The interleukin hypothesis: a quantitative assessment.
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Complement C5↗
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Spur cells in patients with alcoholic liver cirrhosis are associated with reduced plasma levels of apoA-II, HDL3, and LDL.
The precise nature and origin(s) of the abnormalities in lipoprotein and apolipoprotein profile associated with severe hepatic dysfunction and the presence of spur cells remain poorly defined. To shed light on this question, we have analyzed the plasma lipoprotein and apolipoprotein profiles in five patients with alcoholic cirrhosis and spur cells, and compared them with those of a group with similar hepatocellular dysfunction, but lacking spur cells, and with that of a control group. Lipoproteins were subfractionated by density gradient ultracentrifugation and their physicochemical properties were determined; apolipoprotein A-I, A-II, and B contents in plasma and the respective subfractions were quantitated by radial immunodiffusion, while the complement of low molecular weight apolipoproteins in each subfraction was analyzed by isoelectric focusing and electrophoresis in alkaline-urea polyacrylamide gels. Spur cell plasma was distinguished by reduced levels of apoA-II and elevated ratios of apoA-I/apoA-II (approximately 13:1 as compared to 3.3-3.9:1 in the other two groups), and by reduced concentrations of HDL3. Gradient fractionation showed the apoA-II content of HDL3 to be dramatically and significantly diminished in spur cell plasma; in addition, apoA-II content was reduced relative to apoA-I in this subclass (4.7:1 as compared to 1:1 in cirrhotics lacking spur cells and 1.9:1 in controls). Spur cell HDL2 was similarly deficient in apoA-II, with elevated ratios of apoA-I:apoA-II (9.8:1 in comparison with 1.9-2.5:1 in the two other groups). Nonetheless, high HDL2 concentrations were seen in both series of cirrhotic patients, irrespective of red cell morphology. Spur cell HDL2 thus appears to consist primarily of particles possessing only apoA-I, with a minor population containing both apoA-I and apoA-II. The free cholesterol content of all lipoprotein subfractions from spur cell plasma was increased, as indeed was the molar ratio of free cholesterol to phospholipid, in comparison with that of corresponding fractions from alcoholic cirrhotics lacking spur cells and of control subjects. LDL levels were reduced in spur cell plasma, thereby distinguishing this group from the cirrhotics without spur cells who displayed elevated LDL levels. Markedly reduced plasma levels of apoA-II, HDL3, and LDL appear characteristic of alcoholic cirrhotics presenting with spur cells. Our findings suggest that apoA-II may be essential to the normal function and metabolism of HDL, one aspect of which may be the transport of free cholesterol and thereby the direct or indirect maintenance of red cell morphology.
Apolipoprotein A-II↗
A simple microassay for computing the hemolytic potency of drugs.
A simple microassay and computer program are described for determining the erythrocyte hemolytic potency of drugs in vitro. This microassay is sensitive for both micro as well as macro ranges of hemoglobin concentration. An ELISA reader has been adapted to read erythrocyte lysis (hemolysis), which reduces the number and culture of replicates. A computer program was developed that calculates parameters such as C50 (concentration of drug causing 50% hemolysis), C100 (concentration of drug causing 100% hemolysis) and beta (slope of the curve) and graphically expresses the hemolytic patterns of various drugs simultaneously. The program can obtain optical densities directly from a 96-well plate ELISA reader by interfacing the microplate reader to the computer or by using a keyboard. This method is useful for screening a large number of hemolytic drugs and requires lower amounts of test compounds. It may also be applicable to quantitative functional assays, such as complement-mediated hemolysis and enumeration of antibody-secreting cells. The program can be obtained from the authors on request.
Animals↗
[Immunochemical analysis of histone H1 and H5 from pigeon erythroid cells].
An antiserum with the antibody titer of 1 : 4096 was obtained by immunization of rabbits with the tRNA-histone H5 complex from pigeon erythrocytes. The specificity of the antiserum was studied quantitatively from the reaction of the complement binding to a homologous antigen (histone H5) and its modifications (I, II, III), differing in the degree of phosphorylation. It was shown that phosphorylation of histone H5 increases the ability of the antigen to bind to antibodies, which is especially well-pronounced at the antiserum dilutions as high as 20480. The comparison of the antigenic properties of histones H5 from pigeon and chicken erythrocytes revealed beside structural differences of the proteins the presence of common antigenic determinants. A similar observation was made when histones H5 and H1 from pigeon erythrocytes were compared. Histone H1 from chicken erythrocytes and histone H1 from calf thymus did not produce criss-cross reactions with antiserum H5.
Animals↗
[Evaluation of left ventricular systolic function using cine MRI--application to hypertrophic cardiomyopathy].
Cine MRI is a useful means to analyse the anatomical and functional changes of hypertrophic cardiomyopathy. The visual evaluation of hypertrophic regions and the motion patterns of their walls is in most cases possible on cine MR images. To complement a subjective method a certain quantitative analysis is necessary. Left ventricular regional systolic function can be quantitatively evaluated using cine MRI images printed on films. There is a good correlation between % thickness of LV wall and its thickness at end diastolic phase. The comparison of % thickness of normal subjects with that of hypertrophic cardiomyopathies shows a tendency of its value being less at the region of more severe hypertrophic change.
Cardiomyopathy, Hypertrophic↗
Immunological adjuvants and their modes of action.
New adjuvant formulations contain a vehicle, which carries antigens to antigen-presenting cells. Examples of vehicles are liposomes, immune-stimulating complexes and microfluidized squalene-in-water emulsions. Adjuvant formulations may contain immunomodulators, which augment cytokine production, such as a synthetic muramyl dipeptide analog or monophosphoryl lipid A. In a primary cascade of cytokine production at the site of antigen + adjuvant injection, TNF-alpha promotes the migration of dendritic cells (DC) to lymphoid tissues while GM-CSF accelerates the differentiation of DC into efficient presenters of antigens to T cells. Adjuvants also up-regulate a secondary cascade of cytokines in lymphoid tissues responding to antigenic stimulation: IL-12 augments the production of IFN-gamma, which favors the production of antibodies of protective isotypes (IgG2a in the mouse). Thus adjuvants can regulate immune responses qualitatively as well as quantitatively. Adjuvant formulations can also activate complement, generating C3d, which binds CD21 on follicular dendritic cells (FDC) and B cells. FDC targeting favors the generation of B lymphocyte memory, which is important for vaccination.
Acetylmuramyl-Alanyl-Isoglutamine↗
UV-induced melanoma cell lines and their potential for proteome analysis: a review.
We have established cell lines from benign cutaneous melanocytic lesions and from melanoma-affected lymph nodes of monodelphis domestica, the laboratory opossum (a South American marsupial now widely maintained in captive colonies for experimental purposes). Unlike melanoma cell lines currently available from humans and other mammals, the opossum lines are derived from cells transformed in vivo by experimentally controlled exposure to ultraviolet B (UVB) radiation of known spectral composition. Differences in the patterns of protein expression among cell lines at different stages of the UVB-induced melanoma cascade can be identified by proteome analysis and will provide a useful basis for comparisons with human and mouse melanoma cell lines. Powerful new two-dimensional (2D) gel electrophoresis technologies and sophisticated bioinformatics programs make it possible to carry out qualitative and quantitative analyses of the entire protein complement expressed by the genome (proteome) of a specific cell type. One area of biology particularly well suited to proteome analysis is carcinogenesis. It is now feasible, for example, to attempt to characterize the full repertoire of proteins, including all the antigenic determinants at the cell surface and in the cytosol, during the carcinogenic cascade from normal progenitor cells, to benign tumor cells, and finally, to highly invasive metastatic cells. Proteome analyses have been initiated with the cell lines from M. domestica.
Animals↗
Cross-reactive, cell-associated antigen on L929 cells infected with temperature-sensitive mutants of sindbis virus.
Temperature-sensitive (ts) mutants of Sindbis virus (SIN) were used to aid in the identification of alphavirus cross-reactive proteins on the surface of infected cells by antibody-dependent, complement-mediated cytolysis. Antisera prepared in rabbits against purified SIN or Semliki Forest viruses were highly cytotoxic for cells infected with wild-type SIN and for cells infected at the permissive temperature with maturation-defective, ts mutants of SIN belonging to several distinct complementation groups. When these SIN mutants were analyzed by antibody-dependent, complement-mediated cytolysis at the restrictive temperature only cells infected with the SIN mutant of complementation group E, ts20, participated in both homologous (with anti-SIN serum) and heterologous (with anti-Semliki Forest virus serum) antibody-dependent, complement-mediated cytolysis reactions. These data and the known defect of ts20 suggested that the cell-associated viral E1 glycoprotein was a functional target antigen for homologous and cross-immunoreactivity in alphavirus-infected cells. At the restrictive temperature there were quantitative differences in antibody-dependent, complement-mediated cytolysis reactivity of ts20- versus wild type-infected cells consistent with the suggestion that ts20-infected cells do not fully express all of the homologous or the cross-reactive antigenic determinants found in wild-type infection. Additional potential sites for antigenic determinants involved in alphavirus-immune cross-reactivity are discussed in relation to events in virus maturation.
Animals↗
Terminal complement complex in blood of patients with neurological diseases.
The local involvement of complement (C) in the pathophysiology of tissue lesions in several neurological diseases is known, but it has never been studied whether or not in neurological disorders the C activity in the circulation is altered as well. This was the aim of the present investigations. We measured in blood plasma, with an automatic device for analysis and quantitation of the haemolytic activity of the terminal complement complex, the variables T1, T2 and T3 which define the latter quantitatively. We did this, on the one hand, in 100 patients who had 46 neurological disorders systematized in 16 nosological groups, and, on the other hand, in a control group of 40 healthy blood-donors. The mean values of all variables found in the patients have not been statistically different from those found in the controls. This demonstrates that in neurological disorders possible activations of C remain restricted to the local tissue lesions and do not occur in blood, probably due to the opposition against C activation of the known inhibitor system.
Complement Hemolytic Activity Assay↗
DNA analysis of human cholesteatomas.
HYPOTHESIS: The hypothesis tested in this article is that if cholesteatomas are a low-grade squamous cell neoplasm, then evidence of genetic instability, in the form of abnormal or aneuploid amounts of DNA, should be evident. BACKGROUND: Cholesteatoma is a destructive lesion of the middle ear and/or mastoid process that produces complications by erosion of the temporal bone. The clinical hallmarks of cholesteatomas, namely invasion, migration, uncoordinated proliferation, altered differentiation, aggressiveness, and recidivism, are traits typically associated with the neoplastic cell. However, there is little evidence to support or refute the speculation that cholesteatomas are a low-grade squamous cell neoplasm. the existence of defects in the genetic complement of the major cellular constituents comprising a cholesteatoma, fibroblasts and keratinocytes, would support the speculation that cholesteatomas are a neoplasm, since cancers commonly manifest quantitative and qualitative alterations in the normal euploid complement of genetic information, resulting in a cell that has an abnormal or aneuploid amount of DNA. METHODS: DNA content (ploidy) within cholesteatoma tissues was measured by flow cytometry and image analysis. RESULTS: The DNA content of 11 human cholesteatomas and nine postauricular skin specimens was analyzed using flow cytometry, while the DNA content of 10 cholesteatoma specimens was analyzed using image analysis. Interpretable data was obtained from 10 cholesteatoma specimens and six postauricular skin specimens. One cholesteatoma specimen demonstrated an abnormal aneuploid DNA content, whereas the remaining nine cholesteatomas and the six postauricular skin specimens demonstrated a normal euploid DNA content. CONCLUSIONS: We conclude that, due to the lack of overt genetic instability, as evidenced by the presence of a normal euploid DNA content, cholesteatomas are not low-grade neoplasms.
Adult↗
Species-dependent immunological differences between various mammalian cardiac tropomyosins.
Antisera were produced from guinea-pigs against purified pig or rat cardiac tropomyosins and antigen-antibody interactions were analyzed by the micro-complement fixation technique. Immunoadsorption with purified tropomyosins coupled with CN Br-activated Sepharose 4B enabled us to establish that these antisera were only specific to tropomyosin and not to other contractile proteins. Direct cross-reactions and competition experiments performed with both the above antisera indicated quantitative differences in the maximum amount of complement fixed by tropomyosins from various heterologous species (man, beef, pig, rabbit, rat and mouse). These data provide direct evidence that mammalian cardiac tropomyosin is species-specific.
Animals↗
Innate immune responses to human malaria: heterogeneous cytokine responses to blood-stage Plasmodium falciparum correlate with parasitological and clinical outcomes.
Taking advantage of a sporozoite challenge model established to evaluate the efficacy of new malaria vaccine candidates, we have explored the kinetics of systemic cytokine responses during the prepatent period of Plasmodium falciparum infection in 18 unvaccinated, previously malaria-naive subjects, using a highly sensitive, bead-based multiplex assay, and relate these data to peripheral parasite densities as measured by quantitative real-time PCR. These data are complemented with the analysis of cytokine production measured in vitro from whole blood or PBMC, stimulated with P. falciparum-infected RBC. We found considerable qualitative and quantitative interindividual variability in the innate responses, with subjects falling into three groups according to the strength of their inflammatory response. One group secreted moderate levels of IFN-gamma and IL-10, but no detectable IL-12p70. A second group produced detectable levels of circulating IL-12p70 and developed very high levels of IFN-gamma and IL-10. The third group failed to up-regulate any significant proinflammatory responses, but showed the highest levels of TGF-beta. Proinflammatory responses were associated with more rapid control of parasite growth but only at the cost of developing clinical symptoms, suggesting that the initial innate response may have far-reaching consequences on disease outcome. Furthermore, the in vitro observations on cytokine kinetics presented here, suggest that intact schizont-stage infected RBC can trigger innate responses before rupture of the infected RBC.
Adult↗
Circulating immune complexes in rheumatoid arthritis with extra-articular manifestations.
Circulating immune complexes (CIC) and complement C4, C3 and CH50 levels in serum were monitored during 6-30 months in 10 patients with rheumatoid arthritis and extra-articular manifestations (EM). A total of 58 observations were made, 17 at times when new EM emerged, 41 at times when the patients were in a steady state. CIC were demonstrated by two methods, viz. a complement consumption test (CCT) and a polyethyleneglycol (PEG) precipitation assay. The precipitates were analysed for their content of IgG, IgM and IgA. The CCT titre decreased significantly at the time of a new EM, whereas PEG precipitates were found most often at this time. Two types of precipitate could be demonstrated. One consisted of IgG only, which was found most often when the patients were in a steady state. The other one was composed of IgG and other immunoglobulins, most often IgA. The latter type was found most often at the time when the patients developed new EM. Subnormal serum complement levels were demonstrated frequently. The level of C4 was significantly lower at the time of a new EM, compared with the level of patients with RA but without EM. The decrease in anticomplementary effect and the signs of complement activation suggest that the qualitative and quantitative changes in CIC observed at the time of new EM were the cause rather than the consequence of the clinical manifestations.
Aged↗
Effect of gangliosides on activation of the alternative pathway of human complement.
Liposomes as defined model membranes were used to quantitatively study the effects of specific sialic acid containing glycolipids on activation of the alternative pathway of human C. Liposomes containing dimyristoylphosphatidylethanolamine, cholesterol, and cerebrosides at molar ratios of 1.0/0.75/0.33 activated the alternative pathway in human serum treated with MgEGTA. Activation was measured by C3 conversion and the deposition of total C3 and functional C3b on the liposome surface. The monosialoganglioside GM1, when incorporated into the activating liposome membrane at molar ratios between 10(-5) and 10(-2), inhibited activation in a dose-dependent manner. Sialosylparagloboside also inhibited activation in human serum, and inhibition was completely reversed after neuraminidase treatment. The degree of inhibition by GM1 correlated with the relative amount of GM1 exposed on the liposome surface. Sialic acid did not directly inhibit the binding of C3b when liposomes containing gangliosides were incubated with the purified components C3, B, D, and P. GM1 did inhibit activation when liposomes were incubated with a mixture of purified C3, B, D, P, H, and I. Binding assays with radiolabeled H showed increased binding of H to liposome-bound C3b in the presence of GM1. These results establish the ability of sialic acid on glycolipids to promote H binding to C3b and thereby regulate alternative pathway activation on a defined lipid membrane.
Binding, Competitive↗
Value of fluid phase complement consumption as a means of 'in vitro' allergen standardization.
Atopic allergens can readily be shown to inactivate hemolytic complement in human serum. This property has been explored as a means of in vitro allergen quantitation and standardization. The purification of house dust allergen, guided by skin tests and complement inactivation, demonstrates that the in vitro technique is clinically relevant. Since human sera differ in their sensitivity to complement inactivation by allergens, it is desirable to prepare a purified house dust allergen as a standard. By referring to this standard, other house dust extracts may then be standardized by using any given serum sample of human origin, provided that such extracts have undergone some degree of purification. Kinetic studies demonstrate that other allergens of the house dust group may be standardized in similar fashion; it was noted that the ratio of the complement inactivating powers of these respective allergens remains constant whatever the serum used. The method is not suited for the quantitation of pollen allergens.
Allergens↗
Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells.
The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.
Ataxia Telangiectasia↗
Complement activating agents in allergenic extracts.
OBJECTIVE: Extracts of environmental allergens, moulds and plant pollens are known to consume haemolytic complement (huC) in human serum in vitro through the antibody-independent engagement of the first component C1 of the classical pathway. The present work was undertaken to establish the nature and characteristics of the complement activating agents in allergenic extracts and to probe their relationship with the IgE-binding allergens. MATERIALS AND METHODS: A large series of different > 10 kDa allergenic products was investigated for their capacity to consume haemolytic complement in the sera of allergic patients with specific anti-allergen IgE antibodies, as well as in normal control sera. UV-spectroscopy was used for categorizing the non-protein components in the extracts. RESULTS: The experiments confirmed that huC is consumed in an antibody-independent fashion and in a qualitatively similar, but quantitatively distinct manner. The ratio of the complement activating potencies among the different allergenic preparations thereby remained constant and independent of the serum source, while an overt relationship with specific IgE-antibodies could not be established. UV-spectroscopy of the allergenic preparations revealed the presence of chemical decomposition products in nearly every extract and roughly in proportion to the complement activating potencies. CONCLUSION: The data support the idea that huC-activation by traditional allergenic extracts is mainly due to by-stander degradation products of the melanoidin (Maillard) or tannin type, which may or may not occur in physical association with the IgE-binding protein allergens.
Allergens↗
Generation of C5-dependent bioactivity by tissue-bound anti-BMZ autoantibodies.
We previously reported that complement-binding antibasement membrane zone (BMZ) autoantibodies can mediate complement-dependent directed migration and adherence of leukocytes to the BMZ in cryostat skin sections and that there is heterogeneity in the ability of anti-BMZ autoantibodies to mediate that response. Those observations suggested that directed migration and adherence of leukocytes to the BMZ might be dependent on the amount of complement-activating autoantibody deposited at the BMZ and the extent to which those antibodies could activate complement and generate C5-derived peptides (C5a, C5a des arg). In this study, we have examined the role of autoantibody concentration and C5 in mediating the adherence response. When cryostat skin sections were pretreated with anti-BMZ autoantibodies and subsequently incubated with neutrophils suspended in fresh serum, neutrophils adhered to the BMZ. Adherence was anti-BMZ autoantibody specific and proportional to anti-BMZ autoantibody concentration. To determine the role of C5 in mediating adherence, neutrophils were suspended in increasing concentrations of: 1) fresh serum, 2) heat-inactivated serum, 3) serum pretreated with antihuman C5, 4) serum pretreated with antihuman IgG, 5) C5-depleted serum, 6) purified C5, and 7) C5-depleted serum reconstituted with increasing concentrations of purified C5. The suspensions were then incubated with autoantibody-treated skin sections. The results showed a dose-dependent requirement for fresh serum and for C5-depleted serum reconstituted with increasing doses of C5. Adherence could be detected with C5 concentrations less than 200 ng/ml, which correspond to a C5a/C5a des arg concentration of 10(-8)-10(-9) molar. These results suggest that complement-dependent neutrophil adherence is a highly sensitive method for detecting and quantitating the ability of tissue-deposited anti-BMZ autoantibodies to activate complement and generate C5-derived bioactive peptides, for estimating the amount of C-activating anti-BMZ autoantibody deposited at the BMZ in vivo, and for evaluating the potential role of C-activating anti-BMZ autoantibodies in the pathogenesis of lesions.
Antibody Specificity↗