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Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) fromPorphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from thefimA-encoded major fimbriae) ofP. gingivalisinduced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein fromBacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins.

The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis -induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.

Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene ( bspA ) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli , purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus -associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.

Fimbrial production by Porphyromonas gingivalis was inactivated by insertion-duplication mutagenesis, using the cloned gene for the P. gingivalis major fimbrial subunit protein, fimA. by several criteria, this insertion mutation rendered P. gingivalis unable to produce fimbrilin or an intact fimbrial structure. A nonfimbriated mutant, DPG3, hemagglutinated sheep erythrocytes normally and was unimpaired in the ability to coaggregate with Streptococcus gordonii G9B. The cell surface hydrophobicity of DPG3 was also unaffected by the loss of fimbriae. However, DPG3 was significantly less able to bind to saliva-coated hydroxyapatite than wild-type P. gingivalis 381. This suggested that P. gingivalis fimbriae are important for adherence of the organism to saliva-coated oral surfaces. Further, DPG3 was significantly less able to cause periodontal bone loss in a gnotobiotic rat model of periodontal disease. These observations are consistent with other data suggesting that P. gingivalis fimbriae play an important role in the pathogenesis of human periodontal disease.

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces. In the present study, a coding region of the fimbrillin gene from P. gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography. The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin. In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P. gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin. The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels. The r-fim 10-337 polypeptide was capable of inhibiting the binding of P. gingivalis 2561 to saliva-coated hydroxyapatite beads. These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P. gingivalis cells to saliva-coated surfaces. We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P. gingivalis fimbrillin subunit from E. coli.

The effect of age on the humoral response to Porphyromonas gingivalis was assessed in groups of adults (25 to 54 years and 55 to 74 years) with periodontal disease and compared with that in age-matched healthy controls. To determine whether there was an antibody response against P. gingivalis, we measured serum antibodies against whole cells of P. gingivalis 381, A7A1-28, and W50. In addition, antibody levels against purified P. gingivalis outer membrane proteins (i.e., the 43-kDa fimbrial protein and a 75-kDa protein) were also evaluated. Elderly subjects showed the same response to P. gingivalis as younger subjects. Immunoglobulin G (IgG) antibodies to both purified proteins were also elevated in both diseased groups as compared with the normal groups. Total serum IgG, IgA, and IgM levels were also determined by an enzyme-linked immunosorbent assay for all four groups. Total serum IgG levels were elevated in older adults with periodontitis and total IgA levels were elevated in both groups of older adults compared with the younger groups of similar disease status. Total serum IgM levels were comparable for the four groups. Antinuclear antibody titers were assessed in the two groups of older adults and were also found to be higher for the group with periodontitis. These studies show that older adults as well as younger adults have markedly elevated specific antibodies of the IgG and IgA classes to antigens of P. gingivalis, a putative pathogen in both groups. Furthermore, older adults with periodontitis have significantly elevated levels of total serum IgG which may possibly be related to higher levels of autoantibodies.

The human immunoglobulin G (IgG) immune response against Porphyromonas (Bacteroides) gingivalis A7A1-28 iron-repressible membrane proteins (IRMPs) and other membrane proteins was examined by immunoblot analysis. Thirty sera from patients with adult periodontitis and 30 sera from periodontally healthy subjects were included. Iron limitation of P. gingivalis was achieved by growing bacteria in brain heart infusion broth supplemented with protoporphyrin IX and 250 microM alpha, alpha'-dypyridyl, a ferrous iron chelator. Iron-sufficient growth was achieved by growing bacteria in the same medium without alpha, alpha'-dypyridyl. Human sera, in particular those from patients with periodontitis who exhibited high levels of IgG against whole cells of P. gingivalis A7A1-28 in serum in an enzyme-linked immunosorbent assay (ELISA), commonly reacted with five membrane proteins with apparent molecular masses of 80, 67.5, 51, 40.5, and 28 kDa and four IRMPs of 46, 43, 37.5, and 22 kDa. More than 80% of the sera from patients with periodontitis and high levels of IgG against strain A7A1-28 in serum by ELISA reacted with the 46-, 43-, and 37.5-kDa IRMPs, and 40% of these subjects expressed immunoreactivity against the 22-kDa IRMP. Sera from patients with periodontitis and low levels of IgG against strain A7A1-28 in serum by ELISA and sera from periodontally healthy subjects exhibited less immunoreactivity against IRMPs and the five membrane proteins of P. gingivalis. The present study indicates that P. gingivalis IRMPs are immunogenic and that these proteins are expressed in vivo.

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum . In this study, a novel adhesin, FadA ( Fusobacterium ad hesin A ), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum , Fusobacterium periodonticum , and Fusobacterium simiae , the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans , Fusobacterium mortiferum , Fusobacterium naviforme , Fusobacterium russii , and Fusobacterium ulcerans . In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

A water-soluble extract of Actinomyces viscosus was tested for its capacity to induce deoxyribonucleic acid synthesis in spleen cells from normal mice and to augment the in vitro antibody-forming cell (AFC) response to the T-cell-dependent antigen, sheep erythrocytes (SRBC) by T-cell-deficient adherent spleen cell monolayers from the same mice. Our results indicated that the water-soluble extract induced an in vitro blastogenic response, as assessed by tritiated thymidine incorporation into mouse spleen cells. The water-soluble extract also augmented the antibody response to SRBC. However, after fractionation of the water-soluble extract on Biogel P-300, not all of the fractions induced a blastogenic response and augmented an AFC response to SRBC. This difference in the ability of A. viscosus fractions to induce in vitro responses suggests that the water-soluble extract has multiple immunological properties. Chemical analysis of the water-soluble extract indicated an enrichment for amino acids and amino sugar common to peptidoglycan.

The gene encoding the fimbrial subunit protein of Bacteroides gingivalis 381, fimbrilin, has been cloned and sequenced. The gene was present as a single copy on the bacterial chromosome, and the codon usage in the gene conformed closely to that expected for an abundant protein. The predicted size of the mature protein was 35,924 daltons, and the secretory form may have had a 10-amino-acid, hydrophilic leader sequence similar to the leader sequences of the MePhe fimbriae family. The protein sequence had no marked similarity to known fimbrial sequences, and no homologous sequences could be found in other black-pigmented Bacteroides species, suggesting that fimbrillin represents a class of fimbrial subunit protein of limited distribution.

The hemagglutinating factor (hemagglutinin) of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and Sephadex G-100 gel filtration. The hemagglutinating activity of the preparation was 53.3 times higher than that of ammonium sulfate precipitate. In electron microphotographs, hemagglutinin appears to have a vesicle or tubelike structure. The hemagglutinating activity of intact cells was completely destroyed by heating at 100 degrees C for 10 min, but the activity of extracted hemagglutinin was heat stable. The activity of hemagglutinin was inhibited by L-arginine and L-lysine and partially inhibited by phospholipase D, but it was not affected by proteolytic enzymes, neuraminidase, hyaluronidase, lipase, phospholipase A and C, or sugars. The B. gingivalis hemagglutinin appeared to be comprised mainly of a 40,000-molecular-weight material. The Fab fragment of immunoglobulin G prepared from rabbit antiserum to whole cells of B. gingivalis and monoclonal antibody against the hemagglutinin bound to the cell surface and inhibited the hemagglutinating activity of both the cells and the purified hemagglutinin.

Actinobacillus actinomycetemcomitans is a facultative gram-negative coccobacillus associated with periodontal disease and nonoral infections. This organism is resistant to serum bactericidal mechanisms but is nevertheless killed by human neutrophils under aerobic and anaerobic conditions. Most of the killing attributable to oxidative mechanisms is inhibited by sodium cyanide, which suggests that the myeloperoxidase-hydrogen peroxide-chloride (MPO-H2O2-Cl-) system may be a key factor in the oxidative killing process. In this report, we examine whether the isolated MPO-H2O2-Cl- system is bactericidal against A. actinomycetemcomitans. We found that three major chromatographic forms of MPO were capable of killing A. actinomycetemcomitans at sublethal concentrations of H2O2 and that both catalase-positive and catalase-negative strains of this organism were sensitive to killing by the MPO-H2O2-Cl- system. We conclude that the isolated MPO-H2O2-Cl- system is bactericidal for A. actinomycetemcomitans independent of other neutrophil granule constituents and may be an important component of the oxygen-dependent bactericidal activity of the neutrophil with respect to this periodontopathic organism.

Capnocytophaga is a newly described genus of gram-negative bacteria which can cause serious oral and extraoral infections in the susceptible host. In the present study, sensitivity of Capnocytophaga spp. to the bactericidal properties of human serum was investigated. Laboratory strains representative of Capnocytophaga sputigena, C. ochracea, and C. gingivalis and seven oral isolates of Capnocytophaga spp. obtained in primary culture were determined to be sensitive to killing by pooled normal serum. In contrast, little or no killing of Capnocytophaga spp. was observed when these organisms were incubated in the presence of hypogammaglobulinemic serum despite evidence for alternative pathway activation. However, hypogammaglobulinemic serum could be reconstituted to bactericidal activity by the addition of the immunoglobulin M fraction of normal serum. Capnocytophaga spp. failed to activate the classical pathway in hypogammaglobulinemic serum, thus ruling out an antibody-independent mechanism of killing. In contrast, good correlation was observed between serum-mediated killing and antibody-dependent classical pathway consumption. These results indicate that complement in the presence of bactericidal antibody may be an important determinant of host resistance to intra- and extraoral infections caused by Capnocytophaga spp.

Fluoretec-M is a polyvalent conjugate used in direct fluorescent-antibody staining for identification of the Bacteroides asaccharolyticus-Bacteroides melaninogenicus group. The Fluoretec-M reagent detected all oral and nonoral test strains of B. melaninogaenicus subsp. intermedius, all test strains of B. melaninogenicus subsp. melaninogenicus, and the nonoral strains of B. asaccharolyticus. However, the Fluoretec-M polyvalent reagent and the monovalent conjugates which constitute Fluoretec-M did not detect the oral strains B. asaccharolyticus. The use of Fluoretec-M can therefore generate false-negative results in studies of specimens from oral cavity and from nonoral sites in which an infection with B. asacacharolyticus of oral origin may have taken place. It is suggested that antibodies reactive with the oral antigenic type of B. asaccharolyticus be included in the preparative procedure of the Fluoretec-M reagent.

Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.

Polymorphonuclear leukocyte (PMNL) chemotaxis studies of 32 patients with localized juvenile periodontitis (periodontosis or LJP), 10 adult patients with a history of LJP (post-LJP), 8 patients with generalized juvenile periodontitis (GJP), and 23 adults with moderate to severe periodontitis were performed: (i) to determine the prevalence of a PMNL chemotaxis defect in a large group of LJP patients; (ii) to study PMNL chemotaxis in patients with other forms of severe periodontal disease; and (iii) to determine if the PMNL chemotaxis defect seen in LJP patients is a cell-associated defect or is mediated by humoral factors. The effect of periodontal treatment on PMNL chemotaxis was studied in nine LJP patients. The chemotactic response was measured with the Boyden chamber procedure, and patient's peripheral PMNL were compared with those of control subjects, using endotoxin-activated serum, bacterial factor, N -formylmethionyl-leucylphenylalanine, and leukocyte-derived chemotactic factor as the standard chemoattractants. Based upon statistical analysis of chemotaxis assays, most carried out on at least two and often three or more separate occasions, 26 of 32 LJP patients, 7 of 10 post-LJP patients, and 5 of 8 GJP patients exhibited cellular defects of chemotaxis, whereas only 2 of 23 of the patients with adult periodontitis exhibited depressed chemotaxis. Elevated PMNL chemotaxis was occasionally found in subjects with juvenile periodontitis (2 of 32 with LJP and two of eight with GJP); however, it was found in a significant number (10 of 23) of patients with adult periodontitis. In eight of nine LJP patients, depressed PMNL chemotaxis was observed before and after periodontal therapy. The results indicate that the PMNL chemotaxis defect observed in juvenile periodontitis is due to a cell-associated defect of long duration. These studies suggest that the PMNL plays a major protective role against periodontal infection and that the cellular chemotactic defects and may predispose subjects to LJP.

Nineteen rhesus monkeys were inoculated with mumps virus by retrograde ductal instillation into the parotid gland. Evidence of infection was obtained in all instances. Virus was isolated from buccal swabbings and parotid biopsies for 1 week after inoculation. A vigorous serum-neutralizing antibody response occurred within 3 weeks, and there was marked monocytic infiltration of the parotid stroma. The monocytic infiltrate comprised as much as 60% of the total gland volume 1 week after infection. The predominant inflammatory cells were non-immunoglobulin-containing mononuclear cells resembling lymphocytes. Plasma cells containing immunoglobulin G (IgG), IgA, IgM, and IgE increased in numbers in the gland after infection, the greatest increase occurring in IgG-containing cells. Neutralizing antibodies and interferon were found in extracts prepared from the infected glands. Neutralizing activity was highest in samples taken 3 weeks after infection but was detectable in samples taken as soon as 36 to 48 h after infection. Interferon activity was detected in significant amounts 36 to 48 h after infection. Challenge of previously infected animals resulted in an increase in the monocytic infiltrate as well as an increase in numbers of immunoglobulin-containing plasma cells. However, reinfection did not occur as evidenced by the inability to culture shed virus after challenge. This model should be useful for in vivo study of biochemical mediators which evoke inflammatory cell infiltration and which may be significant both in protection and in tissue damage.

Using a recently developed assay for glucosyltransferase activity based on 14 C-glucose incorporation into an alcohol-insoluble polysaccharide, we were able to study inhibition by antibody of this enzyme activity. Rabbit antibody was relatively specific for the strain of Streptoccus mutans from which the enzyme was obtained. Absorption studies showed that neither removal of antibodies directed to dextran nor absorption with intact bacteria offset the enzyme-inhibitory capacity of these sera, whereas absorption with partially purified enzyme did result in removal of the inhibitory capacity.