Neutralization of human serum lysozyme by sodium polyanethol sulfonate but not by sodium amylosulfate.

Sodium polyanethol sulfonate (SPS) at 500 microgram/ml, but not sodium amylosulfate (SAS) at 500 microgram/ml, precipitated egg white lysozyme (1 mg and 50 microgram of lysozyme per ml) as determined with the assay strain Micrococcus lysodeikticus ATCC 4698. Fresh and heat-inactivated (56 degrees C, 30 min) human serum (80%, vol/vol) killed M. lysodeikticus (10(4) bacteria per ml at zero time) within 1 to 2 h after exposure. Addition of 250 to 500 microgram of SPS per ml to fresh human serum protected M. lysodeikticus for 22 h as effectively as absorption of either fresh or heat-inactivated human serum with bentonite (10 mg/ml of serum, 10 min, 37 degrees C); the latter procedure is known to remove serum lysozyme. In contrast, SAS at 250 and 500 microgram/ml of serum retarded killing of the assay bacteria for periods of 4 h; after overnight (22 h) incubation, however, the number of M. lysodeikticus survivors had decreased significantly. The finding that SPS, but not SAS, at 250 to 500 microgram/ml effectively neutralized serum lysozyme-mediated killing of a lysozyme-sensitive assay strain may be of relevance with respect to laboratory processing of human blood culture specimens.     

Comparison of sodium amylosulfate and sodium polyanetholsulfonate in blood culture media.

A comparison between sodium polyanetholsulfonate and sodium amylosulfate in unvented vacuum blood culture bottles containing tryptic soy broth was made with 5,800 sets of blood cultures. No statistically significant differences in isolation rates of bacteria were noted.     

Studies on neutralization of human serum bactericidal activity by sodium amylosulfate.

The synthetic anticoagulant sodium amylosulfate (SAS) at concentrations of 125 to 2,000 microgram/ml failed to completely neutralize the bactericidal activity of 80 and 50% (by volume) fresh human serum. Furthermore, SAS failed to inhibit the alternative pathway of complement activation in 80% (by volume) fresh human serum that had been chelated with 0.01 M magnesium ions plus 0.01 M ethyleneglycol-bis(beta-aminoethylether)-N,N-tetraacetic acid. However, SAS at 250 to 1,000 microgram/ml effectively neutralized the bactericidal activity of 20% (by volume) fresh human serum. Therefore, SAS (at 250 to 1,000 microgram/ml) should be used only in blood samples that have been diluted at least fivefold (less than or equal to 20% [by volume]) in suitable broth media.     

Inactivation of classical and alternative pathway-activated bactericidal activity of human serum by sodium polyanetholsulfonate.

Sodium polyanetholsulfonate (SPS) at a final concentration of at least 250 microng/ml (0.025%) was required for inhibition of the bactericidal activity of 80% (vol/vol) of fresh human serum against "promptly serum-sensitive" strains of Serratia marcescens and control strain Escherichia coli C, i.e., for inhibition of the classical pathway of complement activation. In contrast, SPS at 125 microng/ml (0.0125%) was sufficient for neutralization of the bactericidal activity of 80% (vol/vol) fresh human serum against "delayed serum-sensitive" strains of S. marcescens known to activate the alternative pathway of human complement. Addition of up to 500 microng of SPS per ml to 80% (vol/vol) fresh human serum failed to neutralize transferrin-mediated, "late" bacteriostasis against control strain E. coli C, an effect that was demonstrable only after prolonged, i.e., overnight, incubation of the test strain. However, this late inhibitory effect against E. coli C was not observed in SPS-treated 20% (vol/vol) fresh human serum or in 10 or 20% (vol/vol) conventionally heat-inactivated human serum. Immunoelectrophoretic examination disclosed that SPS did not precipitate transferrin from either fresh or heat-inactivated human serum. Thus, SPS, at 250 microng/ml, was demonstrated to be sufficient for the inhibition of both classical and alternative complement pathway-activated bactericidal activity of 80% (vol/vol) human serum. However, SPS at a concentration of 500 microng/ml failed to antagonize one antimicrobial system of 80% (vol/vol) human serum, namely transferrin-mediated bacteriostasis.     

Inactivation of the polyanionic detergent sodium polyanetholsulfonate by hemoglobin.

Sodium polyanetholsulfonate (SPS) has been added to blood culture media for many years. Its incorporation results in a higher yield of positive blood cultures due to its inactivation of antimicrobial cationic compounds. The most active of these cations include complement components, aminoglycoside-aminocyclitol antibiotics, and receptors on polymorphonuclear leukocytes. There have been reports from studies conducted outside patient blood culture bottles that SPS itself may possess antibacterial activity against some isolates of Neisseria meningitidis, Neisseria gonorrhoeae, and Peptostreptococcus anaerobius. Conversely, in patient clinical trials there has been no significant difference in pathogen isolation rates in the presence or absence of SPS. In an attempt to explain this in vitro/in vivo disparity, a search was undertaken to elucidate which variable constituent in blood, heretofore not studied quantitatively, might have a major effect on modulating the activity of SPS. It was found that hemoglobin combined stoichiometrically with SPS with a Kd of approximately 10(-7) mol/liter. Optimum SPS inactivation occurred at an SPS/hemoglobin ratio of 1:6 (wt/wt). SPS-sensitive isolates of N. gonorrhoeae and N. meningitidis were protected by the addition of hemoglobin from the antimicrobial effects of this polyanion in time-kill studies. This protection was directly related to the amount of SPS combined in solution. Therefore, the amount of free hemoglobin in solution must be measured when studying the antimicrobial activity of polyanions or when evaluating the effect of different polyanions on the recovery rates of pathogens in patient blood culture clinical trials.     

Neutralization of bacteria- and endotoxin-induced hypotension by lipoprotein-free human serum.

Normal human serum and a fraction rich in lipoprotein, Cohn fraction IV1, have been shown in previous studies to detoxify native endotoxin by decreasing lethality for mice, fever in rabbits, and by the alteration of the characteristic endotoxin-anti-endotoxin precipitin pattern in gels. These studies are extended herein and document the ability of normal human serum and fraction IV1 to neutralize the induction of hypotension in rabbits by viable gram-negative bacilli. Further fractionation of serum, using an ultracentrifugal flotation method for producing lipoprotein-free human serum and purified high-density lipoproteins, revealed the lipoprotein-free fraction to be capable of inhibiting endotoxin hypotensive activity and to alter diffusion of endotoxin in gels. On the other hand, the purified high-density lipoproteins failed to negate either activity.     

Medium-dependent inhibition of Peptostreptococcus anaerobius by sodium polyanetholsulfonate in blood culture media.

Of 13 species of anaerobic cocci, Peptostreptococcus anaerobius was the only species tested that was sensitive to 0.1% sodium polyanetholsulfonate (SPS). However, the sensitivity of P. anaerobius to SPS varied according to the media in which the cultures were grown. In supplemented peptone (B-D) and brain heart infusion media, most strain of P. anaerobius were not inhibited by SPS. Gelatin and proteose peptone were the medium components which were protective. The minimal inhibitory concentration of SPS for P. anaerobius was approximately 60-fold higher in media. However, the concentration of SPS required to neutralize the bactericidal properties of human serum was only four fold higher in media containing geltain. In a commerical medium containing SPS (0.03%) and gelatin (1.2%), SPS-sensitive strains of P. anaerobius were not inhibited by SPS, and the bactericdal action of human blood on Escherichia coli C and Serratia marcescens SM 29 was eliminated.     

Neutralization of bacterial lipopolysaccharides by human plasma.

To quantify the neutralization of bacterial lipopolysaccharide (LPS) by human plasma, dilutions of Escherichia coli O113 LPS were incubated with plasma, followed by the addition of Limulus amebocyte lysate (LAL). The reaction between the LPS and LAL was monitored spectrophotometrically, and the concentration of LPS resulting in 50% lysate response (LR50) was determined. Analysis of 145 outdated plasma samples yielded a range of LR50 between 6 and 1,500 ng/ml. Pools of plasma with high and low LR50 were prepared. The pool with high LR50 neutralized 166-fold more E. coli 0113 LPS, 190-fold more E. coli 0111B4 LPS, 42-fold more Klebsiella pneumoniae LPS, and 29-fold more Salmonella typhimurium LPS than did the pool with low LR50. Each pool had similar immunoglobulin G (IgG) and IgM antibody levels to homologous LPS, measured by an enzyme-linked immunosorbent assay. Analysis of 212 fresh-frozen plasma units revealed a range of LR50 between 48 and 6,000 ng/ml. Incubation of LPS in a pool of fresh-frozen plasma with high LR50 elicited significantly less fever in the rabbit pyrogen test than did LPS incubated in plasma with low LR50 (fever index, 2.68 +/- 0.61 degrees C X h and 3.52 +/- 0.66 degrees C X h, respectively; P = 0.003). We conclude that there is a 100-fold range in the endotoxin-neutralizing capacity of human plasma and that this variation is not due to LPS-specific IgG or IgM antibodies. Further investigations are needed to determine whether differing susceptibility of patients to the effects of LPS is due to differences in the endotoxin-neutralizing capacity of their plasma and whether plasma screened for high endotoxin-neutralizing capacity may be therapeutically useful in endotoxemia.     

Comparative evaluation of supplemented peptone broth with sodium polyanetholesulfonate and trypticase soy broth with sodium amylosulfate for detection of septicemia.

We compared the yield and speed of detection of clinically important microorganisms from 10,156 paired 5-ml samples of blood cultured in supplemented peptone broth (SPB) with 0.03% sodium polyanetholesulfonate (SPS) or Trypticase soy broth (TSB) with 0.5% sodium amylosulfate (SAS). The atmosphere of incubation (open venting units) and ratio of blood to broth (1:10) were the same for both samples. Only cultures with adequate blood samples (greater than or equal to 80% of stated volume) were compared statistically. Overall, SPB/SPS outperformed TSB/SAS. Bacteroidaceae and Eubacterium were found more often (P less than 0.05) and viridans streptococci were found sooner (P less than 10(-4)) in SPB/SPS than in TSB/SAS. Most importantly, staphylococci were found both more often (P less than 0.03) and sooner (P less than 10(-7)) in SPB/SPS than in TSB/SAS. In a separate experiment, SAS slowed the growth of a clinical strain of Staphylococcus aureus in TSB. Unless important advantages can be confirmed for SAS in controlled clinical trials, SAS cannot be recommended for routine use as an anticoagulant in blood culture media.     

Neutralization of tetanus toxin by human and rabbit immunoglobulin classes and subunits

This investigation found that the human antibody class of importance in neutralizing tetanus toxin in mice was IgG, and that toxin neutralization was retained by the F(ab')2 and Fab' subunits of the human IgG class. Although human IgM and IgA classes appeared to neutralize tetanus toxin at very low levels, evidence was obtained that this neutralization was probably due to IgG contamination. Human Fabmu isolated from the IgM class did not neutralize tetanus toxin. Human antibodies of the IgG, IgM and IgA classes reacted with tetanus toxoid in the indirect haemagglutination (HA) test with IgG giving the highest HA titre. Rabbit antibodies of the IgG class also neutralized tetanus toxin, with neutralization being retained by the F(ab')2 and Fab' subunits of the rabbit IgG class. Absorption of several rabbit antisera to tetanus toxoid with goat-antirabbit Fc which is specific for absorption of IgG from antiserum, rendered them incapable of neutralizing tetanus toxin.     

Release of beta-lysin from platelets caused by antigen-antibody complexes, purified enzymes, and platelet-aggregating substances.

The release of beta-lysin, which followed the intravenous injection of antigen-antibody complexes, did not take place when these complexes were added to citrated whole blood but did occur in heparinized blood. beta-Lysin release in heparinized blood was inhibited by citrate but were reversed by the addition of calcium ions that implicated complement reactions. Fourteen different enzymes were added to platelet-rich plasma (PRP). Streptokinase, neuraminidase, papain, phospholipase C, sulfatase, and trypsin caused platelets to release significant quantities of beta-lysin, whereas elastase, phosphatase, protease, ribonuclease A, hyaluronidase, lipase, and pepsin caused little or no increase in the plasma beta-lysin concentration. One enzyme, fibrinolysin, inactivated beta-lysin faster than it was released. The enzyme-induced release of beta-lysin from PRP was often accompanied by a reduction in the number of platelets. The intravenous injection of streptokinase, neuraminidase, and sulfatase caused in vivo releases of beta-lysin into the plasma. The platelet-aggregating substances collagen, arachidonic acid, and adenosine 5'-diphosphate caused beta-lysin to be released from PRP. The platelet-aggregating substances L-epinephrine, zymosan, fibrinogen, reserpine, and serotonin caused little or no release of beta-lysin from platelets. The results of this study indicate that the release of beta-lysin during antigen-antibody-complement reactions, blood coagulation, phagocytosis, and inflammation could be enzyme mediated.     

Neutralization of HIV-1 primary isolates by polyclonal and monoclonal human antibodies

To examine antibody-mediated neutralization of HIV-1 primary isolates in vitro, we tested sera and plasma from infected individuals against four clade B primary isolates. These isolates were analyzed further for neutralization by a panel of several human anti-HIV-1 mAb in order to identify the neutralizing epitopes of these viruses. Each of the HIV-1+ serum and plasma specimens tested had neutralizing activities against one or more of the four primary isolates. Of the three individual sera, one (FDA-2) neutralized all of the four isolates, while the other two sera were effective against only one virus. The pooled plasma and serum samples reacted broadly with these isolates. Based on the neutralizing activities of the mAb panel, each virus isolate exhibited a distinct pattern of reactivity, suggesting antigenic diversity among clade B viruses. Neutralizing epitopes were found in the V3 loop and CD4- binding domain of gp120, as well as near the transmembrane region (cluster II epitope) of gp41. A mAb directed to the cluster I epitope of gp41 near the immunodominant disulfide loop weakly neutralized one primary isolate. None of the mAb in the panel affected one primary isolate, US4, although this virus was sensitive to neutralization by some of the polyclonal antibody specimens. This isolate was also resistant to neutralization by a cocktail of 10 mAb, most of which individually inhibited at least one of the other three viruses tested. These results suggest that neutralizing activity for this latter virus is present in certain HIV-1+ sera/plasma, but is not exhibited by the mAb in the panel. Thus, effective neutralizing antibodies against primary isolates can be generated by humans upon exposure to HIV-1, but not all of these antigenic specificities are represented in a large panel of human anti-HIV-1 mAb.      

Neutralization of LIGHT ameliorates acute dextran sodium sulphate-induced intestinal inflammation

Emerging data indicate that alterations in the expression of tumour necrosis factor (TNF) superfamily members play a crucial role in the pathogenesis of intestinal inflammation. Recent results demonstrated that sustained transgenic expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT; TNFSF14) induced severe intestinal inflammation, suggesting a specific role of LIGHT-mediated signalling to the intestinal compartment. In order to dissect the role of LIGHT in intestinal inflammation, we used LIGHT-deficient mice in the mouse model of acute dextran sodium sulphate-induced colitis. Interestingly, LIGHT-deficient mice were characterized by strongly reduced signs of intestinal inflammation compared with wild-type mice in this experimental model. Determination of mouse LIGHT mRNA expression in colon tissues of wild-type mice revealed a strong induction of mouse LIGHT mRNA expression during acute DSS-induced colitis. We therefore generated anti-mouse LIGHT monoclonal antibodies in LIGHT-deficient mice which bind specifically to LIGHT and are capable of neutralizing the activity of LIGHT in vitro and in vivo. With these antibodies, we demonstrated that neutralization of LIGHT during acute DSS-induced colitis resulted in reduced signs of intestinal inflammation. These data suggest that LIGHT is an important mediator in intestinal inflammation and may serve as a new target for therapeutic intervention.     

Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.     

Neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide

Endotoxin (lipopolysaccharide [LPS]) is the major pathogenic factor of gram-negative septic shock, and endotoxin-induced death is associated with the host overproduction of tumor necrosis factor alpha (TNF-alpha). In the search for new antiendotoxin molecules, we studied the endotoxin-neutralizing capacity of a human lactoferrin-derived 33-mer synthetic peptide (GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP; designated LF-33) representing the minimal sequence for lactoferrin binding to glycosaminoglycans. LF-33 inhibited the coagulation of the Limulus amebocyte lysate and the secretion of TNF-alpha by RAW 264.7 cells induced by lipid A and four different endotoxins with a potency comparable to that of polymyxin B. The first six residues at the N terminus of LF-33 were critical for its antiendotoxin activity. The endotoxin-neutralizing capacity of LF-33 and polymyxin B was attenuated by human serum. Coinjection of Escherichia coli LPS (125 ng) with LF-33 (2.5 microg) dramatically reduced the lethality of LPS in the galactosamine-sensitized mouse model. Significant protection of the mice against the lethal LPS challenge was also observed when LF-33 (100 microg) was given intravenously after intraperitoneal injection of LPS. Protection was correlated with a reduction in TNF-alpha levels in the mouse serum. These results demonstrate the endotoxin-neutralizing capability of LF-33 in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.     

Inactivation of lysis of oncornaviruses by human serum.

The phenomenon of oncornavirus inactivation by human serum was examined. As judged by 50% inhibition of plaque or focus formation, every fresh human serum tested inactivated eight different oncornaviruses at serum dilution endpoints of 1:16. Fresh human serum released RNA-dependent DNA polymerase (RDDP) activity from each of 10 tested oncornaviruses. Thus, at least one mechanism of the human serum-induced virus inactivation was by viral lysis. The RDDP assay provided us with a simple method to screen the virolytic properties of many sera against a number of viruses. Oncornaviruses were lysed by sera from monkeys and cats, but not by sera from guinea pigs, rabbits, chickens, rats, goats, and mice ($\text{BALB}\text{c}$, CBA, NIH Swiss). Heated normal human sera did not inactivate or lyse oncornaviruses, nor did they sensitize virions to guinea pig serum-mediated lysis. However, serum from a human vaccinated against Rauscher leukemia virus did sensitize the virus to guinea pig serum-mediated lysis. This ability of human sera to lyse oncornaviruses may be a cause of the difficulties in finding oncornaviruses in humans.     

Automated determination of serum BSP. Improvement of dialysis by sodium salicylate]

Because of the great difficulty of BSP dialysis in automatized determinations in serum the authors investigated the effect of sodium salicylate. They proved its good performance with aqueous and proteic solutions of BSP. About 70 mul of patient sera is used to perform the analysis with Technicon Autoanalyser. Comparison of data from about 220 serum evaluated by their proposed method and manual technique are studied.     

Opsonization of yeast by human serum IgA anti-mannan antibodies and phagocytosis by human polymorphonuclear leucocytes.

The ability of sera from 72 patients with liver disease to opsonize yeast for phagocytosis by normal polymorphonuclear leucocytes has been studied. Seven showed defective opsonization. The opsonic activity of all but two sera was decreased markedly by heating at 56 degrees C for 1 h. When the two sera with heat stable opsonic activity were fractionated by gel filtration and by ion exchange chromatography, the activity copurified with IgA, not with IgG. The purified IgA, radiolabelled with 125I was shown to bind in a saturable manner to the yeast. Both sera had high levels of anti-yeast mannan IgA detected by an ELISA. In one case most of the anti-mannan activity was due to monomeric IgA, in the other it was dimeric. This was consistent with the observation of an apparent molecular weight of the opsonin of approximately 180 kD in one serum and 300-400 kD in the other.     

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxins.

Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxin was studied by cytotoxicity assay in tissue culture. The sources of toxin were stools from two patients with pseudomembranous colitis and a culture filtrate of C. difficile isolated from one of the patients. C. sordellii antitoxin was available either in monovalent form or as gas gangrene polyvalent antitoxin. The potency of antitoxins against C. difficile determined by cytotoxicity assay did not correlate with the established values reported for mouse protection tests against C. sordellii toxin. An equivalent zone of optimal neutralization was demonstrated for stool toxin, and a slightly different one for culture toxin. The rate of neutralization appeared to be instantaneous, either at 24 or at 37 degrees C. The efficacy of antitoxin in preventing cytotoxicity in cultured cells preexposed to toxin decreased rapidly with preexposure time. The union between toxin and antitoxin could be readily dissociated by simple dilution or by ammonium sulfate precipitation followed by dissociated by simple dilution or by ammonium sulfate precipitation followed by dilution. Continued incubation of toxin-antitoxin mixture did not increase the firmness of the union; on the contrary, more dissociation occurred. The unusual looseness of the toxin-antitoxin union is probably relatd to lack of serological specificity or affinity. Based on these observations, a practical diagnostic method for antibiotic-induced colitis is outlined.