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Intraocular pressure (IOP) remains the only modifiable risk factor for glaucoma progression. Our previous discovery that stimulation of nuclei within the hypothalamus can modulate IOP, intracranial pressure (ICP), and translaminar pressure difference (TLPD) fluctuations led us to investigate this pathway further. Our purpose was to determine the role of orexin neurons, primarily located in the dorsomedial hypothalamus (DMH) and perifornical (PeF) regions of the hypothalamus, in modulating these pressures.Sprague Dawley rats were pretreated systemically with a dual orexin receptor antagonist (DORA-12) at 30 mg/Kg (n = 8), 10 mg/Kg (n = 8), or vehicle control (n = 8). The IOP, ICP, heart rate (HR), and mean arterial pressure (MAP) were recorded prior to and following excitation of the DMH/PeF using microinjection of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide (BMI).Administration of the DORA at 30 mg/Kg significantly attenuated peak IOP by 5.2 ± 3.6 mm Hg (P = 0.007). During the peak response period (8-40 minutes), the area under the curve (AUC) for the 30 mg/Kg DORA cohort was significantly lower than the control cohort during the same period (P = 0.04). IOP responses for peak AUC versus DORA dose, from 0 to 30 mg/Kg, were linear (R2 = 0.18, P = 0.04). The ICP responses during the peak response period (4-16 minutes) versus DORA dose were also linear (R2 = 0.24, P = 0.014). Pretreatment with DORA significantly decreased AUC for the TLPD following stimulation of the DMH/PeF (10 mg/kg, P = 0.045 and 30 mg/kg, P = 0.015).DORAs have the potential to attenuate asynchronous changes in IOP and in ICP and to lessen the extent of TLPDs that may result from central nervous system (CNS) activation.

In recent years, there have been significant advances in the treatment of breast cancer resulting in remarkably high survival rates. However, treatment options for metastatic triple negative breast cancer (TNBC) are quite limited due to a lack of identifiable, unique markers. Using a phage display-based whole cell biopanning procedure, we developed two human antibodies that bind to tumor cells with a metastatic TNBC phenotype. Our studies further identified domain 1 of HSPG2 (perlecan) protein as the cognate cell surface antigen bound by the antibody. Immunohistochemistry studies utilizing patient tissue samples revealed significant cell surface expression of HSPG2 in both primary tumors and metastatic lesions. Further, higher HSPG2 expression correlated with poor survival in TNBC. The affinity-matured antibody inhibited the growth of triple negative MDA-MB-231 tumors to a greater extent in nude mice than in NSG mice, pointing to the potential role of natural killer cell-mediated antibody-dependent cell cytotoxicity. This mechanism of action was confirmed through in vitro assays using mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). These results suggest that HSPG2 is a promising target in metastatic TNBC and HSPG2-targeted antibodies could represent a potentially novel class of targeted therapeutics for TNBC.

The repair of dermal wounds, particularly in the diabetic population, poses a significant healthcare burden. The impaired wound healing of diabetic wounds is attributed to low levels of endogenous growth factors, including vascular endothelial growth factor (VEGF), that normally stimulate multiple phases of wound healing. In this study, chitosan scaffolds were prepared via freeze drying and loaded with plasmid DNA encoding perlecan domain I and VEGF189 and analyzed in vivo for their ability to promote dermal wound healing. The plasmid DNA encoding perlecan domain I and VEGF189 loaded scaffolds promoted dermal wound healing in normal and diabetic rats. This treatment resulted in an increase in the number of blood vessels and sub-epithelial connective tissue matrix components within the wound beds compared to wounds treated with chitosan scaffolds containing control DNA or wounded controls. These results suggest that chitosan scaffolds containing plasmid DNA encoding VEGF189 and perlecan domain I have the potential to induce angiogenesis and wound healing.

High density chitosan membranes were produced via a novel manufacturing process for use as implantable resorbable surgical membranes. The innovative method utilizes the following three sequential steps: (1) casting an acidic chitosan solution within a silicon mold, followed by freezing; (2) neutralizing the frozen acidic chitosan solution in alkaline solution to facilitate polymerization; and (3) applying coincident compression-dehydration under a vacuum. Resulting membranes of 0.2 – 0.5 mm thickness have densities as high as 1.6 g/cm3. Inclusion of glycerol prior to the compression-dehydration step provides additional physical and clinical handling benefits. The biomaterials exhibit tensile strength with a maximum load as high as 10.9 N at ~ 2.5 mm width and clinically-relevant resistance to suture pull-out with a maximum load as high as 2.2 N. These physical properties were superior to those of a commercial reconstituted collagen membrane. The dense chitosan membranes have excellent clinical handling characteristics, such as pliability and “memory” when wet. They are semi-permeable to small molecules, biodegradable in vitro in lysozyme solution, and the rates of degradation are inversely correlated to the degree of deacetylation. Furthermore, the dense chitosan membranes are biocompatible and resorbable in vivo as demonstrated in a rat oral wound healing model. The unique combination of physical, in vitro, in vivo, and clinical handling properties demonstrate the high utility of dense chitosan membranes produced by this new method. The materials may be useful as surgical barrier membranes, scaffolds for tissue engineering, wound dressings, and as delivery devices for active ingredients.

Periodontitis is characterized by advancement of a narrow band of epithelium (1-10 cells wide) through the collagenous periodontal ligament in response to bacterial accumulation and infection. A modulating role by epithelial cells in the progression of periodontitis was hypothesized due to the close proximity of the advancing epithelium to both the etiological bacteria and to the collagen fibers of the ligament. We demonstrate that rat mucosal epithelial cells and human fibroblasts are similarly stimulated to degrade a collagen type I cellular substrate by thiol-dependent activity released by the major periodontal pathogen Porphyromonas gingivalis. A purified, extracellular bacterial thiol-proteinase from P. gingivalis ATCC 33277 stimulated mucosal epithelial cells to upregulate expression of collagenase and stromelysin, and to degrade a collagen type I fibril matrix. Stimulation of the epithelial cells with this purified proteinase was associated with morphological changes in the cells and with accumulation of secreted latent procollagenase throughout the culture medium. Release of active collagenase was minimal and collagen degradation by the epithelial cells was discreet and localized subcellularly suggesting the possibility that activation of secreted procollagenase was cell-associated. We conclude that a collagen-degrading phenotype can be stimulated in relatively quiescent mucosal epithelial cells and fibroblasts by the presence of bacterial proteinase. These experiments suggest roles for the P. gingivalis thiol-proteinase and the epithelial cell in the pathogenesis of periodontal disease and demonstrate the potential for dysregulation of extracellular matrix remodeling events during healing of other bacterially infected wounds.

Background and Objective: The plasminogen activating system is a protease/inhibitor system central to extracellular matrix remodeling with a suggested role in periodontal disease pathology. A few studies have reported polymorphisms in the genes of plasminogen activator inhibitors to be associated with periodontal disease severity. Two gene polymorphisms – a BamHI restriction fragment length polymorphism in the urokinase plasminogen activator gene (uPA) and a HindIII restriction fragment length polymorphism in the plasminogen activator inhibitor type 1 gene (PAI-1) – have been associated with conditions having a vascular component, and our objective was to assess the association of these gene polymorphisms with alveolar bone loss in chronic periodontal disease of adults. Material and Methods: Genotype was determined by polymerase chain reaction amplification of whole blood, pertinent histories were obtained by interview, and alveolar bone loss was assessed from current radiographs. Results: In 77 elderly patients with a normal distribution of alveolar bone loss, we demonstrated a significant association between levels of alveolar bone loss and these polymorphisms in the uPA and PAI-1 genes. Controlling for the contributions of smoking or diabetes to periodontal bone loss, estimated odds ratios for predicting lower levels of alveolar bone loss, associated with a greater degree of periodontal health, were strongest when defined by the concurrent presence of a homozygous urokinase plasminogen activator genotype and the nuclease-sensitive plasminogen activator inhibitor type 1 (HindIII) allele (odds ratio = 2.6; 95% confidence interval: 5.8–1.3). Conclusion: The urokinase plasminogen activator (BamHI) and plasminogen activator inhibitor type 1 (HindIII) genotypes may serve as useful markers for heritability of bone loss associated with periodontal disease.

Porphyromonas gingivalishas been implicated as a key etiologic agent in the pathogenesis of destructive chronic periodontitis. Among virulence factors of this organism are cysteine proteinases, or gingipains, that have the capacity to modulate host inflammatory defenses. Intercellular adhesion molecule expression by vascular endothelium represents a crucial process for leukocyte transendothelial migration into inflamed tissue. Ligation of CD99 on endothelial cells was shown to induce expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and major histocompatibility complex class II molecules and to increase adhesion of leukocytes. CD99 ligation was also found to induce nuclear translocation of NF-κB. These results indicate that endothelial cell activation by CD99 ligation may lead to the up-regulation of adhesion molecule expression via NF-κB activation. However, pretreatment of endothelial cells with gingipains caused a dose-dependent reduction of adhesion molecule expression and leukocyte adhesion induced by ligation of CD99 on endothelial cells. The data provide evidence that the gingipains can reduce the functional expression of CD99 on endothelial cells, leading indirectly to the disruption of adhesion molecule expression and of leukocyte recruitment to inflammatory foci.

Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.

Periodontitis is a response of highly vascularized tissues to the adjacent microflora of dental plaque. Progressive disease has been related to consortia of anaerobic bacteria, with the gram-negative organismPorphyromonas gingivalisparticularly implicated. The gingipains, comprising a group of cysteine proteinases and associated hemagglutinin domains, are major virulence determinants of this organism. As vascular expression of leukocyte adhesion molecules is a critical determinant of tissue response to microbial challenge, the objective of this study was to determine the capacity of gingipains to modulate the expression and function of these receptors. Given the potential multifunctional role of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the vasculature, the effect of gingipains on PECAM-1 expression by endothelial cells was examined. Activated gingipains preferentially down-regulated PECAM-1 expression on endothelial cells compared with vascular cell adhesion molecule 1 and endothelial-leukocyte adhesion molecule 1, but the reduction in PECAM-1 expression was completely inhibited in the presence of the cysteine proteinase inhibitor TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone). Endothelial monolayers treated with activated gingipains demonstrated progressive intercellular gap formation that correlated with reduced intercellular junctional PECAM-1 expression as determined by Western blotting and immunofluorescence microscopy. This was accompanied by enhanced transfer of both albumin and neutrophils across the monolayer. The results suggest that degradation of PECAM-1 by gingipains contributes to increased vascular permeability and neutrophil flux at disease sites.

The gingipains have been implicated in the pathogenicity of Porphyromonas gingivalis , a major etiologic agent of chronic periodontitis. Mature gingipains often present as a membrane-bound glycosylated proteinase-adhesin complex comprising multiple adhesin domains (HA1 to -4) and a catalytic domain. Using recombinant adhesin domains, we were able to show that patients with chronic periodontitis produce significantly more immunoglobulin G reactive with gingipain domains than a corresponding group with healthy periodontium. Titers were predominantly directed toward the carbohydrate epitopes shared between the gingipains and the lipopolysaccharide of P. gingivalis with little recognition of the peptide backbone of the catalytic domains. Distribution of titers to peptide epitopes of the adhesin domains was as follows: HA4 ≈ HA1 > HA3 ≫ HA2. No correlation was observed between markers of disease severity and titers to individual adhesins within the disease group. Posttreatment titers showed no change or a decrease in titers for the majority of patients except for titers to the HA2 domain which showed marked increases in a few responding patients. Since the HA2 domain is important in hemoglobin binding and acquisition of essential porphyrin, boosting titers of antibodies to this domain may have the potential to control the growth of this organism.

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-γ) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-γ have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogenPorphyromonas gingivalisstimulated monocytes primed with IFN-γ to release IL-12, thereby enhancing IFN-γ accumulation in T-cell populations. P. gingivalisLPS was shown to enhance IL-12 induction of IFN-γ in T cells in a manner independent from TNF-α contribution. The levels of T-cell IL-12 receptors were not affected byP. gingivalisLPS and played only a minor role in the magnitude of the IFN-γ response. These data suggest that LPS fromP. gingivalisestablishes an activation loop with IL-12 and IFN-γ with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-γ accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-γ levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) ofP. gingivaliscould lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.

Previous studies have shown a correlation between the production of certain matrix metalloproteinases (MMPs), especially the gelatinases, by malignant tumors and the progression of these cancers as they invade and metastasize through the extracellular matrix and basement membranes. However, very few of these studies examined this relationship in human oral cancer in vivo, and none addressed the issue of how combinations of the MMPs may further enhance tumor progression. To determine which MMPs are produced in vivo by human oral cancers, we used specific anti-human-MMP antibodies and immunocytochemistry (ICC) methods to examine oral cancer tissue specimens from 20 surgery patients. The ICC data indicated that 72-kDa (72K-GL) and 92-kDa gelatinases (92K-GL) were produced in vivo by discreet clusters of tumor cells and by stromal fibroblasts, vascular endothelial cells (72K-GL), and PMNs (92K-GL). Some stromal fibroblasts near the tumors also appeared to produce fibroblast-type collagenase (FIB-CL), a finding confirmed by Western blot analysis of media conditioned by oral tumor explant cultures. ICC results indicated that 5 of the 20 tumors coincidentally produced all three MMPs. To examine how the two gelatinases and FIB-CL may interact in vitro to degrade fibrillar type I collagen, a major structural component of the extracellular matrix, we used a modified FIB-CL activity assay. Combinations of the gelatinases and FIB-CL were incubated with a 3H-collagen substrate, with the results compared with the combination of stromelysin-1 (SL-1, a superactivator of FIB-CL) and FIB-CL. 92K-GL caused a nine-fold increase in collagenase activity, equivalent to SL-1, while 72K-GL produced a four-fold increase. These results indicate that human oral cancers produce 92K-GL, 72K-GL, and FIB-CL in vivo and that the gelatinases and FIB-CL cooperate to enhance collagen degradation greatly in vitro.

Background Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™). Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.

Background Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. Results By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor. Conclusions With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

Background and Objective: Endothelial cells have a substantial role in maintaining vascular homeostasis, and their dysregulation can contribute to the development of pathology. The plasminogen activators and their inhibitors may, arguably, be the single most important proteolytic system of the endothelium for vascular maintenance by controlling plasminogen activation and other proteolytic cascades that impact on clotting, hemodynamics, angiogenesis and the character of the vascular wall. In chronic periodontal disease, significant changes to the microvasculature occur in association with the severity of the disease. Investigation of the role played by endothelial cells in periodontal health and disease has been limited to in situ immunolocalization or to the use of endothelial cells of nongingival origin, such as human umbilical vein endothelial cells. The objective of this research was to establish a replicable protocol for isolating microvascular endothelial cells from the gingiva. Material and Methods: From inflamed gingiva, isolated cells were characterized by morphology, the expression of factor VIII-related antigen, the expression of UEA-1 ligand, the uptake of acetylated low-density lipoprotein, network formation on Matrigel, and by the expression levels of urokinase plasminogen activator, tissue plasminogen activator, plasminogen activator inhibitor-1 and collagen IV. Results and Conclusion: Gingival endothelial cells were most readily obtained from inflamed gingival tissues, and these endothelial cells, when isolated by the protocol established herein, demonstrated endothelial characteristics and constitutively secreted plasminogen activators and plasminogen activator inhibitor-1 in culture.

SummaryWe previously reported evidence that patients with periodontitis have serum antibodies to oral Gram positive bacteria that are cross-reactive with epithelial antigens. In the present report cross-reactive epithelial antigens including CD24, lactate dehydrogenase A [LDM-A], antioxidant protein 2 [AOP 2] and nuclear factor of activated T cells 5 [NFAT 5], were identified by screening a cDNA expression library with pooled patient sera. Titres of antibodies to CD24 peptide correlated negatively with indices of periodontal disease severity. Strong expression of CD24 in the reactive periodontal epithelium and inflamed gingival attachment contrasted with low to undetectable expression in the external gingival epithelium. In periodontitis, a local action of these auto-reactive antibodies could modulate the regulatory potential associated with expression of CD24 in this epithelium.

The role of Porphyromonas gingivalis cysteine proteinases (gingipains) in the evasion of host cell-mediated immunity has not been fully determined. In this study, modulation by gingipains of accessory and co-stimulatory molecule expression on human CD4 + T cells was evaluated. Arg-gingipain rather than Lys-gingipain binds to resting CD4 + T cells in the presence of serum. The constitutive expression of CD28 on T cells was slightly up-regulated following challenge with gingipains, whereas CD45 and CD3 were not affected. Binding of anti-CD2 and anti-CD4 monoclonal antibodies (mAbs) was reduced after challenge of T cells with gingipains, but restored to 50 and 100%, respectively, of control levels, after 48 h of incubation in medium depleted of gingipains. The induced expression, by anti-CD3 mAb, of CTLA-4, CD25, and CD40 ligand (CD40L) was decreased following incubation of T cells with gingipains which also led to decreased response to anti-CD3 and anti-CD28 mAbs as shown by reduction of interleukin-2 (IL-2) production. Cumulatively, these results indicate that activated gingipains attach to T cells and preferentially cleave CD2 and CD4 molecules, with potential to impair T cell responses at periodontal sites.

Paired subgingival plaque samples representing the most-diseased and least-diseased sites were collected from 34 adult patients with diagnosed chronic periodontitis. The percentage of Porphyromonas gingivalis relative to the total anaerobic and gram-negative bacterial load at each site was determined by real-time PCR. Based on variations in the noncatalytic C terminus of the Lys-gingipain (Kgp), it was reasoned that DNA sequence variation in the 3′-coding region of the kgp gene might determine functional biotypes. Perusal of the available sequence information in GenBank indicated three such forms of the kgp gene corresponding to P. gingivalis strains HG66, 381, and W83. Analysis of patient samples revealed the presence of a fourth genotype (W83v) that showed duplication of a sequence recognized by the W83 reverse primer. The four biotypes, HG66, 381, W83, and W83v, were present in the study group in the ratio 8:11:6:5, respectively. Each subject was colonized by one predominant biotype, and only three patients were colonized by a trace amount of a second biotype.

Background: The hemoglobin-binding domain (HA2) of the Porphyromonasgingivalis gingipains and hemagglutinins strongly binds hemoglobin and hemin and is thought to play a key role in acquisition of this essential metabolite by the microorganism. Methods: In this report, we partially characterized human anti-HA2 humoral antibodies and their relationship to periodontal disease in an analysis of titer and function. Results: Overall, serum anti-HA2 antibodies were relatively low and dominated by the immunoglobulin M (IgM) isotype. Pre-therapy titers had a direct association with periodontal health. Levels of P. gingivalis in the plaque were directly related to pre-therapy anti-HA2 IgG levels, and were an important covariant in a significant direct relationship between pre- and post-therapy anti-HA2 titers. Post-therapy anti-HA2 IgG antibody titers were directly related to the capacity of serum IgG fractions to neutralize hemoglobin binding by Lys-gingipain (Kgp). Further, lower levels of neutralizing activity post-therapy were directly related to severe periodontitis within the patient cohort. Conclusions: These data suggest that anti-HA2 IgG antibodies correspond directly with periodontal health, possibly through their ability to neutralize P. gingivalis hemoglobin capture. The data also suggest that inadvertent or therapeutic inoculation of P. gingivalis in the plaque may contribute to generation of neutralizing anti-HA2 IgG and improvement of periodontal prognosis.

SUMMARY Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process that has been linked with Th2 pathways. Critical in maintaining Th2 activity is the response of B lymphocytes to environmental interleukin (IL)-4, a cytokine that also counteracts Th1-cell differentiation. Here we demonstrate that while the gingipains effectively degrade interleukin (IL)-4 under serum-free conditions, limited hydrolysis was observed in the presence of serum even after prolonged incubation. Gingipains up-regulated CD69 expression directly in purified peripheral blood B cell preparations. Further, the induction of IL-4 receptor expression on B cells by gingipains correlates with B cell activation, which is also manifested by a mitogenic response. These results suggest that the gingipains of P. gingivalis act during the early stage of B-cell growth as a competence signal, whereby sensitized B cells might become more responsive to further challenge in the disease-susceptible individual.

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae , reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.

In a rat periodontitis model, preinoculation with the Porphyromonas gingivalis HA2 binding domain for hemoglobin provided protection from disease. Protection was associated with induced anti-HA2 immunoglobulin G (IgG) humoral antibodies. The IgG subclass ratios suggested that relatively lower Th2/Th1-driven responses were directly associated with protection when rHA2 was administered in saline.

Porphyromonas gingivaliscysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-γ) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-γ production from CD4+T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-γ production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.

Publisher Summary This chapter focuses on two major oral diseases, periodontitis and residual ridge resorption, and reviews current understanding of the etiology, prevalence, and treatment of these diseases. The diagnosis of loss of bone and soft tissue attachment due to periodontitis is accomplished with radiography and periodontal probing. Risk factors in periodontal disease or residual ridge resorption have been determined primarily through epidemiologic or observational research. Such research aids in the identification of high risk patients for progressive bone loss, thereby allowing clinicians to prescribe appropriate preventive care or treatment. Studies indicate that one of the most important risk factors for progressive alveolar bone resorption in periodontitis is the presence of previous destruction. Logistic modeling revealed that the primary risk factors for progressive loss of attachment around the teeth were age and a history of attachment loss. The discussion on pathophysiologic mechanisms in periodontitis implies, treatment of periodontitis could involve either control of the pathogenic plaque bacteria or the destructive host response. Current periodontal treatment is focused primarily on controlling plaque bacteria. Patient's self-administered plaque control and scaling and root planing performed by the dental professional reduces the mass of bacterial plaque. Root planning smoothens the root surface and removes decalcified cementum and bacterial components such as endotoxins. Periodontal surgical procedures provide access to the roots for effective root planing or to the bone for pocket reduction procedures. Regenerative procedures using the principles of guided tissues regeneration or bone grafting materials may be used to aid the restoration of lost periodontal ligament and bone in selected cases.

Cysteine proteinases have been emphasized in the virulence of Porphyromonas gingivalis in chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-γ). Treatment with the cysteine proteinases resulted in a rapid shift in the apparent molecular size of IFN-γ from 17 to 15 kDa, as shown by Western blot analysis, a response which also occurred in the presence of serum. Further, glycosylated natural IFN-γ from human leukocytes and unglycosylated recombinant IFN-γ from Escherichia coli were both digested by the cysteine proteinases. Immunoblot analysis indicated that cleavage within the carboxyl terminus of recombinant IFN-γ correlated with the loss of induction of MHC class II expression as monitored by analytical flow cytometry. No hydrolysis of MHC class II molecules or human IFN-γ receptor by these proteinases was detected by Western blot analysis. These findings suggest that P. gingivalis cysteine proteinases may alter the cytokine network at the point of infection through the cleavage of IFN-γ. Degradation of IFN-γ could have important consequences for the recruitment and activation of leukocytes and therefore may contribute significantly to the destruction of the periodontal attachment.

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis . We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin ( K d = 16 nM), and binding was inhibited by iron-free protoporphyrin IX ( K i = 2.5 μM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain ( K d = 2.1 nM) was also inhibited by protoporphyrin IX ( K i = 10 μM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.

A critical outcome of periodontal disease is degradation of the collagenous periodontal ligament that connects teeth to bone in the dental arch. Periodontal diseases occur in response to bacterial colonization of the teeth, but their molecular pathogenesis is still speculative. One family of enzymes, known as the matrix metalloproteinases (MMPs), has been implicated in the degradation of the periodontal ligament. MMPs, which are also suspected to play a role in many other physiologic and pathologic remodeling processes, can be secreted by epithelial cells surrounding the teeth and are found in relative abundance in tissues and fluids near periodontally diseased sites. Since most MMPs are secreted as inactive zymogens which may be activated by limited proteolysis, it has been suggested that proteinases expressed by the infecting periodontal pathogens might activate latent host MMPs to initiate or accelerate degradation of the collagenous periodontal ligament. The aim of this work was to examine interactions between purified host MMPs and bacterial proteinase. In this article, we demonstrate that a proteinase isolated from the periodontopathogen Porphyromonas gingivalis can activate MMP-1, MMP-3, and MMP-9 and can catalyze the superactivation of MMP-1 by MMP-3. Activation of these MMPs is demonstrated to result from initital hydrolysis within their propeptide. Also, for MMP-1 and MMP-9, the P. gingivalis proteinase cleaves the MMP propeptide following a lysine residue at a previously unreported site which, for both MMPs, is one residue NH2-terminal to the known autocatalytic cleavage site. These data describe a mode of virulence for the periodontopathogen Porphyromonas gingivalis that involves activation of host-degradative enzymes.

Thiol-dependent proteinases that are expressed and released by Porphyromonas gingivalis are considered virulence factors in periodontitis because of their potential to effect matrix degradation and inflammation. A number of P. gingivalis thiol-proteinases have been described, however, with similar biochemical characteristics. In this report we demonstrate that an isolated P. gingivalis proteinase consists of nonco-valently associated peptides and that slight variations in the association pattern of these peptides could result in different proteinases with different affinities and activities. We also describe the co-purification of thiol-proteinase activity with hemagglutinin activity and demonstrate that each type of activity has similar inhibition profiles. With the use of monoclonal antibodies against the P. gingivalis proteinase we follow proteinase released into the culture medium over the course of 10 days and, by Western blot analysis, demonstrate that many of the proteinases with varying molecular weight are related. The identification of a single, immunoreactive, 140 kDa proteinase detected early in the culture and in association with the P. gingivalis cells suggests that multiple proteinases may originate from a single 140 kDa proteinase.

Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn++endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in specific developmental or pathological conditions, including human periodontal diseases.