Comparison of particulate number concentrations in three Central European capital cities.

Number size distributions of atmospheric aerosol particles in the mobility diameter range from 10 to 1000 nm were determined in Budapest, Prague and Vienna for a one-year-long period. Particle number concentrations in various size fractions, their diurnal and seasonal variations, mean size distributions and some properties of new particle formation events were derived and compared. Yearly median particle number concentrations for Budapest, Prague and Vienna were 10.6×10(3), 7.3×10(3) and 8.0×10(3) cm(-3). Differences were linked to the different pollution levels of the cities, and to diverse measurement environments and local conditions. Mean contributions of ultrafine particles (particles with a mobility diameter <100 nm) to the total number concentration were 80%, 84% and 74% for Budapest, Prague and Vienna, thus these particles represent an overwhelming share of all particles in each city. Seasonal variation of particle number concentrations was not obvious. Diurnal variations of particles with a diameter between 100 and 1000 nm (N(100-1000)) exhibited similar shape for the cities, which was related to the time-activity pattern of inhabitants and regional influences. The structure of the diurnal variation for ultrafine particles was also similar. It contained a huge morning peak in each city which was explained by emissions from vehicular traffic. The second peak was shifted from afternoon rush hours to late evenings as a result of the daily cycling in meteorological parameters. The character of the measurement site also influenced the diurnal variation. Diurnal variation of the mean ratio of ultrafine particles to N(100-1000) clearly revealed the presence and importance of new particle formation and subsequent growth in urban environments. Nucleation frequencies in Budapest and Prague were 27% and 23%, respectively on a yearly time scale. They showed a minimum in winter for both places, while the largest nucleation activity was observed in spring for Budapest, and in summer for Prague.

Heat-labile, complement-like factor(s) of animal sera prevent(s) HIV-1 infectivity in vitro.

We studied inactivation of HIV-1 by fresh sera of animals. We found that while fresh sera of humans and chimpanzees (among others) did not have antiviral activity, fresh sera of several other mammals, especially those of rodents and felines, showed a dose-dependent viral-inactivating property against cell-free HIV-1; these sera were also capable of inactivating virus preadsorbed to cells, similar to neutralizing antibody. The activity was destroyed by heating to 56 degrees C, required Ca2+ but no antiviral antibody, and therefore apparently involves the classical complement pathway. The activity could not be ascribed to any single fraction of sera separated on a size exclusion HPLC column. Mouse serum (the only one tested) also inactivated HIV-2. The data are consistent with classical C-mediated pathway inactivation of HIV-1 and HIV-2 by animal sera and is the first report of any "complement-like" antilentiviral serum factor. Elucidation of this mechanism may aid in understanding the lack of activity of human serum against HIV-1 and may prove useful in combined interventive strategies against HIV-1.

Immune complex mediated activation of the classical complement pathway.

The activation of classical C pathway by immune complexes depends on the binding and activation of C1, the first component of C. The Ig in the complex must be of the right class and in the right configuration to accomplish the conversion of precursor (zymogen) C1s to C1s, the active enzyme, whose substrates are C4 and C2. The primary question discussed in this paper is evidence that indicates that epitope distribution and density is a major factor in controlling the configuration of antibodies which in turn controls the activation of bound C1. This evidence confirms earlier findings that binding of C1 is a necessary but not sufficient condition for activating C1.

Further evidence for dilution-dependent dissociation of C1q in human serum.

Dissociation of the C1q subcomponent in native C1 upon dilution was reexamined by using ultracentrifugation in a sucrose gradient and high performance liquid chromatography system with a size exclusion column for separating the dissociated C1q fractions. The antigenic content of C1q in each fraction was detected by ELISA and Western blotting; binding to erythrocyte antibody was also determined. The results confirmed a previous claim that C1q in native C1 dissociated as a function of dilution: up to 14.5% of C1q antigen was in the low molecular weight form (approximate S value: 4-5). Commercial preparations of purified C1q also contained C1q antigen in the low molecular weight form.

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.

Serotherapy of cancer: cellular changes in primary rat mammary carcinomas after infusion of syngeneic sera absorbed with protein A-Sepharose.

Serotherapy and plasma therapy have proved to be effective in the treatment of diverse neoplasms. The mechanisms of the tumoricidal or growth-inhibitory effects are unknown. We previously reported that activation of the alternative pathway of complement in absorbed sera correlated with the presence of anti-tumor activity. Complement components generated during absorption may serve as the initial mediators of cytotoxicity; for example, C5a may function in its role as a chemo-attractant. To further investigate the anti-tumor mechanisms, we undertook a series of sequential histological studies of in vivo changes in tumors following i.v. serotherapy. We found diffuse inflammatory cellular infiltrates in the interstitial compartments of primary mammary carcinomas of rats within 3-4 hr of administration of protein A-Sepharose absorbed syngeneic serum. The number of inflammatory cells was significantly higher in tumors from treated rats: total infiltrating cells (p = 0.002), eosinophils (p = 0.001), neutrophils (p = 0.001), macrophages (p = 0.001), lymphocytes (p = 0.004) and plasma cells (p = 0.001). Also, the mitotic index of tumor cells was significantly lower 4 hr after serotherapy when compared with that of untreated rat tumor cells. C3 in tumor tissue was decreased at 4 hr following serotherapy. Fibrosis was present in tumor nodules with retarded growth 5 weeks after the start of serotherapy. Localization of the infiltrating cells to tumor interstitial compartments prevents direct contact between inflammatory cells and neoplastic cells, making it unlikely that direct cell-cell killing occurs. Indirect cell killing within the tumor bed apparently occurs through several mechanisms involving interactions between serotherapy-initiated humoral mediators and inflammatory cells. The resulting anti-tumor effects include microvascular injury leading to localized ischemia, tumor infarction, and fibroblastic reactions obstructing tumor invasion and growth.

Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes.

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.

Anti-IgM antibody decreases dissociability of cell bound IgM anti-hapten antibodies as measured with fluid phase hapten.

We studied the effect of an IgG anti-IgM on the dissociation of cell bound IgM anti-hapten antibody, as measured with fluid phase hapten. Methotrexate (MTX) covalently bound to the red cells surface was used as hapten; rabbit anti-MTX IgM and rabbit anti-allotype IgG reactive with the IgM were used as antibodies. The amount of cell bound antibodies was measured with 125I-labelled protein A. In the absence of the anti-allotype antibody, most of the anti-hapten IgM was prevented from reassociation by fluid phase hapten. In the presence of the anti-allotype IgG most of the anti-hapten IgM was non-dissociable, with about a 1000-fold increase in the apparent binding constant. We found that (1) dissociability of the anti-hapten IgM depended on the density of the cell bound hapten, (2) decrease in dissociability was a function of the anti-allotype antibody concn and (3) even in the presence of excess anti-allotype antibody dissociability was an inverse function of anti-hapten IgM concn.

Augmentation of hemolytic efficiency of anti-hapten IgG antibody by anti-IgG antibody is primarily the function of the anti-hapten IgG.

IgG anti-cell antibodies are inefficient in inducing cell lysis by complement. C mediated lysis by anti-cell IgG can be augmented by the use of C fixing anti-antibody. We have studied augmentation of rabbit anti-Forssman IgG, rabbit anti-methotrexate IgG and rabbit anti-folinic acid IgG antibodies by rabbit anti-allotype IgG antibodies. Large variability (from a low of 2 to a high of over 100) was found in augmentation efficiency even with the same anti-allotype antibody; the variability was primarily the function of the anti-hapten antibody. It was concluded that quantitative studies on anti-hapten IgG production relying on augmented hemolysis may lead to misleading conclusions.

Serotherapy of primary rat mammary carcinoma: inhibition by ethylenedinitrilotetraacetic acid but not by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

We reported inhibition of growth of primary rat mammary carcinomas after infusions of tumor-bearer plasma absorbed with Protein A-Sepharose or inactivated CNBr Sepharose. Absorbed plasmas were depleted of the third component of complement (C3) (other complement components defined similarly) and C5 but not C1, C4, or C2. These results suggested that activation of the alternative pathway of complement might be involved in the observed antitumor effects. To test this concept sera were treated with ethylenedinitrilotetraacetic acid or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid before absorption with Protein A-Sepharose. Ethylenedinitrilotetraacetic acid, by chelating calcium and magnesium, prevents activation of both the alternative and classical complement pathways. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid, by chelating calcium but not magnesium, permits activation of the alternative pathway but inhibits activation of the classical complement pathway. Sera in the presence or absence of chelating agent were absorbed with Protein A-Sepharose twice at room temperature. After absorption calcium was added to the sera. Rats were treated by i.v. injection of sera twice a week for 2 weeks. Measurements of tumor size were made weekly for 5-7 weeks and then tumor weight was determined. Groups were compared both for size of index and total tumors. The results can be summarized as follows: tumor-bearer sera before absorption did not inhibit the growth of rat primary mammary carcinomas; tumor-bearer sera after absorption with Protein A-Sepharose showed significant consumption of C3 and did inhibit tumor growth; tumor-bearer sera absorbed in the presence of ethylenedinitrilotetraacetic acid did not show a decrease in C3 functional activity and did not inhibit tumor growth; tumor-bearer sera absorbed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid did show a decrease in C3 functional activity and did inhibit tumor growth; sera from normal adult female rats after absorption with Protein A-Sepharose did inhibit tumor growth. The results are consistent with a role for the alternative pathway of complement in the inhibition of growth of rat primary mammary carcinomas observed after treatment with absorbed sera.

Anti-hapten IgG antibodies bound to cell surface hapten: anti-IgG antibody prevents dissociation as measured with fluid phase hapten.

We investigated the dissociation by fluid phase hapten of IgG antibodies bound to cell surface hapten in the presence and absence of anti-IgG antibodies. Dissociation was quantitated with fluid phase hapten, preventing reassociation of the anti-hapten antibodies. More than 90% of the anti-hapten IgG alone was prevented from reassociation by low concentrations of fluid phase hapten (nanogram to microgram range). In contrast, no dissociation of some IgG-anti-IgG complexes could be measured even at 24 hr incubation in the presence of very large excess of fluid phase hapten (100 mg/ml). We excluded aggregate formation between anti-hapten antibodies due to cross-linking by anti-antibodies as a cause for decreased dissociability by 1) performing the experiments in large excess of anti-antibody, 2) showing that the phenomenon was independent of anti-hapten antibody density, 3) showing that decreased dissociation also occurred at 4 degrees C, and 4) showing that aggregation by protein A did induce decreased dissociability, albeit three orders of magnitude lower than the anti-antibody. It was concluded that anti-antibody directly affected the "avidity" of cell hapten bound anti-hapten IgG in an unknown manner.

Effect of fibronectin on the haemolytic activity of complement.

Sheep erythrocytes were coupled with trinitophenyl sulphonate, sensitized with anti-TNP (or-DNP) IgM monoclonal antibodies, and exposed to components of the classical pathway of complement activation. When human fibronectin (FN) was added after C1q, but before addition of C1r and C1s (subunits of the first complement component), inhibition of haemolytic activity was observed which was strictly dependent upon the dose of FN. When FN was added after addition of C1 (reconstituted from C1q, C1r and C1s), the haemolytic activity of complement was not affected by the presence of FN. These data suggest that FN binds on C1q by interfering with C1r and C1s fixation. In addition, FN was unable to displace the activated subcomponents (C1r and C1s) from their binding site on C1q. When using other systems (sheep erythrocytes sensitized with anti-Forssman IgM monoclonal antibodies), the quantity of FN required to inhibit complement haemolytic activity was greater than in the TNP system. In normal plasma, there is a 50-fold excess of FN compared to free C1q.

Plasma therapy of primary rat mammary carcinoma: dependence of consumption of C3 during absorption of plasma with sepharose derivatives on the anticoagulant.

A previous study demonstrated inhibition of growth of primary rat mammary carcinomas after infusion of tumor-bearer plasma absorbed against Sepharose derivatives. In this report we have quantitated changes in individual complement components that occur during absorption of rat plasma with Sepharose derivatives and defined optimal conditions for consumption of the third component of complement (C3) (other complement components defined similarly). The concentration of functionally active C1 to C9 was measured before and after absorption in plasmas from both normal rats and rats with mammary tumors. C3 activity in plasmas from normal and tumor-bearing rats was reduced (consumed) during absorption under appropriate conditions with Sepharose 4B, inactivated CNBr Sepharose, or Protein A-Sepharose. The concentration of functionally active C1 and C4 did not decrease significantly during absorption with Sepharose derivatives. Consumption of C3 in rat plasma was influenced by the anticoagulant and by the time and temperature of incubation with Sepharose derivative. C3 consumption in rat plasma anticoagulated with acid citrate dextrose solution was variable; addition of Mg2+ (5 mM) to plasma anticoagulated with acid citrate dextrose solution augmented C3 consumption. There was no C3 consumption in plasma anticoagulated with ethylenedinitrilotetraacetic acid (a chelator of calcium and magnesium). In contrast, this reduction was observed in plasma anticoagulated with [(ethylenebis(oxyethylenenitrilo)]tetraacetic acid (a chelator of calcium). The results demonstrate optimal conditions for activation of the alternative pathway of complement during absorption of rat plasma with Sepharose derivatives and suggest in vivo experiments to define the role of this pathway in inhibition of growth of mammary tumors.

Activation of hemolytic complement by mouse monoclonal IgG1 antibody: comparison with rabbit IgG.

We investigated the ability of a mouse anti-hapten monoclonal IgG1 antibody (Ab) to bind to cell-bound specific hapten and to fix and activate C1 and thus the lytic sequence of complement (C). In a comparative study with polyclonal rabbit anti-hapten IgG Ab, we found that about 6 times more monoclonal Ab molecules than polyclonal were necessary for the generation of 1 hemolytic site/cell: the data were interpreted to mean that a cluster of four cell-bound monoclonal Ab molecules was necessary to bind C1 and activate C-mediated hemolysis. Experiments performed under conditions of low density of cell-bound hapten and excess of antibody showed that both monoclonal and polyclonal IgG Abs were able to react only with 20-30% of the cell-bound hapten and that both Abs recognized the same hapten specificity. We also found that even though monoclonal IgG1 Ab was able to bind strongly to a protein A-Sepharose column and could be eluted only by a low-pH buffer, the purified Ab, when bound to cell surface hapten, showed a weak ability to react with free protein A.

Efficiency of activation of complement by anti-hapten antibodies at the red cell surface: effect of patchy vs random distribution of hapten.

Binding and activation of complement (C) by anti-hapten IgG and IgM antibodies (Abs) bound to a cell surface are dependent on the density and presumably on the distribution of cell-bound hapten. The purpose of this study was to find out if altering the distribution of the hapten on a red cell surface could modify the ability of anti-hapten IgG or IgM Ab to activate C. To test this we devised methods for comparing the C binding and activating efficiency of Abs bound to a hapten distributed randomly or in patches on cells. Random distribution was achieved by the covalent binding of methotrexate (MTX) directly to sheep red blood cells (E), while patchy distribution was achieved by the convalent binding to E of bovine serum albumin-MTX complexes. We considered bound albumin molecules as patches of MTX molecules. The results showed that, for IgG Ab, the number of hapten/E and the number of anti-hapten Ab molecules/cell required to generate one C-activating IgG complex were about an order of magnitude lower for hapten bound in patches than for randomly bound hapten. In contrast, IgM Ab bound to a hapten distributed in patches on an E surface lacked the ability to activate the lytic sequence of C, although maintaining a full ability to binding C1.

Mouse monoclonal antibodies at the red cell surface--I. Generation of EAC4 and interaction with C1.

C1 and C1 activity measurements were performed with EA and EAC4 prepared with rabbit anti-Forssman IgG or IgM and were compared to measurements with EA and EAC4 prepared with mouse monoclonal IgG2b and IgM anti-DNP antibodies on cells coupled with TNP: the amount of TNP per cell was optimal for antibody activity. No differences were found in the ability of EAC4 made with poly- or monoclonal IgM to measure C1 activity; in contrast, monoclonal IgM was capable of activating only about 30% of C1 when compared to activation by polyclonal IgM. Monoclonal vs polyclonal IgGs behaved in a similar manner but they were detecting only 50% of C1 or C1 activity when compared to IgM of the appropriate class. It was concluded that monoclonal antibodies were capable of generating EAC4 intermediate, and that the ability of monoclonal antibodies in the EAC4 complex to bind C1 and to detect C1 activity is not significantly different from that of polyclonal antibodies but that monoclonal antibodies are less efficient in activating C1 than polyclonal antibodies.

Mouse monoclonal antibodies at the red cell surface--II. Effect of hapten density on complement fixation and activation.

Complement (C)-dependent hemolytic dose-response curves of anti-TNP IgG2b and IgM monoclonal antibodies as a function of TNP density were analyzed: sheep red cells coupled with TNP served as targets. Under conditions when equal numbers of either IgG2b or IgM anti-TNP antibodies were taken up by cells with various TNP densities, both antibodies showed optimal activity at a hapten density of approximately 10(6) TNP/E with regard to C-mediated lysis. These results were similar to those obtained with polyclonal antibodies. The effectiveness of monoclonal antibodies in utilizing guinea pig or mouse C was also investigated. IgM anti-TNP monoclonal antibodies lysed E-TNP in the presence of guinea pig C, but failed to produce lysis in the presence of mouse C. Two monoclonal IgG1 (anti-TNP and anti-SRBC) and an IgG2a anti-TNP antibody failed to produce hemolysis in the presence of guinea pig and mouse C. IgG2b monoclonal antibodies, whether directed against TNP or Forssman antigen, activated guinea pig and, to a lesser extent, mouse C. Finally, monoclonal anti-TNP IgG3 antibodies exhibited a low but measurable hemolytic activity with guinea pig C (10-20 times below that of IgG2b).

Binding and activation of hemolytic complement by IgG antibodies: cooperativity between antibodies of different hapten specificity.

The ability of IgG antibodies with different hapten specificities to fix C1 and activate C as a function of hapten density on a red cell surface was investigated. Rabbit anti-methotrexate and anti-folinic acid IgG antibodies in a mixture were highly efficient in fixing C1 and activating C when cells carried simultaneously high levels of both haptens. We wished to find out whether in a C-activating IgG complex both IgG molecules had to be in a form that could activate C1. By reducing hapten density of one of the haptens on a double labeled cell, complexes were generated where only one in a pair of IgG molecules was in the activating form; such a pair had the same activating efficiency as a pair in which both IgG molecules were in the activating form. It was concluded that cooperative activation of C in C1-binding IgG complexes required only one IgG in the complex to be in the activating form.