Antiviral activity of Epimedium koreanum Nakai water extract against influenza viruses.

Epimedium koreanum Nakai (EKN) is a popular plant in Korean and Chinese medicine for treating a variety of ailments. The aqueous extract of EKN has a significant inhibitory impact on influenza A virus (IAV) infection by directly blocking viral attachment and having a virucidal effect, according to this study. Using fluorescent microscopy and fluorescence-activated cell sorting (FACS) with a green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus, we examined the effect of EKN on viral infection. By viral infection, EKN strongly suppresses GFP expression, and at a dosage of 100 µg/mL, EKN decreased GFP expression by up to 90% of the untreated infected control. Immunofluorescence and Western blot analyses against influenza viral proteins revealed that EKN decreased influenza viral protein expression in a dose-dependent manner. EKN inhibited the H1N1 influenza virus's hemagglutinin (HA) and neuraminidase (NA), preventing viral attachment to cells. Furthermore, EKN had a virucidal impact and inhibited the cytopathic effects of H1N1, H3N2 and influenza B virus infection. Finally, our findings show that EKN has the potential to be developed as a natural viral inhibitor against influenza virus infection.

Daratumumab for delayed RBC engraftment following major ABO mismatched haploidentical bone marrow transplantation.

BACKGROUND: Recent case reports have described the efficacy of daratumumab to treat refractory pure red cell aplasia (PRCA) following major ABO mismatched allogeneic hematopoietic stem cell transplantation (HSCT). In this report, we describe the use of daratumumab as a first-line agent for treatment of delayed red blood cell (RBC) engraftment following a major ABO mismatched pediatric HSCT and provide a review of the literature. STUDY DESIGN AND MATERIALS: We report on a 14-year-old with DOCK8 deficiency who underwent a myeloablative, haploidentical bone marrow transplant from her major ABO mismatched sister (recipient O+, donor A+) for treatment of her primary immunodeficiency. Despite achieving full donor chimerism, she had delayed RBC engraftment requiring ongoing transfusions. Due to iron deposition, symptomatic anemia, and persistence of anti-A iso-hemagglutinins despite discontinuation of immunosuppression, treatment for delayed RBC engraftment with the CD38-targeted monoclonal antibody daratumumab was selected as a less immunosuppressive agent that could more selectively target iso-hemagglutinin producing plasma cells without causing broad B-cell aplasia. RESULTS: Clinical effect with daratumumab was demonstrated by reduced iso-hemagglutinin titer, increased reticulocytosis, normalization of her hemoglobin, and transfusion independence. In the 11-month follow-up period to date, no additional transfusions or immunosuppression have been necessary, despite persistence of low-level anti-A iso-hemagglutinin. CONCLUSION: Our experience suggests that daratumumab was an effective first-line therapy for delayed RBC engraftment and that earlier consideration for daratumumab in treatment of delayed RBC engraftment may be warranted.

2ß-3,4-Unsaturated sialic acid derivatives: Synthesis optimization, and biological evaluation as Newcastle disease virus hemagglutinin-neuraminidase inhibitors.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

In Vitro and In Vivo Antiviral Activity of Nylidrin by Targeting the Hemagglutinin 2-Mediated Membrane Fusion of Influenza A Virus.

Influenza A virus, one of the major human respiratory pathogens, is responsible for annual seasonal endemics and unpredictable periodic pandemics. Despite the clinical availability of vaccines and antivirals, the antigenic diversity and drug resistance of this virus makes it a persistent threat to public health, underlying the need for the development of novel antivirals. In a cell culture-based high-throughput screen, a ß2-adrenergic receptor agonist, nylidrin, was identified as an antiviral compound against influenza A virus. The molecule was effective against multiple isolates of subtype H1N1, but had limited activity against subtype H3N2, depending on the strain. By examining the antiviral activity of its chemical analogues, we found that ifenprodil and clenbuterol also had reliable inhibitory effects against A/H1N1 strains. Field-based pharmacophore modeling with comparisons of active and inactive compounds revealed the importance of positive and negative electrostatic patterns of phenyl aminoethanol derivatives. Time-of-addition experiments and visualization of the intracellular localization of nucleoprotein NP demonstrated that an early step of the virus life cycle was suppressed by nylidrin. Ultimately, we discovered that nylidrin targets hemagglutinin 2 (HA2)-mediated membrane fusion by blocking conformational change of HA at acidic pH. In a mouse model, preincubation of a mouse-adapted influenza A virus (H1N1) with nylidrin completely blocked intranasal viral infection. The present study suggests that nylidrin could provide a core chemical skeleton for the development of a direct-acting inhibitor of influenza A virus entry.

Synthesis, Structure Activity Relationship and Anti-influenza A Virus Evaluation of Oleanolic Acid-Linear Amino Derivatives.

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin-Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure-activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC50 of 2.98 µM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.

Safety and Upper Respiratory Pharmacokinetics of the Hemagglutinin Stalk-Binding Antibody VIS410 Support Treatment and Prophylaxis Based on Population Modeling of Seasonal Influenza A Outbreaks.

BACKGROUND: Seasonal influenza is a major public health concern in vulnerable populations. Here we investigated the safety, tolerability, and pharmacokinetics of a broadly neutralizing monoclonal antibody (VIS410) against Influenza A in a Phase 1 clinical trial. Based on these results and preclinical data, we implemented a mathematical modeling approach to investigate whether VIS410 could be used prophylactically to lessen the burden of a seasonal influenza epidemic and to protect at-risk groups from associated complications. METHODS: Using a single-ascending dose study (n = 41) at dose levels from 2 mg/kg-50 mg/kg we evaluated the safety as well as the serum and upper respiratory pharmacokinetics of a broadly-neutralizing antibody (VIS410) against influenza A (ClinicalTrials.gov identifier NCT02045472). Our primary endpoints were safety and tolerability of VIS410 compared to placebo. We developed an epidemic microsimulation model testing the ability of VIS410 to mitigate attack rates and severe disease in at risk-populations. FINDINGS: VIS410 was found to be generally safe and well-tolerated at all dose levels, from 2-50 mg/kg. Overall, 27 of 41 subjects (65.9%) reported a total of 67 treatment emergent adverse events (TEAEs). TEAEs were reported by 20 of 30 subjects (66.7%) who received VIS410 and by 7 of 11 subjects (63.6%) who received placebo. 14 of 16 TEAEs related to study drug were considered mild (Grade 1) and 2 were moderate (Grade 2). Two subjects (1 subject who received 30 mg/kg VIS410 and 1 subject who received placebo) experienced serious AEs (Grade 3 or 4 TEAEs) that were not related to study drug. VIS410 exposure was approximately dose-proportional with a mean half-life of 12.9 days. Mean VIS410 Cmax levels in the upper respiratory tract were 20.0 and 25.3 µg/ml at the 30 mg/kg and 50 mg/kg doses, respectively, with corresponding serum Cmax levels of 980.5 and 1316 µg/mL. Using these pharmacokinetic data, a microsimulation model showed that median attack rate reductions ranged from 8.6% (interquartile range (IQR): 4.7%-11.0%) for 2% coverage to 22.6% (IQR: 12.7-30.0%) for 6% coverage. The overall benefits to the elderly, a vulnerable subgroup, are largest when VIS410 is distributed exclusively to elderly individuals, resulting in reductions in hospitalization rates between 11.4% (IQR: 8.2%-13.3%) for 2% coverage and 30.9% (IQR: 24.8%-35.1%) for 6% coverage among those more than 65 years of age. INTERPRETATION: VIS410 was generally safe and well tolerated and had good relative exposure in both serum and upper respiratory tract, supporting its use as either a single-dose therapeutic or prophylactic for influenza A. Including VIS410 prophylaxis among the public health interventions for seasonal influenza has the potential to lower attack rates and substantially reduce hospitalizations in individuals over the age of 65. FUNDING: Visterra, Inc.

Selection of aptamers targeting the sialic acid receptor of hemagglutinin by epitope-specific SELEX.

A new SELEX scheme is proposed for the selection of aptamers targeting a specific epitope of a native protein. Anti-sialic acid receptor (SAR) aptamers that inhibit H1 hemagglutination at a low picomole dose are selected accordingly.

Purification and partial characterization of a new pro-inflammatory lectin from Bauhinia bauhinioides Mart (Caesalpinoideae) seeds.

A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for an hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses.

Disease-associated carbohydrate-recognising proteins and structure-based inhibitor design.

The role of carbohydrate-related pathways in a wide range of clinically significant diseases has provided great impetus for researchers to characterise key proteins as targets for drug discovery. Carbohydrate-recognising proteins essential in the lifecycles of high health impact pathogens and diseases such as diabetes, cancer, autoimmunity, inflammation and in-born errors of metabolism continue to stimulate much interest in both structure elucidation and structure-based drug design. For example, advances in structure-based inhibitor design against the mycobacterial enzyme UDP-galactopyranose mutase offer new hope in next generation anti-tuberculosis chemotherapeutics. The appearance of H5N1 avian influenza virus has re-stimulated much research on influenza virus haemagglutinin and sialidase. These latest developments on influenza virus sialidase have provided new opportunity for the development of Group 1-specific anti-influenza drugs. The role of siglecs and galectins in a range of disease processes such as inflammation, apoptosis and cancer progression has also inspired significant structure-based inhibitor design research.

Immune response to influenza vaccine in pediatric patients with inflammatory bowel disease.

BACKGROUND & AIMS: The aim of this study was to compare response to inactivated influenza vaccine in healthy children and pediatric patients with inflammatory bowel disease (IBD). METHODS: A prospective, open-label, controlled clinical trial during influenza seasons of 2002-2004 was performed. Single-dose inactive trivalent influenza vaccine was administered. Immune response to vaccination was measured by pre-immunization and postimmunization hemagglutinin inhibition titers. A postimmunization hemagglutinin inhibition titer of 40 or higher was considered protective against influenza. IBD activity and adverse events were recorded. RESULTS: Eighty subjects were enrolled (29 healthy controls, 51 IBD patients). One patient did not complete the study. Patients were divided into 3 subgroups: infliximab and immunomodulatory (16), immunomodulatory (20), and anti-inflammatory therapy (14). Immunomodulatory therapy included corticosteroids, 6-mercaptopurine, or methotrexate. Overall, there was a statistically significant decrease in immune response in patients compared with healthy controls who received 1 influenza vaccine antigen (B/Hong Kong, P = .0125). Patients receiving infliximab and immunomodulatory therapy were less likely to respond to 2 influenza vaccine antigens (A/New Caledonia/20/99 and B/Hong Kong/330/2001, P = .018 and .0002, respectively). Fifteen subjects (19%) reported 19 mild adverse events: 11 (14%) reported soreness at the site, 4 (5%) reported having a cold, 3 (4%) reported flu-like symptoms, and 1 (1%) reported a headache. The clinical activity of IBD was not affected by vaccination. CONCLUSIONS: The serologic conversion rate to influenza vaccine in patients with IBD ranged from 33% to 85%. Patients on concomitant infliximab and immunomodulatory therapy are at risk of inadequate response to vaccination. The vaccine was safe and did not affect IBD activity.

Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a.

BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis.

Efficacy of novel hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 against human parainfluenza viruses in vitro and in vivo.

Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK(2) cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 micro M in HA inhibition assays and from 0.02 to 20 micro M in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 micro M. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.

Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis.

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg-gingipain (Rgp) and Lys-gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, and (-)-catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 microm. While (-)-epigallocatechin and (-)-gallocatechin moderately inhibited Rgp activity (IC50s, 20 microm), (-) -epicatechin, (+)-catechin, and gallic acid were not effective, with IC50s greater than 300 microm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.

Modulation of Porphyromonas gingivalis proteinase activity by suboptimal doses of antimicrobial agents.

BACKGROUND: Antimicrobial agents are sometimes used as adjuncts for the treatment of aggressive and refractory forms of periodontitis. In this study, we used a culture plate assay to investigate the effect of suboptimal doses of antimicrobial agents on proteinase activity of Porphyromonas gingivalis. METHODS: A culture plate assay using gelatin as the substrate, which allows a semiquantitative determination of proteinase activity, was developed. Suboptimal inhibitory concentrations of tetracycline, minocycline, doxycycline, metronidazole, penicillin G, or chlorhexidine were added to the medium, and proteolysis zones were determined following the growth of three strains of P. gingivalis. The effect of antimicrobials on outer membrane vesicle-associated gingipains also was determined. RESULTS: The gelatin plate assay was a convenient, simple procedure for investigating the effect of suboptimal inhibitory concentrations of antimicrobial agents on proteinases produced by P. gingivalis. The largest reduction (> 75%) in the proteolysis zones produced by three strains of P. gingivalis was obtained with minocycline. Tetracycline and doxycycline also reduced the proteolysis zones. A suboptimal inhibitory concentration of chlorhexidine increased the proteolysis zones by up to 70%. Metronidazole and penicillin G produced no noticeable effect. The suboptimal inhibitory concentrations of minocycline, tetracycline, and doxycyline did not reduce the activity of outer membrane vesicle-associated Arg- and Lys-gingipains. CONCLUSION: Results from this study suggest that sublethal concentrations of some antimicrobial agents in subgingival sites have the potential to affect the physiology of P. gingivalis, notably by increasing or decreasing the proteolytic activity of the bacteria.

Attenuation of the virulence of Porphyromonas gingivalis by using a specific synthetic Kgp protease inhibitor.

The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar K(i) was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens.

Bacteroides forsythus hemagglutinin is inhibited by N-acetylneuraminyllactose.

Bacteroides forsythus, which has been recognized as a pathogen associated with periodontitis, produces a hemagglutinin. The hemagglutinin was localized in the envelope of B. forsythus. The hemagglutinating activity was inhibited by lactose at concentrations as low as 1 mM, and by L-arginine and L-lysine at concentrations of 100 mM. N-Acetylneuraminyllactose (NeuAc-lactose) was at least 100 times more potent an inhibitor than lactose, as it completely inhibited the hemagglutination at concentrations below 10 microM. This is similar to the Helicobacter pylori hemagglutinin. The hemagglutinin was heat-labile, and resistant to treatment with proteases such as trypsin. A specific antibody raised against one of the S-layer proteins that are major species-specific proteins had no inhibitory effect on the hemmaglutination. These results suggest that the NeuAc-lactose-sensitive adhesin of B. forsythus may play an important role in colonization in the oral cavity.

Inhibition of trypsin-like cysteine proteinases (gingipains) from Porphyromonas gingivalis by tetracycline and its analogues.

Extracellular cysteine proteinases, referred to as gingipains, are considered important virulence factors for Porphyromonas gingivalis, a bacterium recognized as a major etiologic agent of chronic periodontitis. We investigated the effect of tetracycline and its analogues, doxycycline and minocycline, on the enzymatic activities of gingipains. Tetracyclines at 100 microM totally inhibited the amidolytic activity of arginine-specific gingipains (HRgpA and RgpB). In contrast, inhibition of Kgp was less efficient and required a somewhat higher concentration of the antibiotic to achieve the same effect. Among tetracycline derivatives, the most potent gingipain inhibitor was doxycycline, followed by tetracycline and minocycline. RgpB was inhibited by doxycycline in an uncompetitive and reversible manner with a 50% inhibitory concentration of 3 microM. Significantly, inhibition was unaffected by calcium, excluding the chelating activity of tetracyclines as the mechanism of gingipain inactivation. In contrast, the inhibitory activities of the tetracyclines were reduced by cysteine, a reducing agent, suggesting an interference of the drug at the oxidative region with the catalytic system of the enzyme. Doxycycline, at 10 microM, significantly inhibited the RgpB-mediated production of vascular permeability-enhancing activity from human plasma, thus proving an effective inhibition of gingipain in vivo. These results indicate a new activity of tetracyclines as cysteine proteinase inhibitors and may explain the therapeutic efficiency of these antibiotics in the treatment of periodontitis.

Changes in immunologic properties of group A RBCs during treatment with an A-degrading exo-alpha-N-acetylgalactosaminidase.

BACKGROUND: Blood group A RBCs theoretically can be converted to universal-donor group O cells by incubation with alpha-N-acetylgalactosaminidase (A-zyme). The purpose of this study was to compare the extent of A-zyme treatment required to abolish several immunologic responses of group B or O recipients to group A RBCs. STUDY DESIGN AND METHODS: A(1) RBCs were incubated for 2 hours at 37 degrees C in buffer alone (control) or with 0.1 to 5.0 units (U) of A-zyme per mL of packed cells. They were then tested for 1) immune adherence (% rosetting) and activation of monocytic cells (% erythrophagocytosis, TNFalpha production), 2) activation of immune hemolysis, and 3) hemagglutination with Dolichos biflorus lectin and pooled human anti-A serum. RESULTS: A 2-hour incubation with < or =5.0 U of A-zyme per mL of packed cells abolished monocytic cell adherence and phagocytosis of group A(1) RBCs coated with IgG(3) A MoAb. Epitopes of A binding to IgG(3) anti-A A005 MoAb and BG-2 MoAb differed in A-zyme sensitivity. TNFalpha production in group B or O whole blood in response to the addition of A(1) cells varied, but it essentially was abolished when group A cells were treated with 0.1 to 1.0 U of A-zyme per mL of packed cells. A epitopes mediating immune hemolysis and hemagglutination with D. biflorus lectin were also cleaved by <5 U of A-zyme per mL of packed cells. In contrast, hemagglutination with polyclonal anti-A typing serum was diminished by only 1 to 2 serial titer dilutions. CONCLUSION: A epitopes mediating immune hemolysis and immune adherence to and activation of monocytic cells are highly sensitive to A-zyme cleavage, as compared to those mediating hemagglutination with monoclonal and polyclonal anti-A.

Suppression of gingival inflammation induced by Porphyromonas gingivalis in rats by leupeptin.

In this study, we developed a procedure to produce gingivitis in rats by inoculation of Porphyromonas gingivalis and studied the contribution of the bacterial cysteine proteinases, Arg-gingipain (Rgp) and Lys-gingipain (Kgp), to the pathology in the gingiva. To adhere the bacterium to periodontal tissues, a cotton thread was inserted between the first and second molar of right maxillary sites of rats. Rats in group A were administered with vehicle alone after bacterial (strain W83) inoculation. In group B, the bacteria were inoculated in combination with leupeptin, a potent inhibitor of Rgp and Kgp, and then leupeptin alone was administered the week after. Rats in group C were administered leupeptin for 6 weeks after bacteria inoculation. All left maxillary gingiva in three groups showed no inflammatory changes. Right maxillary gingiva of group A showed most of the clinical landmarks of gingivitis. Leupeptin exhibited only a little inhibitory effect on this gingivitis in group B, whereas it had a strong inhibitory effect on the inflammation in group C. These results suggest that P. gingivalis-induced gingivitis is attributable to Rgp and Kgp and that leupeptin is more effective in the late phase than the early stage of gingivitis.

Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.