Analyses, carried out with the quantitative micro-complement fixation technique, indicated that the antibodies were specific to heavy meromyosin and myosin and not to other contractile proteins. For each muscle type, the corresponding intact myosin reacted, and the degree of dixation was always lower than with heavy meromyosin (50 and 70% fixation respectively). This vertical shift was the same for the three muscle types, indicating that the heavy meromyosin represent corresponding fragments of the myosin molecule from one muscle to the other. Antisera to pig or human cardiac heavy meromyosin clearly distinguished antigens (heavy meromyosins, myosins, or crude extracts) from the ventricles of various heterologous species. Relative to pig, the immunological distances were 50 for the rabbit, 73 for the rat and greater than 100 for human and mice. Relative to human, these values were 20 for the rat, 60 for the rabbit, 72 for the pig. These data provide direct evidence that mammalian cardiac myosin is species-specific.
The presence of circulating immune complexes was studied in 347 samples of serum from 212 patients with various vascular diseases. Two quantitative methods (complement-consumption assay and C1q-solubility test) were used for the measurement of the concentration of the complexes. Immune complexes were detected in each group of patients tested (coronary arteriosclerosis, myocardial infarction, cerebral artery sclerosis, arteriosclerosis obliterans, phlebothrombosis, pulmonary infarction). A high proportion of positivity was recorded in myocardial infarction (in 43 patients out of the 94 tested) and in arteriosclerosis obliterans (7 out of 11 cases). The possible pathogenic role of the circulating immune complexes is discussed.
Monospecific precipitating sheep antibodies were generated for the first time against the purified, homogeneous alpha-, beta- and gamma-subunits of the Ca2+-dependent protein kinase, phosphorylase kinase, from rabbit muscle. As reference, antibodies against the holoenzyme and the CA2+-transport ATPase of sarcoplasmic reticulum were induced. In all cases antibody titers could be quantitated (standard error 5-10%) by enzyme-linked immunosorbent assay. Differentiation of antibody binding was achieved by quantitative precipitation and complement fixation assays. In general maximal antibody titers were reached 56 days after primary immunization and high titers (approximately 5000) were maintained for several weeks. Anti-alpha, anti-beta and anti-gamma avidly precipitate the denatured subunits employed as immunogens as well as the native enzyme. No cross-reactivity between antibodies against a specific subunit and any of the other heterologous subunits was demonstrable in double immunodiffusion assays providing no evidence for immunologically identical sites on the alpha-, beta- and gamma-subunits. Since anti-alpha, anti-beta and anti-gamma strongly inhibit enzyme activity, it is likely that they do so primarily by sterically interfering with the binding of the large substrate phosphorylase b (Mr 2.0 X 10(5)) to phosphorylase kinase (Mr 1.3 X 10(6)). It cannot be excluded, however, that anti-beta and anti-gamma bind to the active sites on these 2 subunits.
Rabbit antibodies were obtained to nonhistone protein--DNA complexes (dehistonized chromatin) prepared from two human lymphoblastoid cell lines: the Conception line from an American Burkitt lymphoma and NC-37 from a nonmalignant source. Both antisera showed a high degree of specificity for nuclear proteins of their respective cell lines. This specificity was evident in the reactivity of both whole chromatin and dehistonized chromatin using a quantitative micro-complement fixation assay. The results presented here suggest that DNA present in the antigen is necessary for maintaining the structure of the antigenic site.
1. Rabbit antisera against purified serum albumin of Bombina bombina were used to study relationships between B. bombina, B. variegata, and B. orientalis. 2. Quantitative micro-complement fixation tests indicated the albumins of B. bombina from central Poland and Bulgaria were indistinguishable. The albumins from several populations of B. variegata differed very slightly from that of B. bombina. The albumin of B. orientalis was quite distinct from that of B. bombina. 3. Using albumin as a molecular clock, we estimated B. bombina and B. variegata diverged within the last million years, whereas the B. orientalis lineage diverged roughly 10-12 mil yr ago.
The way ahead with the practical development and application of Ergonomic methods is through a better anticipation and appreciation of changes to system effectiveness and human work that will be incurred through the introduction of new technologies to the workplace. These improvements will involve an improved awareness by the system of the working context and environment. The argued future is with improvements in the handling and use of knowledge by systems. The development of suitable Ergonomics methods, or the careful adaptation of existing methods, should accompany any technological revolution. Moreover, future methods are needed that are specifically developed to be applicable to the real time study of work considering both work context and the amalgamation of results from the use of many diverse methods throughout the design and development life cycle of a system. Part of this process will be a necessary complementation of both quantitative and qualitative methods and guidelines. Another focus should be on creating improved Ergonomics participation within multidisciplinary system design and development environments throughout the system's life cycle. Only through this avenue can Ergonomics show a consistent and valued contribution to quality design and its development. In parallel to such a contribution will be an acceptance by other engineering disciplines, managers, and customers that such an application of Ergonomics is cost effective.
The paper describes the development of appropriate antigen and method combinations for the microbiological cancer test using the non-oncolysing strain Clostridium butyricum CNRZ 528 in dogs with spontaneous tumours. The diagnostic rod antibodies could be determined quantitatively by the complement fixation test if a short-time warm fixation and a long-time cold fixation procedure were combined in separate runs and if two different antigens, a rod corpuscular antigen and a rod surface antigen, were used. Since complement-fixing antibodies were not always detected in cases of malignant tumours, we additionally used the passive haemagglutination method after pre-absorbing the sera with cross-reacting clostridial antigens. The efficiency of the microbiological cancer test could not be substantially increased, however, by the method combination the reliability of the evaluation of low seropositive titres was improved.
BACKGROUND & AIMS: Survival of patients with end-stage liver disease is variable and difficult to predict. A two-phase prospective cohort study was conducted at five teaching hospitals to develop and evaluate a model for prediction of death. METHODS: Five hundred thirty-eight hospitalized patients with a history of chronic liver disease and two or more signs of decompensation were studied. RESULTS: The cumulative incidence of death was 30% at 30 days and 50% at 6 months. In 295 patients in phase I, time till death was independently associated (P < 0.01) with five factors measured on study day 3: renal insufficiency, cognitive dysfunction, ventilatory insufficiency, age > or = 65 years, and prothrombin time > or = 16 seconds. These risk factors stratified 243 patients in phase II into three groups with cumulative incidences of death at 30 days of 12%, 40%, and 74%, respectively. Integration of the prognostic model with physicians' predictions led to improved estimates of the probability of death. Although performance of liver transplantation after study entry was independently associated with enhanced survival, the intensity of other acute therapies was not. CONCLUSIONS: Five risk factors were associated with the risk of death in patients with end-stage liver disease and provided a quantitative basis to complement physicians' prognostic estimates.
The unique terminal arabinan motifs of mycobacterial lipoarabinomannan (LAM), which are mannose-capped to different extents, probably constitute the single most important structural entity engaged in receptor binding and subsequent immunopathogenesis. We have developed a concerted approach of endoarabinanase digestion coupled with chromatography and mass spectrometry analysis to rapidly identify and quantitatively map the complement of such terminal units among the clinical isolates of different virulence and drug resistance profiles. In comparison with LAM from laboratory strains of Mycobacterium tuberculosis, an ethambutol (Emb) resistant clinical isolate was shown to have a significantly higher proportion of nonmannose capped arabinan termini. More drastically, the mannose capping was completely inhibited when an Emb-susceptible strain was grown in the presence of subminimal inhibitory concentration of Emb. Both cases resulted in an increase of arabinose to mannose ratio in the overall glycosyl composition of LAM. Emb, therefore, not only could affect the complete elaboration of the arabinan as found previously for LAM from Mycobacterium smegmatis resistant mutant but also could inhibit the extent of mannose capping and hence its associated biological functions in M. tuberculosis. Unexpectedly, an intrinsically Emb-resistant Mycobacterium avium isolate of smooth transparent colony morphology was found to have most of its arabinan termini capped with a single mannose residue instead of the more common dimannoside as established for LAM from M. tuberculosis. This is the first report on the LAM structure from M. avium complex, an increasingly important opportunistic infectious agent afflicting AIDS patients.