Polymer scaffolds fabricated with pore-size gradients as a model for studying the zonal organization within tissue-engineered cartilage constructs.

The zonal organization of cells and extracellular matrix (ECM) constituents within articular cartilage is important for its biomechanical function in diarthroidal joints. Tissue-engineering strategies adopting porous three-dimensional (3D) scaffolds offer significant promise for the repair of articular cartilage defects, yet few approaches have accounted for the zonal structural organization as in native articular cartilage. In this study, the ability of anisotropic pore architectures to influence the zonal organization of chondrocytes and ECM components was investigated. Using a novel 3D fiber deposition (3DF) technique, we designed and produced 100% interconnecting scaffolds containing either homogeneously spaced pores (fiber spacing, 1 mm; pore size, about 680 microm in diameter) or pore-size gradients (fiber spacing, 0.5-2.0 mm; pore size range, about 200-1650 microm in diameter), but with similar overall porosity (about 80%) and volume fraction available for cell attachment and ECM formation. In vitro cell seeding showed that pore-size gradients promoted anisotropic cell distribution like that in the superficial, middle, and lower zones of immature bovine articular cartilage, irrespective of dynamic or static seeding methods. There was a direct correlation between zonal scaffold volume fraction and both DNA and glycosaminoglycan (GAG) content. Prolonged tissue culture in vitro showed similar inhomogeneous distributions of zonal GAG and collagen type II accumulation but not of GAG:DNA content, and levels were an order of magnitude less than in native cartilage. In this model system, we illustrated how scaffold design and novel processing techniques can be used to develop anisotropic pore architectures for instructing zonal cell and tissue distribution in tissue-engineered cartilage constructs.

An improved multimembrane microassay for quantitating the motility of granulocytes and monocytes labeled with chromium-51.

Various modifications of the Boyden chamber chemotaxis assay have been used to screen patients for abnormalities in granulocyte or monocyte motility. In most cases, cell motility has been assessed by quantitating the fraction of cells that migrates from an upper chamber through a filter toward a lower chamber containing chemoattractant. Existing versions of the assay have several shortcomings. They are labor-intensive, require relatively large numbers of cells and lengthy incubation, or they require visual cell counting and do not permit assessment of cells which may drop off the filter into the attractant medium. We have improved the accuracy and efficiency of existing microchamber assays by using 51Cr-labeled cells to eliminate microscopic cell counting, shortening the incubation time, adjusting the assay sensitivity, and accounting for cells which drop off into the attractant well. The modified method uses Neuroprobe multiwell microchambers and two 10 microns polycarbonate filters with 3 microns pores on top of one 100 microns nitrocellulose filter. The optimal incubation period is 60 min, and the assay requires about one-fifth as many cells as the standard Boyden chamber methods. Cell drop-off can be measured accurately by harvesting the attractant wells with detergent, and the assay sensitivity is comparable to that of existing radiometric assays using large chambers. The data indicate that the range of chemotactic and random motility of normal granulocytes and monocytes measured in the modified assay system is comparable to that reported for studies which have used established motility assays.

Serum antibody response to antigens of oral gram-negative bacteria by cats with plasma cell gingivitis-pharyngitis.

The etiology of a form of periodontal disease in domestic cats known as plasma cell gingivitis-pharyngitis is not understood. Actinobacillus actinomycetemcomitans and Bacteroides species have been strongly implicated as the cause of periodontitis in humans and other mammalian species, and most affected patients manifest serum antibodies reactive with the infecting bacteria. We and others have isolated Bacteroides species from the oral flora of cats. Using enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures, we measured serum antibodies in affected and control cats reactive with human isolates of A. actinomycetemcomitans, B. gingivalis, and B. intermedius, and purified lipopolysaccharide (LPS) from these and other species, and Bacteroides of cat origin. Affected cats had serum antibody titers reactive with these Gram-negative anaerobic bacteria that were significantly elevated relative to those of normal control cats. The quantitatively major antigens recognized by cat serum antibodies are proteins; this contrasts sharply with serum antibodies from humans with juvenile periodontitis, where LPS is the quantitatively major antigen fraction. Our data support the idea that plasma cell gingivitis-pharyngitis in cats may have a bacterial etiology, and that Gram-negative anaerobes similar to those that cause periodontitis in humans and other mammals may be involved.

Effect of factors other than pathologic status on responsiveness of peripheral blood mononuclear cells from patients with chronic periodontitis.

Studies were designed to assess factors other than pathologic status of the cell donor which affect the blastogenic responsiveness in vitro of peripheral blood mononuclear cells (PBMs) from normal donors and patients with periodontitis. Cultures were established and activated using phytohemagglutinin-P (PHA) or homogenates of Actinomyces viscosus (AVIS), a gram-positive plaque microorganism, and Fusobacterium nucleatum (FUSO), a gram-negative plaque microorganism. Activation was assessed by measuring the incorporation of labeled precursor into DNA. The effects of incubation time, vessel shape, cell concentration, prostaglandin E2 and indomethacin on blastogenic responsiveness were studied. Blastogenic responsiveness became maximal after 5 to 8 days' activation with the bacterial substances, and after 3 days' activation with PHA. Radioactivity incorporated by cultures in microtest wells with flat, round and conical bottoms was 5.9, 7.8 and 10.6 X 10(3) cpm, respectively. Cultures of cells from all of the patients and normal subjects were activated by PHA, AVIS and FUSO, and cell concentration was a major determinant of the magnitude of the blastogenic response. Responsiveness of cultures from all patients and control subjects activated with AVIS and FUSO was inhibited significantly by prostaglandin E-2 (PGE2) at a concentration of 10 microM. Inhibition was generally 50% or greater. Indomethacin, an inhibitor of prostaglandin production, at a concentration of 0.5 micrograms/ml significantly enhanced responsiveness of AVIS- and FUSO-activated cultures from control donors and patients, indicating that prostaglandins are produced endogenously, and that they affect cell responsiveness. The effect of PGE2 and indomethacin on PHA-activated cultures was more variable and, where present, of a lesser magnitude than that observed for cultures activated with bacterial homogenates. In most cultures the effects were not statistically significant. Our data show that in studies of lymphocyte activation, the incubation time, culture-vessel shape, cell concentration and presence of endogenous inhibitors need to be taken into account.

Effects of treatment on antibody titer to Porphyromonas gingivalis in gingival crevicular fluid of patients with rapidly progressive periodontitis.

Twenty-eight patients diagnosed as having rapidly progressive periodontitis (RPP) were enrolled in a study in which samples of subgingival microflora were harvested from test teeth and assayed for the presence of Porphyromonas gingivalis, and GCF collected and analyzed by ELISA for specific antibody for P. gingivalis. Clinical conditions were measured and recorded, and treatment by scaling and root planing provided at baseline and at 3, 6, 9, and 12 months. Reduction in pocket depth, stabilization of attachment level, and resolution of inflammation were comparable to previously reported values. By 3 months, mean and median specific antibody concentration had decreased, and continued to decrease through 12 months. The proportion of samples in which specific antibody was not detectable increased from 27% at baseline to 73% at month 12. GCF samples from sites at which P. gingivalis was present had greater than 2-fold higher median specific antibody than samples from P. gingivalis-negative sites. At baseline, specific antibody titer of 30-second GCF samples positively correlated with pocket depth, and GCF volume significantly correlated with antibody titer and concentration, and with pocket depth. In addition, change in specific antibody titer of 30-second samples from baseline to both 6 and 12 months correlated positively with pocket depths. Thus sites infected by P. gingivalis manifested high levels of specific antibody, and levels were related to clinical status. Following treatment, antibody levels decreased significantly as pocket depths decreased, attachment levels stabilized, and inflammation resolved.

Humoral immune responses to Porphyromonas gingivalis before and following therapy in rapidly progressive periodontitis patients.

We have performed studies aimed at elucidating the nature of the humoral immune response in rapidly progressive periodontitis (RPP). We analyzed the sera of 36 periodontally normal subjects and 36 RPP patients for titers and avidities of IgG antibodies reactive with the antigens of Porphyromonas gingivalis using ELISA, prior to and following treatment. We used whole-cell sonicate, purified lipopolysaccharide (LPS), and total extractable protein as plate antigens. Twelve of the patients had antibody titers at least 2-fold greater than the median of the controls and were designated as seropositive. The remaining 24 patients had titers that did not exceed twice the median titer of the controls and were designated as seronegative. For both patient groups, antibody titers were highest when whole-cell antigen was used, intermediate for LPS, and lowest for the protein fraction. Following treatment, median titer for seropositive patients decreased from pretreatment values of 241.7 to 76.5, while median titer for seronegative patients increased from 39.5 to 80.1. Avidities of pretreatment sera from both patient groups for all 3 antigen preparations were lower than the median avidities of the control sera. Avidity significantly increased following treatment to levels greater than those for control sera in both patient groups. Thus, some young adults with severe periodontitis mount a humoral immune response and produce high levels of serum IgG antibodies reactive with antigens of P. gingivalis, while others do not. The antibodies produced are of relatively low avidity, and may therefore be relatively ineffective biologically. Therapy, which greatly reduces antigen load, appears to stimulate production of higher avidity IgG antibodies in both patient groups; in the seropositive group, low avidity antibodies appear to be replaced by antibodies of higher avidity. Both the purified LPS and protein fractions contain reactive antigen(s), although LPS binds more antibody. Our data are consistent with the idea that many RPP patients do not produce protective levels of biologically functional antibody during the course of their natural infection, but they may be stimulated to do so by treatment.

Effects of endogenous and exogenous inhibitors on the incorporation of labeled precursors into DNA by human mononuclear cells.

The degree of responsiveness of lymphoid cells to activation by mitogens and antigens is commonly assessed in vitro by measuring radioactive DNA precursor incorporation. Several lines of evidence indicate that artifacts affect the results and that these measurements may not be an accurate reflection of cell activation. Cultures of blastogenically activated lymphocytes contain soluble, noncytotoxic factors that inhibit the incorporation of radioactive nucleosides into DNA by dividing cells without affecting their rate of DNA synthesis. Inhibitors were found in the serum component of the medium and in the bacterial homogenates used to activate the cells, and they were produced by the activated cells. Inhibitor activity in serum has properties expected of a nucleoside such as thymidine, including a molecular weight of less than 10(3). The inhibitor activity present in some bacterial homogenates and that produced by activated cells enzymically degrade labeled DNA precursors, thereby preventing their availability for incorporation. Other bacterial preparations contain DNA precursors, which compete with labeled nucleosides for incorporation, and additional low-molecular-weight inhibitor is produced when the preparations are incubated. Preparations of various bacteria differ greatly with regard to the potency of their inhibitor activity. In some cases incorporation of label in activated cultures is reduced to background levels. Inhibition by these substances leads to erroneous conclusions regarding the proliferative activity of cultured lymphocytes, since the amount of label incorporated does not accurately indicate the true rate of DNA synthesis of the cells.

Enzymatic regulation of glycoprotein synthesis in peripheral nervous system myelin.

The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 micrograms) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5-40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects.

Most juvenile periodontitis patients respond to infection by Actinobacillus actinomycetemcomitans by producing serum antibodies. Specific antigens inducing the humoral immune response have not been identified, nor has the role of the resulting antibodies in disease progression been determined. Adsorbed and unadsorbed sera from juvenile periodontitis patients and normal subjects were analyzed by enzyme-linked immunosorbent assay and Western blots (immunoblots), using digested and undigested bacterial sonicates and French pressure cell fractions to determine the biochemical class, cross-reactivity, and cellular location of the antigens in different A. actinomycetemcomitans serotypes. Antigens detected by using high-titer sera included the following: (i) serotype-specific nonprotein material located on the cell surface, (ii) soluble-fraction proteins showing highly variable antibody binding, (iii) cross-reactive proteins, and (iv) a protein present in soluble and cell wall fractions and immunopositive for all sera tested. In addition, one apparently nonprotein component that was enriched in the cell wall fraction was observed. Sera with high immunoglobulin G titers to one, two, three, or none of the three A. actinomycetemcomitans serotypes were observed. There was a high degree of variation from one patient to another in the humoral immune response to serotype-specific and cross-reactive antigens. As demonstrated by whole-cell adsorption experiments, the serotype-specific surface antigen accounted for approximately 72 to 90% of the total antibody-binding activity for sera with titers greater than 100-fold above background, while cross-reactive antigen accounted for less than 28%. Antibody binding the whole-cell sonicate for high-titer sera was inhibited 90% by lipopolysaccharide from the same serotype, strongly suggesting that lipopolysaccharide is the immunodominant antigen class.

Antigenic variation in Porphyromonas gingivalis ribotypes recognized by serum immunoglobulin G of adult periodontitis patients.

We obtained clinical isolates of Porphyromonas gingivalis of known ribotype from patients diagnosed with adult periodontitis and used Western blot methodology to evaluate profiles of antigens recognized by IgG in heterologous and homologous patient sera. Our aims were to identify isolates belonging to different serogroups, to learn if serogroup membership is related to ribotype to assess variation in IgG responses of patients to antigens is homologous and heterologous ribotypes, and to determine the frequency of shared and variable antigens in different biochemical classes recognized across different serogroups and ribotypes. Blots of separation patterns of 28 isolates were developed in sera from patients and bound IgG was quantified by digital image densitometry. The membership of isolates in different serogroups was determined by correlation and hierarchical cluster analysis of isolate whole-cell IgG binding profiles. Two major isolate clusters, each with two subclusters, were found. Isolates within the same ribotype clustered together in some cases but not others. Homologous isolates ranked high in IgG binding levels relative to those from different patients irrespective of ribotype. Patient subgroups with IgG responses dominant for different ribotypes and serogroups were revealed by correlation analysis. The IgG binding profiles observed for individual protein and proteinase-resistant antigens across both homologous and heterologous isolates were very dissimilar. Furthermore, the frequency of antigens both shared across all ribotypes and recognized by IgG in patient sera was unexpectedly low. Only two protein antigens (Mr 44 kDa and 27 kDa) were strongly recognized across all ribotypes by different sera. We conclude that the IgG response of patients infected with a particular P. gingivalis serotype or ribotype is directed mainly against antigens that are not shared by other potentially infective clonal types.

Treatment outcome for IDDM patients in relation to glutamic acid decarboxylase autoantibodies and serum IgG to periodontal pathogens.

BACKGROUND: Patients with insulin-dependent diabetes mellitus (IDDM) have elevated risk for periodontitis (PD) relative to subjects without diabetes. Whether refractory PD in IDDM patients is related to autoimmunity as indicated by serum glutamic acid decarboxylase autoantibody GAD Ab levels or to host bacterial immunity as reflected by serum antibody titers to periodontal pathogens is unknown. AIMS: To determine if non-surgical periodontal treatment outcome differs between GAD Ab-seropositive and -seronegative IDDM patients by assessing the following parameters: (1) pretreatment serum levels of GAD Ab, (2) pretreatment serum IgG titers to key periodontal pathogens, and (3) changes in periodontal pocket probing depth (PDC) after treatment. METHODS: Before and two months after periodontal treatment of 11 GAD Ab-seronegative and 7 -seropositive subjects, PDC was assessed and serum GAD Ab and IgG to Porphyromonas gingivalis (Pg), Bacteroides forsythus (BJ), and Actinobacillus actinomycetemcomitans (Aa) were studied using established radioligand precipitation and enzyme-linked immunosorbent assays, respectively. RESULTS: The PDC decrease was significantly better for GAD Ab-seronegative subjects than for seropositive subjects (median 1.4 mm+/-0.5 s.d. versus 0.5 mm+/-0.3 s.d., p<0.03, Mann-Whitney). GAD Ab levels and PDC were positively correlated (r=+0.71, p<0.05) for sero-positive subjects but were neutral (r=-0.07) for seronegative subjects. Serum IgG to Pg and GAD Ab levels were positively associated (r2=0.42) in seropositive subjects. Logistic regression analysis confirmed that GAD Ab status was the primary discriminator for PDC (p<0.04). CONCLUSION: Detection of elevated GAD Ab levels in combination with elevated IgG titers to Pg before treatment is indicative of IDDM patients with refractory PD.

Immunoglobulin G response of periodontitis patients to Porphyromonas gingivalis capsular carbohydrate and lipopolysaccharide antigens.

Porphyromonas gingivalis clonal types that participate in periodontal infections express serologically distinct surface antigens. This investigation sought to determine whether serum antibodies titers against the serotype-specific capsular carbohydrate K antigen and lipopolysaccharide antigens of P. gingivalis might reveal which serotypes are most likely to be responsible for subgingival infections in subjects with adult periodontitis. Immunoglobulin G (IgG) titers to purified K antigen and lipopolysaccharide from different P. gingivalis strains were measured by ELISA for 28 healthy controls and 51 patients with periodontal pockets known to be infected with genetically and serologically distinct P. gingivalis clonal types. Titers to purified K antigen from strains W50, HG184, A7A1-28, 49417, HG1690 and HG1691, representing serotypes K1-K6, respectively, and lipopolysaccharide from strains 381, HG1691 and W50, representing serotypes O1-O3, respectively, were measured for all subjects. Chi-square likelihood ratios, Mann-Whitney tests and receiver-operating characteristic sensitivity-specificity plots were used to compare the accuracy with which titer results for different target antigens classified subjects with or without disease. Results from assays targeting K2, K3, K4, K5, O1 and O2 generally gave poor diagnostic accuracy, whether evaluated separately or as summed titer pairs corresponding to the K/O combinations actually expressed by the target antigen parent strains. Exceptions were O3 (from W50) and K5+O2 (both from HG1690), which gave moderate accuracy in classifying subjects. In contrast, highly significant diagnostic accuracy was achieved using individual K1 (W50) and K6 (HG1691) titer data and K1+O3 (W50) and K6+O2 (HG1691) titer sum values. These observations suggest that P. gingivalis clonal types expressing K/O serotypes matching those of W50 (K1/O3) and HG1691 (K6/O2) are more likely than others to participate in periodontal infections in adult periodontitis patients and thus are more likely than others to express relevant virulence factors.

Antigenic cross-reactivity among Porphyromonas gingivalis serotypes.

The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross-reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A-D were 33277, A7A1-28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme-linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross-reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross-reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti-33277 and anti-381 (500-650 mV) but opsonized to a much lesser extent by anti-A7A1-28 and anti-W50 (roughly 125 mV and 350 mV respectively). A7A1-28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti-A7A1-28 (400 mV) and anti-W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens.

Shared antigens of Porphyromonas gingivalis and Bacteroides forsythus.

Periodontitis in humans is caused by a group of predominantly gram-negative, anaerobic bacteria among which Porphyromonas gingivalis and Bacteroides forsythus are prominent. A similar group is present and presumably plays a similar role in experimental periodontitis in the primate Macaca fascicularis. Nevertheless, immunization using a vaccine containing only killed P. gingivalis suppresses the progress of experimental periodontitis in M. fascicularis. We investigated the hypothesis that gram-negative periodontopathic bacterial may share antigens, and immunization with one species may induce antibodies reactive with other gram-negative species. Using enzyme-linked immunosorbent assay (ELISA), Western and dot immunoblots with nonabsorbed and absorbed and immune and preimmune sera we show that monkeys immunized with P. gingivalis produce antibodies reactive not only with antigens of P. gingivalis but also with those of B. forsythus. Similarly, rabbits immunized with P. gingivalis or with B. forsythus produce antibodies that react with antigens of both bacteria. Cross-reactive antibodies bind to epitopes in lipid A and possibly in core carbohydrate of lipopolysaccharide. Using complexes of lipopolysaccharide with polymyxin B, bovine serum albumin and apolipoprotein A1 specificity of binding was documented. Using sera from monkeys immunized with P. gingivalis, cross-reactivity with Actinobacillus actinomycetemcomitans could not be demonstrated by ELI-SA, although binding to lipopolysaccharide but not to lipid A was demonstrated by Western and dot immunoblots. Antibodies to shared lipopolysaccharide epitopes of periodontopathic bacteria may account, at least in part, for the immune protection observed in immunized monkeys, and shared epitopes may have potential as a vaccine for periodontitis in humans.

Defective neutrophil and monocyte motility in patients with early onset periodontitis.

Several studies have documented suppressed polymorphonuclear neutrophil (PMN) chemotaxis in most patients with juvenile periodontitis. In contrast, data regarding PMN chemotaxis in patients with rapidly progressive periodontitis are very limited, and monocyte (MN) chemotaxis and random migration of PMNs or MNs from these patients have not been studied previously. Accordingly, we examined cell motility of PMNs and MNs from 27 patients with rapidly progressive periodontitis, 5 patients with juvenile periodontitis, and 37 normal control subjects by using a microchamber technique and the synthetic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) as the chemoattractant. As a group, PMNs and MNs from patients with rapidly progressive periodontitis manifested significantly enhanced random migration relative to control cells (P less than 0.001), suppressed directed migration (chemotaxis) at FMLP doses of 10(-9) and 10(-8) M (P less than 0.05), and enhanced directed migration at a dose of 10(-6) M FMLP (P less than 0.01). In contrast, PMNs from patients with juvenile periodontitis exhibited normal random migration, and directed migration was significantly suppressed at all doses of FMLP tested (P less than 0.05). An abnormality of either PMN or MN motility was observed in 26 of 27 patients with rapidly progressive periodontitis. Enhanced random migration was seen in PMNs in 63%, MNs in 39%, and both cell types in 26% of the patients. Suppressed chemotaxis was seen in PMNs in 85%, in MNs in 74%, and in both cell types in 69% of the patients. The prevalence and magnitude of abnormalities in motility were somewhat lower in treated than in untreated patients. Thus, most, if not all, of this subgroup of patients with early onset, highly destructive periodontitis have abnormalities in PMN or MN motility, and these defects may differ from those seen in cells from patients with the juvenile form of the disease.

Antigenic variation in Bacteroides forsythus detected by a checkerboard enzyme-linked immunosorbent assay.

Evidence indicating that multiple serotypes of Bacteroides forsythus participate in rapidly progressing periodontal infections has not been reported previously. Our aim was to develop an assay for detecting subsets of B. forsythus clinical isolates which differ in serogroup membership and subsets of patients with immunoglobulin G (IgG) responses which differ in serogroup recognition. A checkerboard enzyme-linked immunosorbent assay (ELISA) was used to assess variation in the IgG binding profiles of 22 clinical isolates in sera from 28 patients with early-onset rapidly progressive periodontitis. To accommodate the maximum number of isolates and sera in a given assay run, a multiplate assay grid with standard 96-well microtest plates was established. Single dilutions of individual sera were placed in rows crossing columns of isolate-coated wells, and antigen-specific IgG immobilized in the wells was measured as ELISA absorbance. Pooled sera and isolates were assayed in parallel to serve as negative controls for variation in IgG binding profiles. Correlation and hierarchical cluster analysis of the absorbance data matrix showed that the isolates could be sorted into at least four clusters based on variations in their IgG binding profiles across different sera. Furthermore, at least two patient clusters were defined by variations in their serum IgG antigen recognition profiles across different isolates. We conclude that multiple serogroups of B. forsythus exist and that different serogroups are dominant in the antibody response of different patients. The method applied here could be used to serologically classify clinical isolates of other species which evoke a serum antibody response in patients.

Ovine periodontitis as a potential model for periodontal studies. Cross-sectional analysis of clinical, microbiological, and serum immunological parameters.

OBJECTIVES: : To investigate infection and host immunity patterns in sheep with naturally occurring "broken-mouth" periodontitis. MATERIALS AND METHODS: : Eight periodontally healthy (HS) and eight periodontally diseased ewes (PDS) were selected. Subgingival plaque and sera were collected and examined for evidence of human periodontitis-associated pathogens. Serum IgG titers were measured by ELISA to multiple strains of Porphyromonas gingivalis, Bacteroides forsythus, Dichelobacter nodosus, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum as well as several purified antigens (cysteine proteases, LPS, K, and fimbriae). RESULTS: : Neither the organism Aa nor antigens to Aa were found in any animal. Most animals were positive for Pg, Bf, and Pi, but DNA probes detected no difference between HS and PDS relative to amounts of pathogens in subgingival plaque. PDS had significantly higher serum IgG titers to all Pg strains, to 50% of Bf strains, to the Pi and Fn strains, and to fimbriae and the two cysteine proteases (p-values ranging from 0.05 to 0.001). Regression analysis demonstrated a significant association between number of teeth lost and serum IgG antibody titers to whole-cell sonicate antigens of P. gingivalis strains (p<0.01) and body weight (p<0.01). CONCLUSIONS: : The presence of pathogens associated with periodontitis was reflected in differences in serum IgG titers between healthy and diseased sheep. This may have influenced animal body weight and might have systemic health and economic consequences. The data suggest that susceptible and non-susceptible sheep can be identified for periodontal research.

Antigenic variation and cross-reactivity in Bacteroides forsythus clinical isolates detected by western blot.

Bacteroides forsythus is one of the etiologic agents of destructive periodontal diseases. Determining which antigenic components of the bacterium are recognized in the immune response of periodontitis patients is an important step in assessing strategies for vaccine development. The aim of this study was to identify the major strain-variable and cross-reactive antigens of B. forsythus clinical isolates recognized by serum IgG from patients with early-onset rapidly progressive periodontitis. Ten patient sera with measurable IgG against antigenic components of the species were identified by Western blot. Positive sera were tested by checkerboard ELISA to identify those most responsive to strain-variable antigens in nine clinical isolates and ATCC strain 43037. Correlation analysis of the ELISA data suggested that different subsets of isolates were preferentially recognized by different sera. Western blots revealed that certain sera also recognized major shared components across all the isolates, but preferential recognition of different isolate subsets by different patients was clearly confirmed. To determine if the variable antigens recognized were nonprotein, proteinase K-digested isolates were compared to undigested controls by Western blot. The main strain-variable antigens were proteinase resistant, while proteins at 200 and 210 kDa were identified as the major shared components. Two-dimensional SDS-PAGE revealed that these proteins are the quantitatively dominant heat-modifiable components of the cell envelope. Even though variable antigens are prominent in the immune response of patients, a cross-protective vaccine based on the shared envelope proteins of B. forsythus seems feasible in light of these observations.

Titer and subclass distribution of serum IgG antibody reactive with Actinobacillus actinomycetemcomitans in localized juvenile periodontitis.

Most patients with localized juvenile periodontitis (LJP) manifest serum IgG antibodies specifically reactive with antigens of Actinobacillus actinomycetemcomitans serotype b (Aa-b). Whether these antibodies are protective, destructive, or irrelevant to the progress of the disease remains unclear. We report results of studies aimed at assessing the subclass IgG responses in 35 LJP patients and 35 periodontally normal control subjects using well-characterized monoclonal antibody subclass reagents in an enzyme-linked immunosorbent assay. Our data show that the mean value for total IgG reactive with antigens of Aa-b was more than sevenfold higher for patients than for normal control sera (2349.6 micrograms/ml for patients vs 332.2 micrograms/ml for controls). Individual patients and control subjects were classified as high- or low-titer, using twice the median value for total anti-Aa-b IgG in control sera as the cutoff. Of 35 patients, 26 (74%) were high-titer, and 9 (26%) were low-titer. This compares to 5 normal control subjects (14%) high-titer and 30 (86%) low-titer. IgG2 accounted for the major quantitative response in both patients and control subjects. Indeed, the mean IgG2 values for both concentration and percentage of total specific IgG were greater than the combined values for specific anti-Aa-b IgG1, IgG3, and IgG4. Of the 26 high-titer sera, IgG2 predominated in 24, with IgG1 and IgG3 predominating in 1 each; IgG2 predominated in only 2 of the low-titer sera.