Hepatocytes produce tumor necrosis factor-α and interleukin-6 in response to Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The liver plays a major role in clearing systemic bacterial infections. In addition, inflammatory cytokines produced in the liver play a critical role in systemic cytokine levels. The aim of this study was to investigate the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by hepatocytes in response to periodontal pathogens. MATERIAL AND METHODS: The mouse hepatic carcinoma cell line Hepa-1.6 and the mouse macrophage-like cell line RAW 264 were co-cultured in Transwell insert plates. Cells were stimulated with bacterial extracts prepared from Porphyromonas gingivalis and the induction of TNF-α and IL-6 was measured using real-time PCR and ELISA. RESULTS: After stimulation with bacteria, the induction of TNF-α and IL-6 was observed in RAW 264 cells and Hepa-1.6 cells. Significant reduction of TNF-α mRNA expression in Hepa-1.6 cells was observed after treatment with antibody to TNF-α. CONCLUSION: The results obtained in the present study show that P. gingivalis extract induces TNF-α and IL-6 in an in vitro liver model and that macrophage-derived TNF-α mediates the induction of TNF-α in hepatocytes.

Detection of periodontopathic bacteria and an oxidative stress marker in saliva from periodontitis patients.

We assessed the salivary levels of periodontopathic bacteria and 8-hydroxydeoxyguanosine (8-OHdG) in patients with periodontitis. The salivary levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia (formerly Bacteroides forsythus) were assessed using real-time polymerase chain reaction. The 8-OHdG levels were determined using an enzyme-linked immunosorbent assay. The salivary levels of 8-OHdG, P. gingivalis, and T. forsythia in the periodontitis patients were significantly higher than those in healthy subjects. By contrast, the A. actinomycetemcomitans level in healthy subjects was higher than that in periodontitis patients. 8-OHdG was significantly correlated with P. gingivalis. Statistically significant decreases in the levels of P. gingivalis, probing depth, bleeding on probing, and 8-OHdG were observed after initial periodontal treatment. These results suggest that the 8-OHdG levels in saliva reflect the load of periodontal pathogens. 8-OHdG could be a useful biomarker for assessing periodontal status accurately, and for evaluating the efficacy of periodontal treatment.

Differential cytokine induction by two types of Porphyromonas gingivalis.

The presence of Porphyromonas gingivalis with type II fimA is strongly associated with adult periodontitis. However, the importance of specific fimA types in the immune response is unknown. Two types of P. gingivalis (type I and type II) and Actinomyces naeslundii were assessed for their degree of cytokine induction in the macrophage-like human cell line U937. Real-time reverse transcriptase polymerase chain reaction was used to determine mRNA expression of 12 cytokines. Significant levels of interleukin (IL)-8 induction and a similar cytokine expression pattern were observed at 6 h postinfection for all three bacterial strains. However, type II P. gingivalis infection showed statistically higher levels of IL-1beta, IL-8, IL-12 and tumor necrosis factor-alpha mRNA induction than those of control at 24 h postinfection, whereas type I P. gingivalis and A. naeslundii showed no significant induction of these cytokines. These data suggest that compared with A. naeslundii and type I P. gingivalis, type II P. gingivalis prolongs the cytokine response. Although other factors may also be involved, the sustained cytokine response induced by type II P. gingivalis may play an important role in enhanced periodontal tissue inflammation and destruction.

Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines.

An MHC class I restricted cytotoxic T lymphocyte (CTL) activity assay has recently been established for rainbow trout. MHC class I restricted cytotoxicity probably plays a critical role in immunity to most viral diseases in mammals and may play a similar role in fish. Therefore, it is very important to investigate what types of vaccines can stimulate this immune response. Although logical candidates for vaccine components that can stimulate an MHC class I restricted response are live attenuated viruses and DNA vaccines, these materials are generally not allowed in fish for commercial vaccine use due to potential safety issues. In mammals, however, a number of interesting vaccination strategies based on exogenous antigens that stimulate MHC class I restricted cytotoxicity have been described. Several of these strategies are discussed in this review in the context of fish vaccination.

Dominant C2 domain epitope specificity of inhibitor antibodies elicited by a heat pasteurized product, factor VIII CPS-P, in previously treated hemophilia A patients without inhibitors.

From June, 1990, to November, 1991, in The Netherlands and Belgium, 16 previously treated severe hemophilia A patients (PTP) developed inhibitors after exposure to factor VIII CPS-P, a new heat pasteurized product. A previously untreated patient (PUP) also developed an inhibitor to CPS-P. In inhibitor neutralization assays with recombinant fVIII C2 and A2 domain polypeptides, plasmas from 14 PTPs were > or = 79% neutralized by C2 and < 10% by A2, but the PUP plasma was partially neutralized by C2 (48%) and A2 (28%). Immunoprecipitation assays of the PTP and PUP plasmas with the fVIII heavy chain and with recombinant C2 and A3-C1 polypeptides confirmed that the C2 dominant immune response to CPS-P was found only in the PTPs. Competition of the binding of 2 inhibitors to 125I-CPS-P by unlabeled CPS-P and another plasma fVIII was similar, demonstrating that the antibody response was not directed to epitopes only present in CPS-P. We propose that the immunogenicity of the CPS-P C2 domain was altered by heat pasteurization.

Successful treatment of two brothers with congenital afibrinogenemia for splenic rupture using heat- and solvent detergent-treated fibrinogen concentrates.

PURPOSE: This report describes life-threatening spontaneous splenic rupture in two brothers with congenital afibrinogenemia. PATIENTS: Two brothers, aged 11 and 14 years, had intra-abdominal bleeding due to splenic rupture confirmed by ECHO ultrasonography and computed tomography scans. RESULTS: Splenectomy was performed after administration of heat- and solvent-detergent treated fibrinogen concentrates. CONCLUSIONS: Hemostatic treatment for splenic rupture using heat- and solvent detergent-treated fibrinogen concentrates was effective. Careful attention must be paid to the risk of splenic rupture during the growth spurt in physically active children with this rare coagulation disorder.

Anti-factor VIII inhibitor alloantibodies recognizing the A2 domain in the human factor VIII heavy chain poorly bind to porcine factor VIII.

Anti-factor VIII (FVIII) inhibitor alloantibodies from 11 patients with hemophilia A, along with five anti-FVIII neutralizing monoclonal antibodies, were examined for differences in their reactivities with the A2 and C2 domains of human and porcine FVIII. None of the patients had been previously treated with porcine FVIII. Six inhibitors which specifically recognized the human FVIII C2 domain bound to both the 76-kDa porcine FVIII light chain and its 69-kDa proteolyzed fragments, showing cross-reactivity against porcine FVIII between 33 and 100%. Two A2-specific inhibitors did not react with porcine FVIII. The cross-reactivity was low (0-0.5%). The inhibitors recognizing both C2 and A2 reacted with the 76- and 69-kDa bands of porcine FVIII light chain, with cross-reactivity of between 11 and 33%. Monoclonal antibodies recognizing A1 (C-5) and A2 (JR8) did not react with the porcine FVIII. No anti-porcine FVIII neutralizing activity was detected in these antibodies. Monoclonal antibodies to the amino-terminal portion of A3 (NMC-VIII/10 and C-2) poorly reacted with the 76-kDa band, the cross-reactivities being 0 and 0.5%, respectively. NMC-VIII/5 recognizing C2 which competes with the C2-specific inhibitor, reacted with both the 76- and 69-kDa fragments showing cross-reactivity of 13%. These findings suggest that porcine A2 is antigenically different from human A2. The A2-specific inhibitor is a useful indicator for therapy with porcine FVIII.

Factor IX inhibition and epitope localization of factor IX inhibitor antibodies in haemophilia B patients with anaphylactoid reactions.

We describe the results of immunological studies, reaction kinetics and epitope localization of six inhibitor antibodies to factor IX (FIX) developed in severe haemophilia B patients. Three of the six patients had suffered recent anaphylactoid reactions to FIX concentrates, two others had in the past and one had none. All six inhibitors rapidly inactivated FIX activity in vitro, and the prominent immunoglobulin (IgG) subclass of the antibody was IgG4 when analysed with ELISA. Interestingly, we found an additional IgG1 component in the antibody samples from the patients who had recently experienced anaphylactoid reactions to FIX. When analysed with Western blotting in these three patients, the IgG4 antibody bound with enhanced affinity to the heavy chain or the light chain of FIX, and in two of the three the IgG1 antibody also bound strongly to the FIX heavy chain. The results suggest that the heavy chain of FIX might play a more significant role than the light chain in the pathogenesis of anaphylactoid reactions in haemophilia B patients with FIX inhibitors.

[Successful induction of immune tolerance and novel hemostatic effects in a hemophilia A with high-responder inhibitor by regular infusions of factor VIII].

A hemophilia A patient with high responder inhibitor had been treated by (activated) prothrombin complex concentrates (A) PCC and activated factor VII until the occurrence of intracranial bleeding at the age of 6 years. Since the inhibitor titer was decreased less than 1 Bethesda Units/ml, high dose of factor VIII was given followed by the infusions of factor VIII concentrates (100 units/kg) three times a week. In spite of previous episodes of anamnestic responses by factor VIII products before, the inhibitor titer did not increase and disappeared completely 6 months after the FVIII infusion therapy. The specific anti-factor VIII IgG subclasses of the inhibitor were IgG2 and IgG4. The inhibitor recognized both light and heavy chains. He have no bleeding episode for 6 months since the beginning of the prophylactic with factor VIII concentrates.

Measurement of anti-factor IX IgG subclasses in haemophilia B patients who developed inhibitors with episodes of allergic reactions to factor IX concentrates.

We have established a simple enzyme-linked immunosorbent assay (ELISA) for the detection of anti-factor IX IgG subclasses in haemophilia B patients with inhibitors. The assay was performed using immobilized purified factor IX. Specific IgG subclasses were detected by peroxidase-conjugated anti-human IgG1,2,3 and 4. Ten plasma samples from 6 haemophilia B patients with inhibitors ranging from 1.0 to 253 Bethesda Units/ml were analyzed. All samples were positive for IgG4. Six out of 10 samples were positive only for IgG4. Three samples were positive for IgG2. Five of the 6 patients had previously had allergic reactions to factor IX concentrates. Three patients had allergic episodes within the past month. Three samples from these latter patients taken on the day when the allergy had occurred showed positive also for IgG1. In later samples, however, taken at 4 days and 4 weeks respectively from two of these same patients. IgG1 was not detected. In two of the five patients in whom allergic reactions had occurred more than one month previously IgG1 was not detected. The results suggested that allergic reactions in patients with haemophilia B treated with factor IX concentrates were associated with the development of the specific IgG1 subclass of antibody to factor IX.

Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor.

Factor VIII (FVIII) inhibitor alloantibodies obtained from seven severe haemophilia A patients were examined for their binding regions and their effects on FVIII binding to von Willebrand factor (vWF). Immunoblotting analysis with a panel of recombinant fragments demonstrated that the binding regions of antibodies in cases 1-5 were contained in the C2 domain of the light chain. Antibodies from cases 1 and 2, which recognized an epitope within residues 2248-2312, completely inhibited FVIII/vWF binding in an ELISA (IC50: 5.0 and 9.0 micrograms/ml, respectively). Antibodies from case 3 recognizing 2170-2312 and case 5 recognizing 2170-2327 also inhibited FVIII/vWF binding (IC50: 110 and 400 micrograms/ml, respectively). Case 4 antibodies recognizing 2218-2307 showed barely detectable inhibition and cases 6 and 7 antibodies recognizing the 44 kD heavy chain, did not inhibit. Our results demonstrate that all anti-C2 alloantibodies with epitopes that extend to the residue 2312 inhibit vWF binding and that an overlap of the inhibitor epitope with residues 2308-2312 is critical for maximal inhibition of vWF binding. Prevention of FVIII/vWF binding appears to be a common property of anti-C2 domain inhibitor alloantibodies.

Measurement of anti-factor VIII IgG, IgG4 and IgM alloantibodies in previously untreated hemophilia A patients treated with recombinant factor VIII. Kogenate Japanese Clinical Study Group.

Using enzyme-linked immunosorbent assay (ELISA), we measured anti-factor VIII immunoglobulin G (IgG), IgG4 and IgM in serum samples obtained from hemophilia A patients treated with a recombinant factor VIII (rFVIII) preparation (Kogenate). Twelve pre- and post-treatment serum samples from 3 previously untreated patients who developed an inhibitor (alloantibody) were evaluated. Both rFVIII and plasma-derived factor VIII (pdFVIII) were used as antigen. Serum samples were diluted 37.5-fold. For the measurement of IgM antibody, protein A Sephadex suspension was added to the patients' serum samples and the supernatant was assayed. Anti-human IgG, IgG4, and IgM monoclonal antibodies labelled with peroxidase were added and absorbance at 450nm was determined. The reactivity of the IgG antibody with rFVIII and pdFVIII was extremely low. Samples containing the IgG4 inhibitor with a neutralizing activity of approximately 7.5 Bethesda units (BU)/ml resulted in absorbance values of 0.451-0.551, thus demonstrating a considerably high degree of sensitivity. The correlation between the neutralizing activity and reactions of the IgG4 antibody with rFVIII and pdFVIII antigens was high, with correlation coefficients of 0.912 and 0.966, respectively. Furthermore, the correlation coefficient between the measured absorbance values for the antibody reacted with pdFVIII and rFVIII was 0.961. No correlation was found between the reactivity of IgM to rFVIII and pdFVIII.

Changes in polypeptide-specific factor VIII antigen levels during whole blood clotting.

The time-course of factor VIII (FVIII) proteolysis during whole blood coagulation was monitored using polypeptide-specific enzyme-linked immunosorbent assays (ELISAs) developed with monoclonal antibodies to FVIII. Such assays of whole blood from normal subjects revealed that, within 30 min after coagulation had started, levels of the amino-terminal region of FVIII light chain and in the middle region of FVIII heavy chain had decreased to baseline in parallel with factor VIII procoagulant activity, but that levels of the 70 kDa thrombin digest decreased more slowly, with 75 U/dl of FVIII antigen (FVIII:Ag) remaining even after 2 h. Similar analysis of the blood of a patient with congenital hypoprothrombinemia indicated a lag phase of 20 min before proteolysis started. No significant change was observed until 1 h after calcium chloride was added to prothrombin-depleted plasma. On the other hand, in the presence of tissue thromboplastin, levels of FVIII:Ag in all ELISAs decreased rapidly. These results indicate a requirement for thrombin generation in the proteolysis of FVIII during the process of whole blood clotting.

Epitope localization of monoclonal antibodies against factor VIII light chain which inhibit complex formation by factor VIII with von Willebrand factor.

We obtained three clones of monoclonal antibodies against factor VIII by immunization with purified human factor VIII. The anti-factor VIII procoagulant activity of these antibodies ranged from 2 to 53 Bethesda units/mg of IgG. According to an immunoblotting study, all antibodies reacted with the 80 kDa light chain but not with 72 kDa peptides derived from thrombin digestion of factor VIII. We attempted to localize the antigenic epitopes of these antibodies by a competitive blocking assay using synthetic peptides and recombinant fragments of the amino-terminal region of factor VIII light chain. In the former assay, a 50 microM peptide containing the fifteen amino acid residues from the Val1670-Glu1684 completely inhibited the binding of the three monoclonal antibodies to immobilized factor VIII. In the latter experiment, 13 reactive recombinant peptides were obtained. Sequences of these peptides revealed fourteen overlapped amino acid residues from Glu1675 to Pro1688. All three antibodies at a final concentration of around 10 micrograms/ml completely inhibited the binding of 125I-labelled factor VIII to immobilized von Willebrand factor (vWF). We conclude that ten amino acid residues, 1675EDFDIYDEDE1684 in the factor VIII light chain are important for complex formation with vWF.

[Properties of factor VIII/von Willebrand factor in two highly-purified factor VIII concentrates, Hemofil M and Monoclate, using monoclonal antibodies].

Properties of F. VIII/vWF in highly-purified factor VIII concentrates were examined using monoclonal antibodies. The F. VIII: C levels obtained with the chromogenic assay agreed with those obtained with the one-stage clotting method. The ratio between F. VIII: Ag and F. VII: C in Hemofil M and Monoclate was 1.09 and 1.34, respectively. The F. VIII: Ag levels assayed by monoclonal ELISAs were the same as those assayed by polyclonal ELISA, except that those assayed by C 5-ELISA tended to be higher. The ratio between F. VIII: Ag and vWF: Ag in Hemofil M and Monoclate was 105 and 45, respectively. Both concentrates lacked the large multimers of vWF and showed the intensification of the satellite bands. SDS-PAGE patterns showed almost no contamination. Immunoblot analysis revealed that F. VIII in both concentrates could react with 6 kinds of monoclonal antibodies to F. VIII. These results suggest that the fundamental structure of F. VIII molecule for coagulant activity in both concentrates are preserved.