Activity Of Normal Human Serum Research Articles

Decreased C5b67-inhibitor activity in two families with hereditary functional deficiency of the eighth component of complement.

In addition to its role in hemolysis and host defense against Neisseria infection, the eighth component of human complement (C8) is one of several plasma proteins that are C5b67-inhibitors (C5b67-INH). The recent identification in our laboratory of two new families with hereditary deficiency of C8 provided an opportunity to study further the role of C8 as a C5b67-INH. Based on mixing and reconstitution experiments, the deficiency of C8 seemed to be due to a selective lack of the C8 beta-chain in one family and the C8 alpha-gamma subunit in the other family. Sera from individuals homozygous for the C8 abnormality were substantially deficient in C5b67-INH activity as well as totally deficient in hemolytic activity. Sera from control individuals possessed approximately 2500 C5b67-INH U/ml, whereas sera from the C8-deficient individuals had markedly depressed C5b67-INH activity, with a mean of only 428 U/ml. C5b67-INH activity was completely reconstituted in C8-deficient serum by the addition of purified human C8. We conclude that C8 constitutes the substantial majority of the C5b67-INH activity of normal human serum.

Human neutrophil chemotactic response to group A streptococci: bacteria-mediated interference with complement-derived chemotactic factors.

The influence of M protein on the capacity of group A streptococci to generate neutrophil chemotactic activity in normal human serum was examined. Incubation of serum with M- bacteria for up to 10 min led to the production of chemotactic activity. In contrast, incubation of serum with M+ bacteria did not elicit serum chemotactic activity over a 1-h period, even though complement was activated to completion. Further experiments revealed that both M+ and M- bacteria could inhibit the chemotactic activity of serum preexposed to zymosan. However, the M+ bacteria possessed a 130-fold-greater inhibitory capacity in this regard than the M- bacteria. This antichemotactic property was not detectable in the fluid phase of serum incubated with bacteria, thereby ruling out neutrophil-directed effects. Treatment of the bacteria with trypsin resulted in the release of the inhibitory molecule, suggesting that proteins are involved in its maintenance at the cell surface. However, the resistance of the chemotactic factor inactivator to pepsin and trypsin indicated that the protease-sensitive M protein was not involved. These results demonstrate a heretofore uncharacterized activity of group A streptococci that may contribute to virulence through modulation of the host chemotactic response.

Examination of the bactericidal and opsonic activity of normal human serum for a mucoid and nonmucoid strain ofPseudomonas aeruginosa

The bactericidal and opsonic activity of fresh human serum (FHS) for a mucoid strain ofPseudomonas aeruginosa, 144M, and its spontaneous nonmucoid revertant, 144NM, was examined. Strain 144M was sensitive to the bactericidal activity of FHS, but strain 144NM was not. This bactericidal activity was due to the combined interaction of IgG and IgM with complement, activated through both pathways. Neither 144M nor 144NM was ingested by human polymorphonuclear leukocytes (PMNL) without FHS. Whereas maximal phagocytosis of 144M required only 5% FHS, comparable ingestion of 144NM required 25% FHS. Maximal phagocytosis of either 144M or 144NM required IgG, IgM, and complement. However, 144M required a heat-sensitive opsonic IgG, whereas 144NM required a heat-resistant IgG. Using selective absorption techniques, the targets for bactericidal and opsonic immunoglobulins on 144M and 144NM appeared to be different, suggesting that the variant 144NM had one or more altered, absent, or inaccessible cell surface components that account for differences in response to FHS and PMNL.

Serum sensitivity of strains isolated and antibodies against O antigen in gram-negative bacteraemia.

The sensitivity of blood culture isolates to the bactericidal activity of normal human serum (NHS) has been studied in 101 patients with gram-negative sepsis. These results were compared with clinical status and outcome, and to the presence of specific IgG or IgM antibodies to O antigens of bacteraemic strains in autologous serum. 23% of the strains were markedly resistant, 27% markedly sensitive and 50% intermediately sensitive to the bactericidal activity of NHS. Shock or death occurred more frequently in immunocompromised patients and those infected with serum-resistant strains. IgG antibody titres to O antigens were significantly lower in patients with serum-resistant organisms regardless of their immune status. Resistance to natural bactericidal antibodies and low immunogenicity of the infecting organism, plus immunodeficiency in the host, may account for apparent increased virulence of some gram-negative bacilli.

STUDIES ON NEISSERIA GONORRHOEAE STRAINS FROM TEST‐OF‐CURE SPECIMENS

Sixty-seven out of 88 Neisseria gonorrhoeae strains isolated from test-of-cure (TOC) specimens during a five-months' period were included in the study. For 62 patients sufficient information was obtained in order to distinguish between relapse (34 ptt) and re-infection (28 ptt). For comparison with strains from these two groups of patients, 63 urogenital and 21 pharyngeal gonococcal strains isolated during the same period of time were randomly selected. The distributions according to susceptibility to penicillin for TOC strains and control strains corresponded to those found for the total number of TOC strains (275) and other strains (3,345) tested in 1979, respectively. The TOC strains did not differ from the control strains in sensitivity to the complement-dependent. The TOC strains did not differ from the control strains in sensitivity to the complement-dependent bactericidal activity of normal human serum. However, gonococcal strains less susceptible to penicillin in vitro (MIC values within the range 0.1-2.0 microgram/ml) were significantly more sensitive to the complement-dependent activity of normal human serum (P less than 0.01) than strains fully susceptible to penicillin (MIC less than 0.01 microgram/ml.) Penicillin-resistant strains (MIC greater than 2.0 microgram/ml) did not differ from strains susceptible to less than 0.1 microgram penicillin/ml and were slightly more serum-resistant than the less susceptible strains (P less than 0.05). No difference in serum-sensitivity of urogenital and pharyngeal isolates could be demonstrated. The level of bactericidal activity of homologous convalescent serum was unrelated to the presence of antibodies either to gonococcal pili or crude gonococcal antigen preparations. The sensitivity to normal human serum of a certain strain was not correlated with sensitivity to homologous convalescent serum.

RELIABILITY OF THE PLAQUE ASSAY TO DETERMINE THE SENSITIVITY OF NEISSERIA GONORRHOEAE TO THE BACTERICIDAL ACTIVITY OF NORMAL HUMAN SERUM

A plaque assay for determination of the sensitivity of Neisseria gonorrhoeae to the complement-dependent bactericidal activity of normal human serum was evaluated. The method was found to be rapid and easy to perform. All strains, which gave a positive result by the plaque assay, were serum-sensitive in a quantitative assay, whereas only 78% of strains giving negative results by the plaque assay were true serum-resistant. The growth of a few gonococcal strains was inhibited, i.e. formed plaques, after exposure to genuine as well as to heat-inactivated normal human serum.

Pyocin-resistant lipopolysaccharide mutans of Neisseria gonorrhoeae: alterations in sensitivity to normal human serum and polymyxin B.

Pyocins from Pseudomonas aeruginosa were used to select several lipopolysaccharide (LPS) mutants of Neisseria gonorrhoeae strain FA19. Three classes of LPS mutans were found in the initial group selected for study. The LPS of one class lacked galactose. That of a second group lacked the typical heptose found in the parental LPS, was reduced in glucose, galactose, and N-acetylglucosamine content, appeared to contain a new unidentified sugar component, and consisted of two species of LPS separable on sodium dodecyl sulfate-polyacrylamide gels. The LPS of a third strain lacked the heptose, glucose, galactose, and N-acetylglucosamine found in the oligosaccharide portion of parental FA19 LPS. The minimal inhibitory concentration for polymyxin B of the mutant strains was 3 to 4 times that of the parental strain. The strains lacking only galactose were as resistant as the parent to the bactericidal action of normal human serum, but cells of the other two classes were quickly killed by serum. Gonococcal LPS thus appears to be important in determining phenotypic properties of the cells.

Immunological basis of serum resistance of Neisseria gonorrhoeae.

The immunological basis for resistance of certain strains of Neisseria gonorrhoeae to the bactericidal action of normal human serum was studied by investigating the potential role of factors which are known to interfere with each of the sequential steps that result in immune lysis of Gram-negative bacteria. Strains of N. gonorrhoeae were characterized as serum-sensitive (sers) or serum-resistant (serr) on the basis of their sensitivity to lysis by the sera of six normal individuals. Neither intrinsic resistance to the lytic action of activated human complement nor inaccessibility of the cell membrane to C5b accounted for serr. Outer membrane lipopolysaccharide (LPS) was the target antigen for lytic antibody in normal human sera. The gross chemical composition and molecular size of the LPS of the strains were heterogeneous and no consistent patterns of differences between those extracted from serr and from sers strains were found. Neither IgA nor IgG 'blocking' antibody in normal human serum was responsible for serr. We conclude that serr results from the absence from the LPS of the strains of antigenic loci for the lytic antibody in most normal human sera, or, expressed as a function of the host, the absence from the sera of most normal humans of lytic antibody directed against LPS antigenic loci for immune lysis.

Genetics of serum resistance in Neisseria gonorrhoeae: the sac-1 genetic locus.

A genetic locus affecting susceptibility to the bactericidal activity of normal human serum has been designated sac-1. This locus was shown to be closely linked to, but not identical with, a second locus (designated nmp-2) that affects protein 1 of the outer membrane. The sac-1 locus could be linked to known antibiotic resistance markers on the gonococcal chromosome by genetic transformation.

The 5'-nucleotidase activity in normal human serum. Electrophoretic patterns and substrate specificity

Both the electrophoretic patterns and substrate specificity of the total 5'-nucleotidase activity at pH 7.4 in serum from 10 normal adults (19–49 years) were identical using various purine and pyrimidine mononucleotides as substrates. Different purified alkaline phosphatases were studied at the same time in the same manner. The serum enzymes showed substrate specificity qualitatively similar to that of the bovine liver enzyme. The electrophoretic study showed two fractions for both the liver enzyme and the serum enzymes with the different substrates, indicating that each of these are dephosphorylated by the same two, mutually different, enzyme molecules. Each of the other enzymes showed only a single fraction, which was identical for each enzyme with the various substrates.

Carbohydrate alters the trypanocidal activity of normal human serum with Trypanosoma brucei.

Three categories of carbohydrates are distinguished based on their effects on the trypanocidal activity of normal human serum on Trypanosoma brucei. The first category includes carbohydrates such as glucose, mannose and fructose which are utilized by parasites as an energy source. These carbohydrates inhibit the trypanocidal activity in a concentration-dependent manner. The third category, represented by xylose and mannitol, are not energy sources to the parasites but inhibit the trypanocidal activity in a manner similar to the first category carbohydrate. However, at very high concentrations (approximately 140-150 mM), the inhibition is reversed. Glycerol is the only known carbohydrate belonging to the second category. Glycerol enhances the trypanocidal activity of normal human serum and of those sera which normally display no cytotoxic activity, although glycerol serves as an excellent energy source. On the basis of these results, the mechanisms by which these three categories of carbohydrate could alter the trypanocidal activity are discussed in terms of the interaction between a trypanocidal factor and the surface receptor.

In vitro studies on complement inactivation by snake venoms

Snake venoms of the families Elapidae, Viperidae and Crotalidae were investigated concerning their effects on the human complement system. Twenty-two of 26 venoms inhibited complement activity in normal human serum. Most efficient were the venoms from Naja naja, Agkistrodon rhodostoma, A. halys, Vipera lebetina and Trimeresurus okinawensis. Consumption of the complement factors C2, C3 and C4 was observed with most venoms. Factor B and C3 were transformed to physiological conversion products. Purified C3 was converted by most venoms, suggesting a direct effect on this factor. Conversion was inhibited by EDTA, but could in some cases be restored by the addition of Mg 2+ or Ca 2+. We conclude that viperid and crotalid venoms inhibit the complement system through proteolytic action on several of its components, in contrast to active elapid venoms of the genus Naja, which activate the system owing to ‘cobra venom factor’. In no other case could a clear specific attack on a single pathway be demonstrated. However, some distinctive features of the multipoint attack made possible a grouping of the venoms into three categories which might be valuable in future studies of the complex factors involved in complement activation.

Ca2+ is essential cofactor for trypanocidal activity of normal human serum.

Normal human serum has been known to exert a cytotoxic effect on Trypanosoma brucei subspecies for nearly 80 yr. But in spite of many attempts, no trypanocidal factor was found in human or baboon serum, until Rifkin demostrated a high density lipoprotein (HDL) in normal human serum with trypanocidal activity. The conclusion that this was the trypanocidal factor was supported by the report that serum from patients with Tangier disease, characterised by a severe deficiency of HDL, lacked trypanocidal activity. We report here that Ca2+ is an essential cofactor for the trypanocidal activity of normal human serum, in which alpha2 macroglobulin (alpha 2) might function as a Ca2+-carrier. We further show that D-glucose, D-fructose and D-mannose can suppress the trypanocidal action of normal human serum, whereas glycerol has the opposite effect.

Susceptibility of Isolates of Bacteroides to the Bactericidal Activity of Normal Human Serum

Seventy-one strains of species from the Bacteroides fragilis group, including 46 isolates of B. fragilis, were tested for susceptibility to the bactericidal effect of serum from healthy subjects. Twenty-seven (38%) of the isolates were killed by serum. Isolates from feces were significantly more sensitive to serum than were isolates from patients with clinical infections. Killing of bacteria required heat-labile serum components and was an exponential function of serum concentration. Among the various species tested, B. fragilis was clearly the most resistant to bactericidal activity of serum. These observations may be important to the understanding of infections caused by the B. fragilis group, which contains the anaerobes of greatest clinical importance.

Immunoglobulin Class Responsible for Gonococcal Bactericidal Activity of Normal Human Sera

Abstract “Natural” gonococcal bactericidal antibody is present in the serum of most normal adults and may prevent the systemic dissemination of serum sensitive strains of Neisseria gonorrhoeae. The bactericidal activity of normal human serum for gonococcal strain F62 resides entirely in the IgM serum fraction and is destroyed by treatment with 2-mercaptoethanol. Its absence from cord, but not paired maternal sera, provides additional evidence that this activity is mediated by IgM. Serum bactericidal activity is low and comparable to cord serum for the first 4 weeks of life, rising progressively thereafter to 25, 50, and 100% of the adult geometric mean titer at ages 6 months, 1 year, and 2 years, respectively. The sera of eight patients with x-linked agammaglobulinemia were deficient in gonococcal bactericidal antibody. In contrast, sera from a single patient with x-linked immunodeficiency with increased IgM and nine patients with ataxia telangiectasia possessed normal bactericidal activity for strain F62. The development of gonococcal bactericidal antibody may be delayed in the Wiskott-Aldrich syndrome, since bactericidal activity was low for age in two patients studied at age 2 years, but normal in a third child studied at age 12. The magnitude and nature of the antibody response to bacteriophage antigen and the IgM serum concentration were correlated with the serum bactericidal activity of 22 patients with variable immunodeficiencies. The characterization of the natural occurrence of gonococcal bactericidal antibody should serve as a useful baseline in studies of the immune response to gonococcal infection or immunoprophylaxis.

The stability of erythroid stimulating activity in normal human serum

Erythroid stimulating activity (ESA) in normal human serum has been shown to be labile to storage even at temperatures as low as −20°C. The addition of phenol to a final concentration of 0.1% preserves the ESA. Serum should be stored with the addition of 0.1% phenol prior to assay for ESA. Because of the variability in the assay procedures two sera cannot be considered to differ significantly in their ESA content unless the measured titers differ by at least a factor of two.

Enhancement of Eosinophil Leukocyte Chemotaxis by a Factor in Normal Human Serum (SEF)

An activity in normal human serum is described which enhances the migration of eosinophils towards neutrophil-derived eosinophil chemotactic factor (ECF). The serum-enhancing factor (SEF), in contrast to the inhibitors in serum, is expressed primarily during weak chemotaxis, acts in a cell-directed fashion, increases the chemokinesis of eosinophils and, in the presence of ECF, enhances chemotaxis in a synergistic fashion. SEF is found in sera of several mammalian species, affects human and guinea pig eosinophils, and it is heat-labile (56 degrees C). On Sephadex column chromatography, SEF has a molecular weight of approximately 800,000 daltons. In some sera, a second activity with a molecular weight of approximately 200,000 daltons is eluted. SEF does not enhance eosinophil migration once the cells have been deactivated in vitro by their chemotactic factor. Because of its wide distribution, SEF may play an important modulating role both in in vitro assay systems and in vivo.

Trypanosoma brucei: Some properties of the cytotoxic reaction induced by normal human serum

The trypanocidal activity of normal human serum has been studied in vitro using Trypanosoma brucei as the test organism. The variables affecting the rate and extent of lysis, such as time, temperature, serum concentration, and pleomorphism of trypanosomes, are described. Trypanocidal titers of serum and serum fractions were quantitatively determined under standardized incubation conditions. Inactivation and/or removal of components of both the classical and alternate pathways of complement activation had no effect on the trypanocidal properties of human serum. The active factor was nondialyzable, present in plasma at equivalent levels to that in serum, and not removed by absorption with IgG fractions of antisera against human IgM or α 2-macroglobulin. The trypanocidal factor could be inactivated by heat (65 C), dithiothreitol, urea, and trypsin. Gel filtration studies indicated that the trypanocidal activity eluted as a single protein with a molecular weight of about 500,000.

Trypanosoma brucei: New radioisotope assay for quantitating cell lysis

A new radioisotope technique for quantitatively determining the extent of cell lysis of trypanosomes is described. Trypanosoma brucei was pulse-labeled with [ 3H]leucine and then incubated at 37 C in lytic (human serum) or nonlytic (rabbit serum) media. The release into the incubation medium of radioactively labeled intracellular macromolecules was then determined. A linear relationship was found between the release of TCA-precipitable radioactivity and lysis, as determined by conventional hemacytometer counting. This method is used to quantitate the trypanocidal activity in normal human serum.

Nonimmunologic Complement Activation in Normal Human Serum Induced by Radiographic Contrast Media

Two different radiographic contrast media (RCM), iothalamate and iodipamide, induced the activation of several complement (C) components in normal, genetically C2-deficient and agammaglobulinemic human sera in vitro. This activation was dose dependent and demonstrable by a reduction in whole C as well as C4, C2, C3, and C5 hemolytic activities. C6, C8, and C9 hemolytic activities were unaffected. Concommitant with the loss of C3 hemolytic activity was the appearance of C3 proteolytic cleavage products that were identified by immunoelectrophoresis. Both the loss of C3 hemolytic activity and the production of C3 fragments occurred in the presence of 10 mM EDTA, indicating RCM-induced C3 cleavage occurred without participation of the multicomponent C3/C5 convertases of either the classical or alternative C pathways. Furthermore, loss of C3 hemolytic activity was not due to the direct alteration of the C3 molecule by RCM because purified C3 was unaffected upon incubation with RCM at a concentration that induced 80% reduction in the C3 hemolytic activity in normal human serum. Serum samples obtained from 40 patients, before and 30 min after undergoing i.v. pyelography, revealed no significant change in total hemolytic C activity; 34 patients received sodium and methylglucamine diatrizoate and six received sodium iothalamate. Hemolytic C3 levels were also determined for the six patients before and 30 min after administration of sodium iothalamate and no significant change in activity was detectable.