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Development of a murine model of actinobacillus actinomycetemcomitans-induced pathogenesis Colfer, Ellen Lee 2000

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DEVELOPMENT OF A MURINE MODEL OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS-INDUCED PATHOGENESIS B y E L L E N L E E C O L F E R D D S , University of Toronto, 1996 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Oral Biological and Medical Sciences) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A December 10, 1999 © E L L E N L E E C O L F E R In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I - agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. D e p a r t ™ , o, / g / ^ ^ j ^ o ^ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT Animals have been used in health sciences for over 100 years to study the mechanism and virulence of bacterial pathogens. Many variations of the abscess model of inflammation have been developed but they do not provide the ability to easily quantify the resulting pathology. The aims of this study were to 1) develop a quantitative method of grading an inflammatory response to Actinobacillus actinomycetemcomitans-mduc&d (A. actinomycetemcomitans) inflammation using non-viable bacteria, 2) determine the reproducibility of the inflammatory and immunohistologic response and 3) determine the virulence of three strains of A. actinomycetemcomitans using the proposed grading system developed in the model. During the study, C D - I mice were injected s.e. in the ear and/or abdomen with two serotypes strains (three strains) of A. actinomycetemcomitans bacteria, JP2 (serotype b), A T C C 43718-Y4 (serotype b) and I D H 1705 (serotype e). Two grading systems were proposed for histological and immunohistological evaluation of the tissues. The histological evaluation was based on the area of the inflammatory spread within the tissues and number of inflammatory cells counted using an optic grid. The immunohistologic response was determined for the three antibodies; F4/80 anti-monocyte/macrophage antibody, anti-neutrophil antibody and anti-CD3 T cell antibody, by the number of cells found in the tissues. The reproducibility of the kinetic study was ii deterirrined by nonparametric analysis of variance using the Kolmogorov-Smirnov statistic. The results of the study did revealed that non-viable A. actinomycetemcomitans could elicit both an inflammatory and an immunohistologic response. The quantitative analysis for both the Histologic Score and the Immunohistologic Score suggested that there was a difference in virulence between the strains that was also dependent on dose (dilution) and time. The results demonstrated a strong trend towards a graded virulence and immunological responses of JP2 >_Y4 >_IDH 1705. iii T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E O F C O N T E N T S iv LIST O F T A B L E S vii LIST O F FIGURES viii A C K N O W L E D G E M E N T S x DEDICATION xi C H A P T E R O N E - L I T E R A T U R E R E V I E W .1.1 PERIODONTAL DISEASE 1 1.1.1 Definition of Periodontal Disease 1 1.1.2 Classification of Periodontal Disease 1 1.1.2.1 Gingivitis 2 1.1.2.2 Periodontitis 5 1.1.3 Categories of Periodontal Disease 6 1.1.3.1 Adult Periodontitis 8 1.1.3.2 Early Onset Periodontitis 8 1.1.3.2.1 Prepubertal Periodontitis 9 1.1.3.2.2 Juvenile Periodontitis 9 1.1.3.2.3 Rapidly Progressive Periodontitis 11 1.1.3.3 Periodontitis Associated with Systemic Disease 11 1.1.3.4 Necrotizing Ulcerative Periodontitis 15 1.1.3.5 Refractory Periodontitis 16 1.1.4 Pathogenesis of Periodontal Disease 17 1.1.4.1 Microbial Challenge 18 1.1.4.2 Microbial Virulence 19 1.1.4.3 Protective Host and Immune Response 20 1.1.4.4 Destructive Immune Response 22 1.1.4.4.1 Tissue and Extracellular Matrix Destruction ....23 1.1.4.4.2 Destruction of Bone 25 1.2 ACTINOBACILLUS ACTINOMYCETEMCOMITANS IN PERIODONTITIS 1.2.1 Biotypes, Serology and Strains 27 1.2.2 Virulence Characteristics 28 1.2.3 Host Susceptibility 32 1.3 ANIMAL MODELS IN PERIODONTAL DISEASE 36 iv 1.4 O B J E C T I V E S O F T H E S T U D Y 40 CHAPTER TWO - MATERIAL AND METHODS 2.1 A N I M A L S 42 2.2 B A C T E R I A 42 2.3 I N J E C T I O N S 43 2.4 T I M E I N T E R V A L S 46 2.5 M U R I N E V I R U L E N C E M O D E L S 46 2.6 H I S T O L O G I C A L P R E P A R A T I O N A N D P R O T O C O L 47 2.7 I M M U N O L O G I C A L M E T H O D S A N D P R E P A R A T I O N 51 2.7.1 Antibodies 51 2.7.2 Staining Method 52 2.8 S T A T I S T I C A L A N A L Y S I S 53 CHAPTER THREE - RESULTS 3.1 M E T H O D O L O G Y F O R T H E E V A L U A T I O N O F H I S T O L O G I C A L A N D I M M U N O H I S T O L O G I C A L S C O R E 54 3.1.1 Histologic Score 54 3.1.2 Immunohistologic Score 61 3.2 C O M P A R I S O N O F S U B C U T A N E O U S I N J E C T I O N S O F E A R A N D A B D O M E N F O R E L I C I T A T I O N O F I N F L A M M A T O R Y R E S P O N S E 67 3.3 D O S E D E P E N D E N C Y O F I N F L A M M A T O R Y R E S P O N S E I N E A R A N D A B D O M E N (JP2) A T D A Y 15 72 3.4 K I N E T I C A N A L Y S I S O F I N F L A M M A T O R Y R E S P O N S E O F T H R E E S T R A I N S O F A. ACTINOMYCETEMCOMITANS 81 3.5 DOSE DEPENDENCY OF INFLAMMATORY RESPONSE OF THREE STRAINS OF A. ACTINOMYCETEMCOMITANS 87 3.6 REPRODUCIBILITY OF THE KINETIC RESPONSE 90 3.7 IMMUNOHISTOLOGICAL ANALYSIS OF THE CELLULAR INFILTRATE MEDIATED BY THREE STRAINS OF A. ACTINOMYCETEMCOMITANS 93 CHAPTER FOUR - DISCUSSION 4.1 INFLAMMATORY RESPONSE OF VIABLE BACTERIA COMPARE TO NON-VIABLE BACTERIA IN MICE 99 4.2 NON-VIABLE BACTERIAL MURINE MODEL COMPARED TO THE MURINE ABSCESS MODEL 101 4.2.1 Histologic Response in the M A M and Non-viable Bacterial Model 102 4.2.1.1 Time/Dose Effects on the Histologic Response 103 4.2.2 Immunohistological Response in the M A M and Non-viable Bacterial Model 105 4.2.2.1 Time Effects on the Immunohistologic Response 105 4.3 STRENGTHS AND WEAKNESSES OF THE A. ACTINOMYCETEMCOMITANS-INDVCED MODEL OF INFLAMMATION 106 CHAPTER FIVE - CONCLUSIONS 109 REFERENCES 110 vi LIST O F T A B L E S T A B L E 1 - Immunohistologic Score of F4/80, Neutrophil and CD3 Antibodies at day 5 and 15 for undilute JP2 95 vii LIST OF FIGURES Figure 1(a) Injection of Mouse Ear 45 1(b) Injection of Mouse Abdomen 45 Figure 2 Histologic Score (magnification XlO and X40) 2(a) Grade 1 - IDH 1705 - day 5 57 2(b) Grade 2 - A T C C 43718 - day 15 58 2(c) Grade 3 - A T C C 43718 - day 5 59 2(d) Grade 4 - JP2 - day 5 60 Figure 3 Immunohistologic Score (magnification XlO and X40) 3(a) Grade 1 - IDH 1705 - CD3 - day 15 63 3(b) Grade 2 - A T C C 43718 - Neutrophil - day 5 64 3(c) Grade 3 - JP2 - Neutrophil - day 15 65 3(d) Grade 4 - A T C C 43718 - F4/80 66 Figure 4(a) Inflammatory Response in Ear - PBS Control and JP2 - day 5 70 4(b) Inflammatory Response in Abdomen - PBS Control and JP2 - day 5 71 Figure 5 Inflammatory Response of Varying Dilutions of JP2 - (Graph) - Ear and Abdomen - day 15 74 Figure 6 Inflammatory Response of Varying Dilutions of JP2 -Ear - day 15 (XlO) 6(a) 0.05 X10 9 bacterial equivalents/ml .76 6(b) 0.5 X l O 9 bacterial equivalents/ml 76 6(c) 1.0 X l O 9 bacterial equivalents/ml 77 6(d) 5.0 X l O 9 bacterial equivalents/ml 78 Figure 7 Inflammatory Response of Varying Dilutions of JP2 - Abdomen - day 15 (XlO) 7(a) 0.05 X l O 9 bacterial equivalents/ml 79 7(b) 0.5 X l O 9 bacterial equivalents/ml 79 7(c) 1.0 X l O 9 bacterial equivalents/ml 80 7(d) 5.0 X l O 9 bacterial equivalents/ml 80 Figure 8(a) Kinetic Response of 3 Undilute Strains - day 5, 15, 25 - Ear 84 8(b) Kinetic Response of 3 Undilute Strains - day 5, 15, 25 - Abdomen 84 8(c) Inflammatory Response of 3 Undilute strains - day 5, 15, 25 - Ear (X40)..85 8(d) Inflammatory response of 3 Undilute strains - day 5, 15, 25 - Abdomen (X40) 86 Figure 9 Inflammatory Response of Varying Dilutions of 3 Strains - day 15 -Abdomen 89 viii Figure 10 Mean Histologic Scores of 3 Strains - (0.5 X l O 9 bacterial equivalents/ml)- day 15 92 Figure 11 Immunohistologic Response of 3 Antibodies -day 5 and 15 (XlO) 11(a) Immunologic Response of Antibody F4/80 for JP2 at day5 and 15 96 11(b) Immunologic Response of Neutrophil Antibody for JP2 at day5 and 15...97 11 (c) Immunologic Response of Antibody C D 3 for JP2 at day 5 and 15 98 ix A C K N O W L E D G E M E N T S To Dr. Doug Waterfield, my thesis supervisor, thank you for your guidance, support, kindness and most of all your patience during the development of my thesis. To the members of my thesis committee, Dr. E . Putnins, Dr. H . Larjava and Dr. J. Uitto, thank you for your insight and constructive participation throughout this project. To Dr. Rendy Yan, thank you for your laboratory expertise and help with the histology and immunology in this study. D E D I C A T I O N This paper is dedicated to the late Dr. John Charles Leishman, D D S (1947-1993), who's professionalism, ethics, and kindness to others has influenced me forever. John, you were my employer, my mentor, and most of all , my friend. Thank you for believing in me. xi CHAPTER ONE - LITERATURE REVIEW 1.1 PERIODONTAL DISEASE 1.1.1 Definition of Periodontal Disease Periodontal diseases have afflicted mankind around the world for centuries. Skulls from ancient civilizations indicate that alveolar bone loss and destructive periodontal disease were a common dental finding. Early writings are replete with descriptions of the disease of the gums as well as primitive remedies and methods of prevention of this disease (Shklar & Carranza, 1996). Periodontitis is an inflammatory condition of the periodontal tissues, involving the periodontal ligament and loss of the bony support around the tooth (Burt & Eklund, 1992; Susuki, 1988; Page & Schroeder, 1976). Genco (1988,1996) refers to periodontal diseases as infections, resulting in periodontal pocket formation, loss of gingival and periodontal connective tissue attachments and loss of tooth-supporting alveolar bone. The term periodontal disease actually describes a group of soft tissue diseases that involve complex bacterial-host cell interactions, with particular forms of the disease becoming more understood (Burt & Eklund, 1992; Meyer & Fives-Taylor, 1997). 1.1.2 Classification of Periodontal Diseases Gingivitis is an inflammatory condition restricted to the gingiva itself (Listgarten, Schifter & Laster, 1985; Suzuki, 1988; Ranney, 1993) whereas the inflammations extending to involve the periodontal ligament, cementum, or alveolar bone are considered Periodontitis (Listgarten, 1986; Suzuki, 1988; Ranney, 1993; Kornman, Page & Tonetti, 1997). 1 1.1.2.1 Gingivitis Gingivitis is caused initially by bacteria that colonize the gingival sulcus and adjacent tooth surfaces (Loe, Theilade, & Jensen, 1965; Theilade, Wright Jensen & Loe, 1966; Lindhe, Hamp & Loe, 1973, 1975). As the disease becomes more severe, there is a more apical extension of the inflammation over time. Although this plaque-associated gingivitis has been shown to be related to bacterial accumulations, the composition of the flora is not very specific (Moore et al, 1987). The severity of plaque- related gingivitis could be aggravated by coexisting systemic conditions resulting in clinical manifestations from the gingival margin to the mucogingival junction. In this case, gingivitis can be attributed to drugs such as calcium channel blockers (nifedipine and oxodipine), anticonvulsants (phenytoin or diphenylhydantoin), or immunosuppressants (cyclosporin). In these cases the clinical findings include diffuse swellings of interdental papillae, or a marginal collar or festooning of the tissue around the clinical crowns of the teeth (Suzuki, 1988). The connective tissue fibroblasts respond with abnormal rates of mitosis when these medications are taken. The amount of associated inflammation is a function of the bacterial plaque accumulations. Gingivitis can also be seen in patients taking oral contraceptives (Pearlman, 1974) or associated with pregnancy (Loe, 1986) and altered hormonal balances, due in part to the effects of progesterone on the micro vasculature of inflamed connective tissue (Hugoson, 1970). Estrogens also play a role in amplifying the clinical inflammatory changes of gingivitis. Steroid hormone therapy use may result in enhanced gingival tissue responses similar to the overall systemic response seen in the reproductive organs and mammary glands (Suzuki, 1988). These clinical signs and symptoms include acute mflammation around one or more plaque retentive areas, with spontaneous bleeding being a common finding. Pyogenic granulomas may be the outcome o f more severe gingival lesions. 2 When severe gingivitis is seen without severe plaque accumulations, especially in children, other systemic factors are suspected such as blood dyscrasias, vitamin deficiencies, leukemia, and granulomatosis. There is also a necrotizing form of gingivitis that presents as ulcerated margins or punched-out papillae, often accompanied by pain, lymphadenopathy, elevated temperature, malaise, malodor and a sloughing grayish white psuedomembrane over the affected gingiva. The extent of these ANUG (acute necrotizing ulcerative gingivitis) lesions may include isolated interproximal areas or may be generalized throughout the entire dentition (Suzuki, 1988). This necrotizing gingivitis has been associated with bacterial invasions of spirochetes, Bacteroides (Porphyromonas or Prevotella), Fusobacterium sp. and other bacteria (Loesche et al, 1982) as well as possible mental or physical stress (Goldhaber, 1964; Stevens et al, 1984), and human immunodeficiency virus (HIV) (Rowlands etal, 1991). Rowlands et al, 1991 suggest that H I V infection should be suspected when the signs of necrotizing ulcerative gingivitis are present. Gingivitis is also associated with dermatological diseases including lichen planus, mucous membrane pemphigoid, pemphigus and other vesiculobullous disorders, including oral manifestations of epidermolysis bullosa and ectodermal dysplasia. Desquamative Gingivitis was once thought to be a nonspecific oral manifestation of a variety of metabolic disturbances (McCarthy, McCarthy & Shklar, 1960; Scopp, 1964; Glickman & Smulow, 1965) based on clinical and histologic findings. The use of electron microscopy and immunofluorescent techniques led to a better understanding of the diseases involved, allowing more accurate diagnosis and treatment (Rogers, Sheridian, & Jordan, 1976; Markopoulos et al, 1996). In an early paper by Weinmann (1941), several theories of progression from gingivitis to periodontitis are discussed. One such theory suggested that inflammation from gingivitis spread into the periodontal membrane (Black G V , 1936; Talbot E S , 1899; Fish E W , 1939) as 3 "the lymphatics within the periodontal membrane form the natural route for the movement of infection.. .from the gingiva through the periodontal ligament to the apex of the tooth."1 Another theory suggested that the inflammation extended into the external surface of the alveolar process and into the bone marrow spaces (Noyes FB, 1927; Coolidge ED, 1931). A third theory proposed that " the path of invasion passes through the gum-corium to the alveolar bone.. .and later, the periodontal membrane."2 Weinmann (1941) showed that inflammation actually followed the blood vessels but there was still great controversy of how gingivitis progressed to periodontitis. Progression was commonly associated with destruction of the connective tissue attachment to the tooth and apical migration of the junctional epithelium beyond the cemento-enamel junction (Hull, Soames & Davies, 1974). Further disease involved bone loss and progressive gingival involvement (Listgarten, 1986). Early studies indicated that human periodontal disease started in childhood and progressed steadily with increasing age (Gad, 1968). In a study of untreated periodontal disease, Becker, Berg and Becker (1979) report that although annual deterioration scores were related to patient age, there was an inverse relationship between pocket depth increase and patient age; as age increased, mean annual deterioration scores decreased. Hirschfield and Wasserman (1978) demonstrated in their study of 420 treated patients, followed from 15-50 years, that bone loss did occur among all patients but to varying degrees and patients were classified as "Well-maintained", "Downhill" or "Extreme Downhill" depending on tooth loss over the period of the study. Animal studies using squirrel monkeys, or beagle dogs, demonstrated that it could take years for progressive soft tissue destruction and eventual bone loss to be apparent (Gad, 1968; Hull, 1 Black GV. Operative Dentistry Vol. 4: 165. Chicago, Medico-Dental Publishing Co., 1936. In Weinmann JP Progression of gingival inflammation into the supporting structures of the teeth. J Periodont. 12(2): 71-81, 1941. 2 James WW and Counsell A. Brit Dent J. 48: 1237, 1927. In Weinmann JP. Progression of gingival inflammation into the supporting structures of the teeth. J Periodont. 12(2): 71-81, 1941. 4 Soames, Davies 1974; Soames, Entwisle & Davies, 1976; Heijk, Rifkin, Zander, Schei, 1976), confirming human findings. Lindhe, Hamp and Loe (1975) demonstrated in their beagle dog study of plaque-induced periodontal disease that as was found in humans, not all gingivitis progressed to periodontal disease (Anerud A , Loe H , Boysen H & Smith M , 1979; Listgarten, Schifter & Laster, 1985). Two out of 20 dogs in the study had no roentgenographical signs of alveolar bone loss. 1.1.2.2 Periodontitis: Progression The clinical progression from gingivitis to periodontitis reflects a histopathologic progression from an established lesion to a more advanced stage of the disease. Three plausible hypotheses for this occurrence have been advanced: 1. Bacterial tissue destruction (plaque); 2.Hyperresponsiveness of the immune system resulting in immune complexes, lymphocyte blastogenesis and/or activation of the complement pathways; 3. Ffyporesponsiveness or immune deficiencies involving neutrophil functions (such as chemotaxis, phagocytosis), neutropenia, or autologous mixed lymphocyte response (Suzuki, 1988). Page and Schroeder (1976) considered four stages of inflammatory periodontitis: the Initial, Early, Established and Advanced stage(s) based on the histologic presentation of the lesions. The first three categories are descriptions of gingivitis, and the fourth stage describes the more advanced periodontal lesion. The Initial lesion, which appears clinically normal, occurs within 2-4 days, and is characterized by vascular dilation and increased permeability to fluid and lymphocytic cells. There is 5 increased P M N extravasation and migration, alterations of the junctional epithelium and loss of perivascular collagen. The Early lesion, occurring approximately 4-7 days later and up to 21 days, is identified by a lymphocyte-rich infiltrate adjacent to the junctional epithelium, with increased collagen loss. At this stage there are cytotoxic effects on the fibroblasts and the lesion may be subclinical or show clinical signs of gingivitis. The Established lesion is a further extension of the early lesion with plasma cells rjecoming the dominant cell o f the inflammatory infiltrate. There is further collagen loss, junctional epithelial cell proliferation and pocket formation. Lymphocytes can be seen in the connective tissues -particularly B cells. This stage is protective and reversible and/or pockets may persist for years with no further destruction. The Advanced lesion has all the signs and symptoms of chronic gingivitis plus attachment loss, bone destruction, pocket formation and apical migration of the junctional epithelium. There may be fibrosis of bone marrow and further collagen loss. P M N s and B-cells continue to infiltrate the lesion that is characterized by periods of quiescence, but with the signs and symptoms of chronic inflammation and bursts of activity resulting in further attachment loss. 1.1.3 Categories of Periodontal Disease Various forms of periodontitis have been described based primarily on clinical observations and basic scientific research over the years. The term "pyorrhea alveolaris" 3 was used by F . H . Rehwinkl (1823) who was associated with the Baltimore College of Dental Surgery and brought 6 the term from Germany when he emigrated to the United States. Later that century, John W. Riggs also of Baltimore College of Dental Surgery, became the leading authority on periodontal disease and alveolar pyorrhea became later known as "Riggs Disease". 4 Merritt (1931), Black (1936), and Fish (1939) described periodontitis as "chronic suppurative pericementitis" 5 . Other synonyms not in current use included "schmutz pyorrhea"(Gottlieb), "paradentitis" (Weski, Beck), "paradentosis", "periodontoclasia", "pericementitis", "parodontitis", and "alveoclasia" 6 The clinical diagnosis of progressing periodontitis was described in early works of Schei (1949), Belting, Massler and Schour (1953), and Marshall-Day and Shourie (1949). The loss of bone was considered a "cardinal symptom" of periodontal disease, as was the resorption of the alveolar crest evaluated roentgenologically. Marshall-Day and Shourie (1949) provided an early system of measuring bone loss radiographically, using percentages of maximum bone height measured with the aid of a specially developed translucent ruler. There are several accepted classifications of periodontal disease. A l l include the recognition of several types of periodontal disease "that have differences among them in etiology, natural history, progression and response to therapy".7 The following descriptions are based on the disease categories established by the World Workshop in Clinical Periodontics, 1989. 3 CarranzaF.A. and Newman M.G. Clinical Periodontology, 8th edition: 6. W.B. Saunders Company, 1996. 4 Carranza F.A. and Newman M.G. Clinical Periodontology, 8th edition: 6. W.B. Saunders Company, 1996. 5 Black G.V. Operative Dentistry 4, Chicago, Medico-Dental Publishing Co., 1936. In Weinmann JP. Progression of Gingival Inflammation into the supporting structures of the teeth. J. Periodont. 12(2): 71, 1941. 6 Carranza F.A and Newman M.G. Clinical Periodontology, 8th edition: 59. W.B. Saunders Company, 1996. 7 1.1.3.1 Adult Periodontitis Adult periodontitis is the most prevalent form of periodontitis clinically recognized after the age of 35. Adult periodontitis has a slow rate of progression (Listgarten M A , 1986), either occurring over time in random bursts of disease activity (Goodson et al, 1982) or, as proposed by Jeffcoat & Reddy (1991), in a continuous, slow progression. The bacteria associated with adult periodontitis include P. gingivalis, P. intermedia, P. micros, Fusobacterium species, B. forsythus, C. rectus, T. denticola, A. actinomycetemcomitans, Capnocytophaga, black-pigmented Bacteroides, spirochetes, and Staphyloccoccus species. There is however, no consistent significant associations of given species with episodes of progression (Zambon JJ, 1996). 1.1.3.2 Early-Onset Periodontitis When severe periodontal disease occurs in very young patients (preteens), teenagers and young adults to age 35, it is considered to be early-onset periodontitis. The American Academy of Periodontology (1989) divides early-onset periodontitis into Prepubertal Periodontitis, Juvenile Periodontitis, and Rapidly Progressive Periodontitis. Prepubertal and Juvenile Periodontitis are further subdivided into localized (LJP) and generalized (GJP) forms of the disease based on intraoral distribution of affected sites and severity. 7 Ranney RR. Classification of periodontal diseases. Periodontology 2000. 2: 16, 1993. 8 1.1.3.2.1 Prepubertal Periodontitis The age of onset of prepubertal periodontitis is 4 years or earlier although it may not be detected until age 7 to 9. It can also be divided into a localized and generalized form. In the localized form, many or only a few primary teeth are affected. The gingival tissues are only slightly inflamed and microbial plaque, i f present, is minimal. Deep pocket formation with destruction of alveolar bone is more rapid than that found in adult periodontitis, but slower than the generalized form o f prepubertal periodontitis. The generalized form o f the disease demonstrates fiery red inflammation of the marginal and attached gingiva combined with gingival proliferation, cleft formation and recession. Alveolar bone as well as tooth roots may resorb, at an extremely rapid rate. The disease can begin with tooth eruption and all primary teeth may be lost by age 2 or 3 years old. In both the localized and the generalized form of prepubertal periodontitis, otitis media and upper respiratory tract infections may be present. Another trait in common for both forms of the disease is that these children appear to have defective peripheral blood leukocyte function, either neutrophil or monocyte chemotaxis and adherence (Genco et al., 1986; Van Dyke & Hoop, 1990), phagocytosis or both (Altman et al, 1980; Bowen et al., 1982). Prepubertal periodontitis may be followed by a normal permanent dentition or by severe periodontitis in the permanent teeth (Page etal., 1983). 1.1.3.2.2 Juvenile Periodontitis This disease affects teenagers and young adults who are otherwise healthy (Schenkein & Van Dyke, 1994), and is also divided into a localized and generalized form. 9 Localized Juvenile Periodontitis is typically limited to severe loss of alveolar bone around the first molars and incisors, although involvement of 1-2 teeth other than first molars or incisors is allowed within the disease definition (Burmeister et al, 1984; Van Dyke et al, 1980). This pattern of disease can persist into the mid-to late twenties or later in many of the cases (Ranny, 1991). These patients rarely demonstrate accumulations of plaque or calculus despite the severity of the disease (Baer, 1971; Waerhaug, 1977). The microbial flora associated with localized juvenile periodontitis (LJP) lesions are predominantly anaerobic and capnophilic rods (Slots, 1976). A. actinomycetemcomitans is the bacteria most strongly associated with L J P (Socransky 1977; Slots 1979; Slots & Dahlen 1979, 1984; Christersson etal 1983, 1985; Zambon 1985), along with some Capnocytophaga strains (Gjermo, 1986). A high percentage of JP patients (77%) demonstrate decreased neutrophil chemotactic response and depressed phagocytosis (Astemborski et al, 1989). Generalized Juvenile Periodontitis (GJP) is found in older juveniles and young adults (Page, 1983), with the disease often involving the entire dentition. These patients differ from L J P patients in that they usually display generalized severe inflammation and heavy accumulations of plaque and calculus (Page, 1983). Astemborski et al (1989) described Form A type GJP, characterized by minimal plaque and an age range from 14 to 26 years old, involving females more than males, and having a decreased neutrophil chemotactic defect. Form B type GJP was characterized as having significant plaque accumulations and an age range from ages 26-35, of undetermined sex distribution with normal or decreased neutrophil chemotactic response (Astemborski et al, 1989). The microbiota associated with GJP includes high percentages of nonmotile, facultative anaerobic, Gram-negative rods such as Porphyromonas gingivalis (Slots, 1982; Wilson, Zambon Suzuki & Genco, 1985), along with Prevotella intermedia, Capnocytophaga, Eikonella corrodens and Neisseria are common isolates in GJP. A. 10 actinomycetemcomitans is present but in low numbers (Newman & Nisengard, 1988). 1.1.3.2.3 Rapidly Progressive Periodontitis Rapidly progressive periodontitis is a disease form characterized by relatively rapid loss of clinical attachment and alveolar bone in young adults. The early stages of R P P may involve localized moderate to deep periodontal pockets and early bone loss. However, the later stages of RPP, which can take only a few months to develop, demonstrate generalized moderate to deep pocket formation, and severe loss of attachment and bone (Suzuki, 1988). Page (1983) described the disease as rapid, with periods of acute, often painful tissue inflammation displaying hemorrhage and exudate. Mobil i ty and tooth loss were seen in the later stages. Suzuki (1988) described another type of RPP (Type B) occurring in older patients, 26-35 years of age, with moderate to heavy plaque deposits. There is, however, little clinical significance of Type B as it may be confused with early adult periodontitis or GJP. Neutrophil chemotaxis is depressed in approximately 66% of patients (Levine, 1997; Van Dyke, 1982), as is the autogenous mixed lymphocyte reaction ( A M L R ) . This may account for the increased rate of pathogenesis with R P P (Suzuki, 1988). 1.1.3.3 Periodontitis associated with systemic disease Periodontitis and tooth loss has been associated with several systemic diseases (Suzuki, 1988; Watanabe, 1989; Ranny, 1993). These include Leukocyte Adhesion Deficiency ( L A D ) , Hypophosphatasia, Papillon-Lefevere Syndrome, Neutropenia, Leukemias, Chediak-Higashi 11 Syndrome, A I D S , Insulin-dependent Diabetes Mellitus, Down's Syndrome, and Ehlers-Danlos Syndrome (TypeVl 11). Pre-pubertal periodontitis has been associated with most of these systemic disorders. Leukocyte Adhesion Deficiency is characterized by severe acute inflammation, rapid bone destruction and proliferation of the gingival tissues (Waldrop et al., 1987; Ranney, 1993). These patients can display other disorders such as respiratory tract infections or otitis media, septicemia, and impaired pus formation (Meyle, 1994; Carranza & Newman, 1996). Page et al. (1983, 1987), noted that patients with L A D had abnormalities of leukocyte chemotaxis, or function. They also contributed to the severity and rapidity of the periodontal progression in children with prepubertal periodontitis to the abnormally low levels of adherence glycoproteins on leukocyte cells, not seen in JP, R P P or A P (Page et al, 1987). Waldrop et al. in that same year published data that demonstrated, using monoclonal antibodies against Mac-1, L F A - 1 (leukocyte function antigen-1), and p l50 , 95 adhesion molecules, that generalized prepubertal periodontitis was an oral manifestation of L A D . Hypophosphatasia is a rare skeletal disease seen in patients with a quantitative deficiency of alkaline phosphatase, resulting in a failure to develop a periodontal ligament due to a disturbance in cementogenesis. Other clinical manifestations include rickets, poor cranial bone formation, craneostenosis and premature loss of teeth (Carranza & Newman, 1996). Leukemia can result in severe periodontal changes such as the infiltration of the gingiva and alveolar bone with leukemia cells, resulting in leukemic gingival enlargements. The gingiva appears red or bluish, cyanotic, rounded, and swollen, and bleeding can be spontaneous. 12 Neutropenia, characterized by a reduction in the number of circulating granulocytes, results in severe inflammations such as necrotizing ulcerative gingivitis, gingival hemorrhage, and periodontal destruction. , Chediak-Higashi Syndrome is a rare disease that effects the production of organelles in almost every cell, producing mild bleeding disorders and recurrent bacterial infections (Carranza & Newman, 1996). Functional neutrophil defects include decreased chemotaxis, degranulation, and microbicidal activity. Oral manifestations include severe periodontitis and ulcerations. Papillon-Lefevre Syndrome is characterized by rapid alveolar bone loss of both the primary and permanent dentitions. Neutrophils have decreased chemotaxis and there is a possible increase in circulating natural killer cells (Carranza & Newman, 1996). The syndrome includes hyperkeratosis of the palms and soles of the feet, possible calcification of the cerebrum tentorium and choroid plexus, increased susceptibility to infections, hyperkeratotic plaques on cheeks, eyelids, labial commissures, legs, thighs, knees and elbows, as well as severe periodontal disease (Hart & Shapira, 1994). Ehlers-Danlos syndrome is an uncommon disorder, clinically characterized by joint hypermobility and skin hyperextensibility due to a decreased amount of collagen hydroxylysine, and/or a failure of procollagen conversion to collagen. There are eight variants of this syndrome; the periodontal form (EDS type VIII) is characterized by rapidly progressing periodontal disease resulting in complete tooth loss by early adulthood (Regezi & Sciubba, 1993). Dental findings can include deep anatomical grooves and excessive cuspal height of the molars and premolars, stunted or dilacerated roots, irregular dentine and enamel hypoplasia. Severe prepubertal and juvenile periodontitis seen in these patients usually leads to tooth loss. 13 Diabetes Mellitis patients have been shown to have a significantly higher incidence of gingivitis and a greater loss of attachment than non-diabetics (Belting et al., 1964; Cohen et al., 1970; Cianciola et al., 1982; Rylander et al., 1987; Hugoson et al., 1989). Neutrophils from diabetic patients with severe periodontal disease have a lower chemotactic response than neutrophils from diabetic individuals with mild periodontal disease or healthy patients with mild or severe disease. Insulin used to treat diabetes has a marked effect on both glucose and lipid metabolism, which can impair phagocytosis and kil l ing by P M N s . Impaired wound healing appears to be the result of abnormalities of collagen cross-linking and reduced collagen synthesis (Salvi et al., 1997). Down's Syndrome (Trisomy 21) patients have an increased prevalence of infections and periodontal disease compared with normal age-matched individuals or other mentally handicapped patients. The oral manifestations of Down's syndrome (DS) patients are consistent with those of juvenile periodontitis, characterized by onset at a young age, a rapid rate of disease progression and destruction of periodontal tissues (Cichon, Crawford &Gr imm, 1997). P M N s and monocytes have demonstrated reduced chemotaxis, diminished phagocytosis, and abnormal bactericidal activity (Barkin et al, 1980; Izumi et al, 1989). Immunological defects such as deficient T-cell function may contribute to the severe disease progression. It is suggested that the T-cells have a diminished ability to recognize and respond to specific antigens, and do not express cell-surface T-cell receptor (Mehta et al, 1993). A n increased frequency of A. actinomycetemcomitans, P. gingivalis, Fusobacterium species, P. intermedium, B.forsythus and P. micros has been associated with periodontitis in Down's syndrome patients (Meskin, Farsht & Anderson, 1968; Barr-Agholme et al., 1992; Cichon, Crawford &Grimm, 1997). The outcome of the pathogenic microflora, immune deficiencies and 14 poor oral hygiene result in the severe periodontal destruction seen in Down's syndrome patients. AIDS - related periodontal findings range from an intensely erythematous marginal gingivitis, a severe form of acute necrotizing ulcerative gingivitis, a severe localized periodontitis, to a destructive necrotizing infection of the gingiva, spreading to the surrounding tissues and alveolar bone (Murray, 1994). C D 4 : C D 8 ratios for HIV-associated gingivitis are in the range of 0.9-1.7, whereas HIV-associated periodontitis is in the range of 0.1-0.9. The absolute CD4+ T-cell counts in H I V periodontitis is also lower than healthy patients or HIV-associated gingivitis patients. A complex host-parasite relationship as well as a compromised immune response accounts for the increase risk of severe and aggressive periodontal disease in infected patients (Murray, 1994). 1.1.3.4 Necrotizing Ulcerative Periodontitis HIV-associated peridontitis (HIV-P) comprises a wide range of conditions including conventional adult periodontitis (Friedman, et al., 1991), rapidly progressive periodontitis (Reichart et al, 1987), and periodontitis with soft tissue loss and irregular bone destruction in an otherwise healthy mouth (EEC-Clearinghouse & W H O , 1993). H I V - P also includes H I V -associated gingivitis with necrosis of gingival/periodontal attachment apparatus and rare spontaneous resolution (Winkler & Robertson, 1992). In the initial stage of acute necrotizing ulcerative periodontitis ( A N U P ) , the clinical manifestations are changes in gingival contour, such as interproximal necrosis, ulceration and cratering. A fetid odor is present in most cases. Severe deep pain, localized in the jawbone, is considered an important feature of HIV-associated A N U P . Another prominent feature of the disease is bleeding on probing and spontaneous bleeding of the involved sites. One of the most distinguishing features is soft tissue necrosis and 15 rapid destruction of periodontal attachment and alveolar bone (Winkler JR et ai, 1990). The disease usually affects several localized areas independently, but severe cases can affect all teeth. Dire to the extensive gingival necrosis that often coincides with loss of crestal alveolar bone, deep periodontal pocketing is not usual, but exposed bone may become sequestrated, creating craters (Holmstrup & Westergaard, 1994). Immunological studies have shown complete absence of T-cells in the gingival tissues of HIV-infected individuals with periodontitis, suggesting that the impairment of local immune defenses could explain the rapidly progressive nature of periodontitis in these patients (Steidley et ai). 1.1.3.5 Refractory Periodontitis Patients referred to as having refractory periodontitis are those patients who seemingly fail to respond predictably to conventional therapy of scaling and root planing, flap surgery and/or antibiotics (Caton, 1989). Treated sites continue to lose attachment in a rapid manner despite the type or frequency of treatment applied (Hirschfeld & Wasserman, 1978; McFall, 1982; Haffajee, 1985). Hernichel-Gorbach et al. (1994) conceded that there was no real clear biological profile of these patients and no consistent microbial or host response patterns could be detected. The term "refractory" then is only defined in terms of therapy employed and the rate of progression of the disease over time (Haffajee et al, 1988). There does not appear to be a significant difference in the microbiology in patients with refractory periodontitis, but higher levels of B. forsythus, F. nucleatum, S. intermedius, E. corrodens and B. gingivalis were found in those subjects who responded poorly to therapy (Haffejee et al., 1988). Magnusson et al. (1991) found a higher frequency of large and small 16 motile rods in active sites, but no significant differences in the level of any other species. They in fact found very little plaque in these patients and no difference in the amount of plaque in sites gaining or losing attachment. The losing sites demonstrated a higher frequency of bleeding and suppuration than those gaining periodontal attachment. Current studies in periodontal disease have suggested that the disease process may reflect the immunoregulation that dictates the balance between protective and destructive aspects of the host response to bacteria (Seymour, 1991, 1993, Ranny, 1991; Gemmell & Seymour, 1994, Hernichel-Gorbach, 1994). In their 1994 study of patients with refractory periodontitis, Hernichel- Gorbach et al. found that there was a greater release of I L - i p and PGE2&om monocytes in patients with gingivitis. They also note a decreased C D 4 / C D 8 T-cell ratio. This recent evidence suggests that refractory patients demonstrate differences in their host immune response that could influence treatment outcomes, although more research is necessary. 1.1.4 PATHOGENESIS OF PERIODONTAL DISEASE The study of the pathogenesis of periodontal disease is one of immense diversity, involving the role of microorganisms, host responses, immunology, environmental risk factors, and genetics. Extensive work has been done in these areas and this paper w i l l briefly outline areas of research, rather than attempt to discuss any particular aspect or contributing factor to pathogenesis in detail. For comprehensive information, refer to the works of Page & Schroeder, 1976; Page, 1991; Genco, 1992; Tonetti, 1993; Offenbacher, 1996; Page & Kornman, 1997; Fives-Taylor et ai, 1999. 17 1.1.4.1 M i c r o b i a l Challenge Bacteria can attach to an acquired pellicle formed from proteins and glycoproteins in the saliva and crevicular fluid. Coaggregation (attachment of one bacteria to another based on metabolic needs) facilitates the specific interactions that increase the number and species of bacteria found in the maturing dental plaque (Whittaker et ai, 1996). Streptococcus and Fusobacterium are two early colonizers and play a major role in plaque biofilm formation. This microbial biofilm provides an ecological habitat, with a primitive circulation system for the provision of nutrients, oxygen and the passage of metabolic wastes needed for the survival of the varied species of bacteria found in dental plaque (Costerton et al, 1994, 1995). In gingival health, the bacterial load is quite low (10 2-10 3 isolates), being predominantly gram-positive Streptococci and Actinomyces (Syed & Loesche, 1978; Tanner et al, 1997). Experimental gingivitis studies have documented the role of increased bacterial load (10 4 — 10 6 isolates) and decreased oral hygiene in the development of gingivitis (Theilade, et al, 1966; Syed & Loesche, 1978; Van Dyke et al, 1999). As well as an increased bacterial load (>108 isolates), multiple bacterial complexes (species) are implicated in the development of periodontitis (Socransky, Haffajee & Dzink, 1988; Haffajee & Socransky, 1994). In health, the dental plaque is characterized by predominantly gram-positive coccoid microbiota. The gingivitis-associated microbiota is characterized by a relative increase in the numbers and proportion of gram-negative bacteria, usually motile rods and filaments. In adult periodontitis, the microbiota consists of predominantly gram-negative microorganisms coaggregating in "test-tube brush" formations (Listgarten, 1994). Although the plaque of juvenile periodontitis is less abundant, there is an increase in the numbers of A. actinomycetemcomitans, and gram-negative 18 fusiform bacteria resembling Capnocytophaga (Berthold & Listgarten, 1986). In deep subgingival sites, anaerobic, gram-negative bacteria ultimately dominate the plaque microbiota. The 1996 World Workshop on Clinical Periodontics has determined that "most human periodontitis is caused by "Porphyromonas gingivalis, Bacteroides forsythus and A. actinomycetemcomitans" 8 . Epidemiological studies have shown, however, that the presence of these specific microbes is not necessarily predictive of disease (Beck et ai, 1990, 1992; Christersson et al, 1992; Wheeler et al, 1994). Although periodontitis, like any infectious disease, requires the presence of an etiological agent (bacteria), the bacteria act in concert with many other host and environmental factors, such as smoking and immune deficiencies which can alter the disease prevalence and predictability. 1.1.4.2 M i c r o b i a l Virulence Microbial virulence properties enable organisms to successfully colonize and compete with other bacteria for nutrients, and confer the ability to evade the host defenses. In general, virulence traits include the expression of fimbriae (Holt etal, 1980; Fives-Taylor, Meyer, Mintz, 1995), proteases, leukotoxin capable of killing the host leukocytes (Baehmi et al, 1979; Ohta et al, 1996), endotoxins such as lipopolysaccharide (LPS) (Fives-Taylor, Meyer, & Mintz, 1996), hemolysins (Kimizuka, 1996), H2S, (Holt, etal, 1999), and the production of short-chain fatty acids toxic to neutrophils (Tsai et al, 1984; Taichman et al, 1987). 8 Page RC & Kornman KS. The pathogenesis of human periodontitis: an introduction. Periodont. 2000. 14: 9, 1997. 19 If the organism does not possess all of the necessary genetic elements to evade the host defenses, a periodontally healthy site may be colonized with a pathogen that is unable to induce tissue destruction. As mentioned earlier, a minimum threshold number of a particular microorganism is likely needed to initiate disease. There have been major advances in the development and use of various procedures such as polymerase chain reaction and other DNA-based technologies to demonstrate that most species of peridontopathic bacteria such as P. gingivalis and A. actinomycetemcomitans involve a large number of genetically distinct clonal types (Zambon et al, 1996; Darveau et al, 1997). Recent research has shown that virulence is not equal in all clonal types of a pathogenic species (Van Steenbergen, 1987; Grenier & Mayrand, 1987; Reddy et al, 1995; Spitznagel et al, 1995) allowing some clonotypes or altered pathogens to be avirulent. 1.1.4.3 Protective Host and Immune Responses Gram-negative bacteria release outer membrane vesicles containing lipopolysaccharide (LPS), lipid and protein, which can have direct effects on host cells to produce inflammatory mediators such as cytokines, chemokines or cellular adhesion molecules (Reddi et al, 1995). Indirect effects include the activation of neutrophils or monocytes by bacteria to produce cytokines such as IL-1 p, which activate fibroblasts, endothelial or epithelial cells to elicit inflammatory mediators such as matrix metalloproteinase (MMP), prostaglandin E2, and tissue necrosis factor (Page, 1991; Darveau, Tanner & Page, 1997). This bacterial shedding is important for the host to respond to the bacterial challenge, allowing the host to resist colonization of the bacteria. 20 The bacterial challenge induces early changes in the junctional epithelium to facilitate vascular permeability and an influx of neutrophils. IL-8, a chemotactic cytokine, is found to be present in gingival tissue (junctional epithelium) and may play a role in directing neutrophils to the gingival sulcus area (Kornman, Page & Tonetti, 1997). The cell turnover rate of the sulcular epithelium increases, with the greatest number of infiltrating cells found in the coronal junctional epithelium. There is an increase in vascular permeability, leukocyte cell adhesion molecules and a release of specific leukocyte- activating agents in early inflammation. The connective tissue is infiltrated when there is increased leakage of plasma components, including acute-phase proteins (a2-macroglobulin) and leukocyte extravasation into the crevicular fluid. Neutrophils exit the inflamed venules, and migrate along the gradient of chemoattractants (intracellular attachment molecule 1 and chemokines) to the junctional epithelium to form the first line of defense. With a change from health to infection there is an alteration in host inflammatory mediators (arachidonic acid metabolites), neutrophil enzymatic activities, and tissue degradation products. The bacterial products and epithelial derived cytokines also activate the local tissue mononuclear cells that influence the local immune response. In diseased sites, mononuclear cells dominate the vascular exudate. Small lymphocytes consisting of T and B cells predonunate in the tissue infiltrate, which continue to enlarge and replicate in the presence of antigen and various cytokines. Macrophages are also activated by the bacterial challenge to secrete cytokines and express cell surface receptors that influence the antigen-specific immune response to the bacteria. These cytokines include interferon y, tumor necrosis factora transforming growth factor p\ I L - l a a n d p\ IL-6, IL-10, IL-12, IL-15. Chemoattractant cytokines (chemokines) such as M C P (monocyte chemoattractant protein), M I P (macrophage inflammation protein) and R A N T E S (regulated on activation, normal T-cell expressed and secreted) are also secreted as are the matrix metalloproteinases and prostaglandin 21 E 2 (Kornman et al, 1997). Thus, these activated macrophages can act as chemoattractants to bring additional monocytes and lymphocytes to the area. Inflammatory mediators are found in severe periodontitis at elevated concentrations, causing complement activation (the products of which are chemotactic for neutrophils), production of matrix metalloproteinases, increased prostaglandin production, upregulation of Fc receptors on neutrophils and monocytes, upregulation of the major histocompatability complex expressed by B and T cells to increase clonal expansion and immunoglobulin production (Kornman et al, 1997). Prostaglandins and leukotrienes are also potent chemoattractants for neutrophils, causing vasodilation. It is by these host-parasite interactions that the host tissues are protected by the neutrophil activity in the sulcus, and antibodies that are produced both locally and systemically. 1.1.4.4 Destructive Immune Responses Macrophages and neutrophils are important for the defense of the host against bacterial invasion. Neutrophils respond to chemoattractants to form a "wal l " separating the advancing plaque front from the sulcular epithelium and are critical in minimizing the destructive effects of the bacteria. With increased accumulations of bacteria and inflammation, a high concentration of chemotactic factors and cytokines occurs in the connective tissue. The connective tissue then becomes exposed to neutrophil degranulation and the releasing of neutrophilic enzymes, including collagenase, potent proteases, prostaglandins (bone resorption) and other proinflammatory agents, causing tissue destruction and furthers inflammation. Abnormalities of neutrophil function or numbers can alter host defenses and are seen in such disorders as agranulocytopenia, cyclic neutropenia, leukocyte adhesion deficiency syndrome, chronic granulomatous disease, 22 Chediak-Higashi syndrome, and early onset periodontitis, all of which involve severe periodontitis (Dennison & Van Dyke, 1997). Activated macrophages also create an environment that favors collagen degradation, and can cause C D 4 + T-lymphocytes to differentiate to cytokine producing T-cells that are capable of initiating B-cel l differentiation and antibody production (Ebersole et al., 1993). However, not all antibodies are protective. B cells have been shown to produce IL-1, which has a potent effect on tissue destruction and bone loss (Birkedal-Hansen, 1993) and which can stimulate fibroblasts to produce collagenases (Richards & Rutherford, 1988 in Gemmell et al, 1997). Destructive periodontitis then is likely due in part to the manifestation of a hypersensitivity-type reaction with the elicitation of tissue-destructive mediators from the host cells (Ebersole & Taubman, 1994). Under continued bacterial challenge, cytokines may not be degraded, resulting in diminished clearance and causing host tissue destruction. With a continued bacterial challenge, some pathogens can produce molecules which mimic host cytokines and can trigger an abnormal inflammatory or immunolmodulatory response (Offenbacher, 1996). Thus, the length of time the immune response continues to be activated may contribute to its destructive effects. 1.1.4.4.1 Tissue and Extracellular Matrix Destruction Connective tissue remodeling is a normal event in growth and development and is tightly regulated by a complex interplay of cell-cell and cell-matrix interactions, involving the 23 production of enzymes, activators, inhibitors, and regulatory molecules such as cytokines and growth factors (Meikle, Heath & Reynolds, 1986). In disease, bacteria, polymorphonuclear leukocytes, macrophages and host cells, release proteinases. Everts et al. (1996) have suggested that when there is a high turnover o f matrix in inflammation, the metalloproteinases are important in extracellular degradation. They have also suggested that an intracellular pathway for resorption, particularly for collagenases, exists when turnover is low. Matrix metalloproteinases (MMPs) , synthesized by macrophages and many cells of the periodontal tissues, are induced by cytokines and prostaglandins to initiate degradation of periodontal ligament and bone resorption. M M P s such as stomelysin and collagenase, and serine proteases such as urokinase-type plasminogen activator, when induced by IL-1 , can digest all the macromolecules of tissue matrices (Leizer et al., 1987). Collagenases ( M M P - 1 , -8, -13) degrade interstitial collagens type / , / / , / / / . Proteoglycanase (stromelysins, M M P - 3 , -10, -11 and the membrane-bound group, M M P - 1 4 , -15, -16, -17) degrade Type I V collagen and a number of matrix glycoproteins such as fibronectin, osteonectin and laminin (Murphy et al, 1981; Birkedal-Hansen, 1993; Galloway et al, 1983). Gelatinases ( M M P - 2 , -9), can degrade Types I V and V collagens as well as denature interstitial collagens (Murphy et al, 1981). Recent studies have demonstrated the importance of these metalloproteinases and other inflammatory mediators (IL-1,11-1(3 and tumor necrosis factor a) in tissue destruction (Page, 1991; Alexander & Damoulis, 1994). In inflammation, arachidonic acid products (thromboxane A2 , prostaglandins, prostacyclins), complement and other plasma proteases can perpetuate and amplify the process. IL-1 can be 24 synthesized by macrophages, blood platelets, fibroblasts, keratinocytes and endothelial cells. Besides its chemoattractant and stimulating effects on neutrophils and monocytes, IL-1 can induce the PGE2 response by macrophages and gingival fibroblasts, with resulting effects on increased bone resorption. Together, these mediators of inflammation can synergistically digest all of the major structural elements of the connective tissue in states of inflammation or disease (Meikle, et al, 1986). 1.1.4.4.2 Destruction of Bone Two major cell types exist in bone; the osteoblast (osteocyte when mineralized) is responsible for synthesizing the organic matrix components and directing mineralization and the osteoclast, which is responsible for demineralization and resorption of the organic matrix of bone. These cells act together in a process known as "coupling" to remodel and maintain bone through life. Remodeling, the process of bone resorption and formation is homeostatically balanced in normal healthy adults (Schwartz et al, 1997). During resorption, osteoclasts release local factors (osteotropic cytokines) from the bone that stimulate further osteoclast formation (resorption) and cause inhibition of osteoblasts (bone formation). In inflammation leukocytes, mesenchymal cells, osteoclasts and gingival fibroblasts release P G E 2 (El Attar & Lin, 1981; Offenbacher, Heasman, Collins, 1993). In low doses, PGE 2can 25 stimulate bone formation, but in the high doses found in inflammation, bone resporption is enhanced (Dewhirst et al., 1983). Osteotropic cytokines such as EL-1, IL-6, tumor necrosis factor (TNF), lymphotoxin and hormones (parathyroid hormone, l,25-(OH)2D3, calcitonin, estrogens and androgens) can have an additive effect on bone resorption (Dewhirst et ai, 1987). IL-1 a, I L - i p are potent stimulators of bone resorption, by stimulating PGE2 release and acting directly on osteoclasts. IL-6 production is increased in response to hormones and cytokines by osteoblasts (Bellido et al., 1995) and is responsible for the formation of an osteoclastic phenotype (Bertolini et al, 1994). Tumor necrosis factor acts by inhibiting osteoblast phenotypes, differentiation and function and stimulates osteoblastic bone resorption. Sex hormones have also been reported to have effects on osteoclastic bone resorption (Bellido et al, 1995). 26 1.2 A. ACTINOMYCETEMCOMITANS AND ITS ASSOCIATION TO PERIODONTAL DISEASE 1.2.1 Biotypes, Serology and Strains A. actinomycetemcomitans bacteria are anaerobic, non-motile coccobacilli (round ended rods) measuring approximately 0.7 ±0.1 by 1.0 ±_0A um. They are found singly, in pairs or in clumps. When grown on selective media they form smooth, circular, convex and translucent colonies with a star-shaped internal morphology. A. actinomycetemcomitans has been subgrouped into 3-10 biochemical groupings known as biotypes based on the variable fermentation of such sugars as dextran, galactose, maltose, mannitol, and xylose (Slots et ai, 1980; Pulverer & K o , 1970). Specific serotypes and bacterial antigens have been identified using agglutination assays. Pulverer & K o (1972) identified 1-6 agglutinating antigens on each strain. These antigens are large molecular weight, heat-stable carbohydrates, which are easily detected by immunofluorescent techniques (Zambon et ai, 1983). Initially three serotypes of oral A.a (serotype a, b, c) were reported by Zambon et al. (1983), while serotypes d and e were described later (Asikainen et al., 1991; Saarela et al., 1992; Gmur et al., 1993). Approximately 3-8% of clinical isolates are non-serotypeable. Serotype c is reported in healthy individuals but serotype b is predominantly associated with localized juvenile periodontitis (Zambon et al., 1983; Asikainen et al, 1991). Within the five serotypes there are major genetic differences, resulting in strains of varying virulence capabilities, particularly as it relates to the leukotoxins that are produced (DiRienzo & 27 M c K a y , 1994; Poulsen et al, 1994, Haubek et al, 1995). Serotype b strains were detected more frequently in U S and Finnish L J P patients than serotype a and c strains (Zambon et al, 1983; Asikainen et al, 1991). Serotype b strain JP2 is thought to be particularly virulent, and found in younger, more disease active L J P patients (Tsai & Tachman, 1986). Leukotoxins are one of the major virulence factors contributing to the pathogenic potential of A, actinomycetemcomitans. 1.2.2 Virulence Factors of A . actinomycetemcomitans The virulence factors of bacteria enable them to colonize and overcome the host defenses and initiate disease. For a comprehensive review, refer to Fives-Taylor et al, Periodont. 2000 20: 136-167, 1999. Fimbriae of A. actinomycetemcomitans (cell surface appendages) have been considered important for colonization and adhesion to epithelial cells, fibroblasts and salivary hydroxyapatite (Rosan et al, 1988). Evidence has shown however that non-fimbriated strain of Aa. also exhibits the ability to colonize and adhere, indicating other nonfibrillar elements function in adherence (Meyer & Fives-Taylor, 1994). Vesicles or "blebs" originating from the outer membrane consist of highly toxic lipopolysaccharides. A. actinomycetemcomitans have an outer membrane covered by a carbohydrate microcapsule (Zambon et al, 1983) as well as membrane vesicles, similar to lipopolysaccharide vesicles (Holt et al, 1980). They also contain bone resorption activity, bactericidins and adhesins. Highly leukotoxic strains exhibit more vesicles than minimally leukotoxic strains. 28 Extracellular Amorphous Material is a cell surface component that mediates adherence to other cells as well as exhibiting bone resporption properties (Wilson, Karnin, & Harvey, 1988). It has both protein (glycoprotein) and carbohydrate, its expression being determined by culture conditions. Bone Resorption Factors include lipopolysaccharide, proteolysis-sensitive factors in microvesicles, and surface associated materials. Lipopolysaccharide has been shown to effect osteoclasts and cause the release of calcium from bone (Kiley & Holt, 1980). Bactericides are lethal proteins produced against other bacteria, to facilitate colonization. One such protein, actinobacillin, is especially active against S. sanguis, S. uberis and A. viscosus (Hammond, Lil lard & Stevens, 1987) causing increased permeability to the cell membrane resulting in leakage of D N A , R N A and macro molecules essential for growth (Fives-Taylor et al., 1999). Collagenase is an important virulence factor in the enzymatic degradation of collagen in periodontal disease. Collagenolytic activity is associated with P. gingivalis and A. actinomycetemcomitans (Robertson et al., 1982), but not with other indigenous oral flora such as Fusobacterium, Actinomyces, Capnocytophaga and Selenomonas. Lipopolysaccharide(LPS) or endotoxin of A . actinomycetemcomitans has been widely studied due to its tissue destructive effects. It can directly cause Schwartzmann reaction skin necrosis, bone resporption, platelet aggregation as well as activate macrophages to produce IL-1 a and IL-1(3, T N F and other proteins (cytokines) involved in tissue inflammation and bone resorption 29 (Saglie era/., 1990). Chemotactic Inhibitors are advantageous for the infecting organism as they w i l l disrupt neutrophil recruitment to the site of bacterial challenge. A. actinomycetemcomitans has been shown to be able to inhibit chemotaxis (Van Dyke et al, 1982). Cytotoxic Factors produced by A. actinomycetemcomitans can inhibit human fibroblast proliferation. This inhibition of fibroblast D N A and R N A is recognized by the decrease in collagen synthesis seen in periodontal disease (Shenker, Kushner, Tsai, 1982; Gibbons RJ , 1989). Immunosuppressive Factors of A. actinomycetemcomitans include a protein capable of suppressing R N A , D N A and protein synthesis of antigen activated T-cells (Shenker, McArthur & Tsai, 1982). This immunosuppressive protein is capable of inhibiting IgG and I g M production in B-cells by interfering with the early stage of cell activation. Kurita-Ochiai, Ochiai and Ikeda (1992) demonstrated the immuno inhibitory effects of A. actinomycetemcomitans via depressed C D 4 / C D 8 ratios in Aa sensitized mice. Fc Binding Components (outer membrane proteins) are produced and released by A. actinomycetemcomitans (Tolo & Helgeland, 1991). These components can bind to the Fc part of IgG molecules, reducing phagocytosis by 90%. Bacterial clearance depends on Fc-receptors and C3b receptors on phagocytic cells. In periodontal lesions, the Fc binding components of A. actinomycetemcomitans interfere with phagocytic activity, complement function and downregulation of the B-cell response. 30 Epithelial Cell Penetration as well as bacterial penetration into gingival connective tissue and bone has been reported. Christersson et al: (1987) have demonstrated bacterial infdtration of 80% of tissue biopsies studied from periodontal lesions. Epithelial invasion by A. actinomycetemcomitans is a multistep process that involves entry into the cell, escape from the vacuole, rapid multiplication, exit from the host cell (intracellular spread) and cell-to-cell spread via host microvill i (Sreenivasan, Meyer & Fives-Taylor, 1993; Fives-Taylor, Meyer & Mintz, 1995; Meyer et al, 1996, 1997). It is this dynamic process of invasion of epithelial cells that enables the spread of Aa to the gingival and connective tissues and initiates the destruction associated with periodontal disease. Antibiotic Resistance of A. actinomycetemcomitans to tetracyclines used to treat the infections of localized juvenile periodontitis and the ability to transfer resistance to other A. actinomycetemcomitans strains and Haemophilus species suggests that current antibiotics may not be effective (Roe et al, 1995). Leukotoxin produced by A. actinomycetemcomitans is a member of the R T X (repeats- in -toxin) family of toxins and has been widely studied. As a virulence factor, it is able to bind to neutrophils, monocytes and some lymphocytes forming pores in their membranes, resulting in cell death (Tsai et al, 1984; Taichman et al, 1991). A. actinomycetemcomitans strains are not equivalent in terms of leukotoxin production (Lally etal, 1996). Non-leukotoxic strains have comparatively few extracellular vesicles (which contain endotoxin, bone resorption activity, bacteriocins and adhesins) compared to more toxic strains. Brogan et al. (1994) have shown a difference in the genetic structure of the highly toxic strains such as JP2 and the minimally toxic strains such as 652. The leukotoxin 31 gene (ItxA) along with three other genes (ItxB, C, and D) which encode for activation and transport of the toxin, make up the ltx operon. The promotor region of the highly toxic JP2 strain has a 530-bp deletion that may be important in repressing leukotoxin expression. Zambon et al. (1996) were able to demonstrate that highly toxic JPs-type strains were found only in localized juvenile periodontitis patients and not in healthy individuals or those patients with adult periodontitis. 1.2.3 Host Susceptibility to A. actinomycetemcomitans The etiology of periodontal disease has been associated with several specific bacteria, including P. gingivalis, A. actinomycetemcomitans, P. intermedia, B. forsythus, W. recta, F. nucleatum and spirochetes. The number and type of bacteria required to exceed an individual's disease threshold defines host susceptibility. As well as bacterial colonization, some of the pathology of periodontitis results from host-mediated responses triggered by bacterial infection. Susceptibility may be influenced by a number of genetic and non-genetic determinants as well as various risk factors, environmental and systemic factors, which affect the protective and destructive host responses and the pathogenic flora. Host response and virulence factors have been discussed earlier. At this time, the discussion wi l l focus on the humoral immune responses that enhances host susceptibility, particularly as it relates to infection with A. actinomycetemcomitans and localized juvenile periodontitis. 32 There are two immune responses: cell-mediated and the humoral immune response. Both components react cooperatively to protect the host. Cell-Mediated Immune Response - Innate immunity, a component of the cell-mediated immune response, refers to the first line of defense against pathogens. The innate immune system includes complement, monocytes/macrophages and neutrophils that can engulf and destroy bacteria. They do not, however, always eliminate the pathogenic bacteria, depending on the virulence characteristics of the organism. The adaptive immune response mediated by lymphocytes with specific receptors of the immunoglobulin superfamily can be activated to improve the efficiency of eUminating the invading microorganisms. Innate and specific immunity work "hand in hand". Phagocytosis (innate immunity) usually requires opsonization (coating of the bacteria for recognition) with antibody (specific immunity) and/or complement (innate immunity). Moreover, phagocytic cells produce cytokines that are involved in activation of specific T and B-cells (Zadeh, Nichols & Miyasaki, 1999). Many functional and biochemical polymorphonuclear leukocyte defects have been associated with periodontal disease, such as decreased integrin expression affecting transendothelial migration and decreased receptor expression for complement seen in Leukocyte Adhesion Deficiency, type 1 ( L A D ) . Other P M N defects related to decreased number, decreased chemotaxis, decreased oxidative kil l ing mechanisms, and pleomorphism of the Fc receptor responsible for binding of I g G l , 2 and 3 by PMNs , are manifested in periodontal problems seen in agranulocytosis, neutropenia, Down's Syndrome, specific granule deficiency, Chediak-Higashi syndromes, L A D , and chronic granulomatous disease. A. actinomycetemcomitans is able to evade the early host defenses by its virulence capabilities already discussed. Polymorphonuclear leukocytes need antibodies to ingest and k i l l A. 33 actinomycetemcomitans. Antibodies and complement are absolutely required for the opsonization of A actinomycetemcomitans to overcome the dense cell-surface outer membrane carbohydrates (Baker & Wilson, 1989). Humoral Immune Response - Early-onset periodontitis (EOP) can cluster in families, suggesting a genetic predisposition to develop periodontal disease early in life. There can be marked differences in the clinical expression related to several factors, but differences in microbial flora and in the ability to respond immunologically are among the important variables (Tew et al, 1996). A. actinomycetemcomitans is strongly associated with E O P which can be supported by the high antibody titer reactive with serotype-specific antigens (especially to serotype b) in most E O P patients (Ebersole et al., 1982; Genco Zambon & Murray, 1985). Approximately 40-90% of L J P patients are seropositive for A actinomycetemcomitans serotype b. The immunodominant antigen for A. actinomycetemcomitans Y 4 (serotype b) is a serospecific carbohydrate. The immunodominant antigen of serotypes a and c is polysaccharide. During periodontal infections, some patients respond by producing serum antibodies against the pathogenic bacteria, whereas other patients do not. Patients resistant to periodontitis likely produce sufficient levels of high affinity antibodies (IgG) to clear the infection, where susceptible patients mount no humoral immune response or produce antibodies of low affmity/avidity that allow the disease to appear and progress (Ishikawa et al, 1997.) IgG demonstrates four subclass isotypes produced during the antibody response, depending on the nature of the antigen. The four subclass isotopes have various defense mechanisms including opsonic activity, complement activation and toxin inactivation (Tsai et al, 1981; Baker & Wilson, 1989). The IgG2 subclass predominates in response to L P S (carbohydrate) derived from A. actinomycetemcomitans serotype b. 34 Seymour, Powell and Davies, 1979 have suggested that progressive lesions demonstrate predominantly B-cells, whereas T-cells are found in advanced resting lesions. Conversion from a T-cell lesion to a B-cell lesion then is destructive to the host. In B-cel l lesions there is evidence for polyclonal activation of B-cells while superantigens have been shown to activate T-cells in T-cell lesions. Polyclonal B-cel l activators can stimulate B-cells in a T-cell independent manner (Sveen & Skaug, 1992). It has also been shown that T-cells and macrophages must be present for bacterial extracts to activate B-cells in a T-cell dependent manner (Tumang et al, 1990). B -cells can be activated by both mechanisms. The humoral antibody response can be detrimental to the host as well as protective. Polyclonally activated B-cells can make large quantities of IL-1 , which can in turn, activate osteoclasts and initiate bone resorption. T-cells activated by superantigens can also lead to the release of a number of cytokines. T-cells express either C D 4 or C D 8 molecules on their surface and are referred to as helper and suppressor/cytotoxic T-cells respectively. C D 4 T-cells demonstrate 3 subsets, depending on cytokine production ( T h l , Th2, ThO). T h l cells produce IL-2, IFN-y and function in delayed-type hypersensitivity reactions. Th2 cells secrete IL-4, IL-5, IL-6 and IL-10 and provide help for IgG, IgA and IgE responses (Mosmann et al, 1986). ThO produces IFN-y, IL-2, IL-4 and IL-5 (Firestein, 1989). It is hypothesized by Zadeh et al, 1999 that T-cell subsets take part in the regulation of the immune response and have protective or destructive consequences. Several models of T-cell involvement in periodontal disease have been constructed. For further explanation and in depth coverage of these models, see Ebersole & Taubman (1994), Yamamoto et al, (1997) and Seymour et al. (1979). 35 1.3 ANIMAL MODELS IN PERIODONTAL RESEARCH Animals, notably the primates, beagle dog and rat have been widely used in periodontal research. Beagle dogs (Rosenberg, Rehfeld & Emmering, 1966; Saxe et al, 1967; Hul l & Davies, 1974; Lindhe, Hamp & Loe, 1975), were often used to study the prevalence, etiology and pathogenesis of periodontal disease as they resemble humans in periodontal anatomy, microbiology and pathophysiology (Madden & Caton, 1994). When plaque is allowed to accumulate, naturally occurring periodontitis usually develops by 4-7 years of age (Lindhe, Hamp & Loe, 1975). Clinical measurements of periodontal disease in dogs may include plaque scoring, calculus, gingivitis scoring, attachment loss, pocket depth and width of gingiva (Sorensen, Loe & Ramfjord, 1980). These parameters are also used in scoring clinical periodontitis in humans. Periodontal virulence studies usually require histological gingival biopsies to examine the inflammatory /bacterial cells in the connective tissues. A measure of disease activity can also be determined by analyzing the cells or biochemical markers found in the gingival crevicular fluid (Attstrom & Engelberg, 1970; Kryshtalskyj, Sodek & Ferrier, 1986). Host immune responses, cytokine activity and altered bone metabolism has been studied in the dog model to determine virulence of periodontal disease. A n important feature of any animal model used to study human infections is that it should simulate an infectious process in humans while mimicking the natural pathogenesis to the greatest extent possible (Holt et al, 1999). Primates used in periodontal research are the cynomolgus monkey (Macacafasicularis), squirrel monkey (Saimiri sciureus), rhesus monkey (Macaca mulatta), baboon (Papio anubis) and the 36 marmoset. Adult monkeys are susceptible to naturally occurring periodontal disease or disease induced by any number of ligature devices designed to accumulate plaque. Caton and Zander (1975), using a primate model, studied bone and attachment loss clinically and histologically. Heijl , Rifkin and Zander (1976) used squirrel monkeys to determine histologic changes following the conversion of gingivitis to peridontitis when silk ligatures were used for plaque accumulation. Holt et al. (1988) modified this model by injecting P. gingivalis into monkeys after ligature-induced plaque accumulation, to demonstrate its pathogenicity. Although primates most closely resemble humans, the expense of maintenance and safety issues limits the number of monkeys used in any study. The mouse, for over 100 years, has been and continues to be used as a research tool in the study of infections and diseases. Research in bacterial diseases as early as the 1880s used mice routinely for isolation of bacteria, as well as determination of pathogenesis of disease and immunization studies (Madden & Fujiwara, 1982). Mice were chosen for the experiments in these studies because of their availability, ease of handling and low maintenance costs. Genetically inbred mice assure the homogeneity of the test subjects to facilitate the study of inflammation. Murine models have been used repeatedly to demonstrate virulence capabilities of such periodontal pathogens as P. gingivalis, P. intermedia, W. recta and A. actinomycetemcomitans (Kastelein et al, 1982; Van Steenbergen et al, 1982; Grenier & Mayrand, 1987; Neiders etal, 1989; Ebersole etal, 1989; Kesavalu etal, 1990). Evaluation of the virulence of a bacterial strain is based on the gross pathology induced following inoculation of the animals. The mice are typically inoculated with a graded dose of different strains of live bacteria, using either implanted subcutaneous chamber models (Genco & Arko, 1977) or subcutaneous site injection, and the pathology compared. The use of chamber 37 models ensures a ready obtainable sample of the transudate which contains bacterial cells, host cells and inflammatory products and allows the researcher to examine the interactions during the progression of infection (Genco et al, 1997). Injections of bacteria at specific sites establish an abscess model that is used to grade soft tissue destruction (Van Steenbergen etal, 1982, 1987; Ebersole et al, 1995; Joiner et al, 1997). The abscess model can also be used to determine the phenotype of lymphocytes and macrophages recruited to the site (Kesavula et al, 1991; Gremmel E , Bird PS, Bowman JJD, et al, 1997) and to study the effects, i f any, of immunization (Chen et al, 1987; Kesavalu et al, 1991; Gemmell etal, 1997). In many papers, the grading of the soft tissue pathology is a subjective, quasi-quantitative evaluation of the extent of the abscess formation. Van Steenbergen et al. (1982) described histological sections by: 1) diameter of a localized abscess formation, < 1 mm > 4 mm, 2) spreading of the inflammatory cells between subcutaneous muscle and the injection site, 3) mean diameter of the abscess progressing 1-2 mm in lateroventral direction 4) area of phlegmonous abscess at the ventral side of the mouse, including skin necrosis. Other evaluations included semi-quantitative estimates of inflammatory cells and bacteria in the tissues (Kesaluva et al, 1991), as well as descriptions of the size and consistency of the lesions at the injection site with presence, location and appearance of a secondary lesion (Chen et al, 1987). Further detail of the appearance of the mice, such as cachexia, ruffling, general erythema and phlegmonous, ulcerated, necrotic lesions and death (Genco et al, 1991) were used as pathologic criteria. These and other papers evaluated histopathology according to clinical signs, 38 localization or spreading of lesions, and/or ultimate death of the animal. There did not appear to be a consistent, reproducible grading system to evaluate the extent of the inflammatory response induced by bacterial invasion. In these abscess models, only live bacteria were used. Studies evaluating the inflammatory response using dead bacteria or bacterial products were not readily found. 39 1.4 OBJECTIVES OF THE STUDY The research objectives of the Early-Onset Periodontitis Workshop includes defining the current status of periodontal diseases and making recommendations for future directions, goals and approaches. One of their future research goals is to find a good animal model for periodontal disease (Van Dyke & Schenkein, 1996). It is hoped that this study may in some way advance those goals. 1. To develop a quantitative method of grading an inflammatory response in a murine model of inflammation using non-viable A. actinomycetemcomitans. Current models of inflammation using live bacteria have assessed virulence capabilities both clinically (by the size and severity of abscess, malaise and eventual death of the animal) and histologically. A few models of inflammation have tried to quantitate the observed pathology, but no standardized method has routinely been used in the literature. In this study we report a new system of quantifying the inflammatory responses in a murine model of inflammation using 3 strains of non-viable Aa 2. To evaluate the reproducibility of an inflammatory reaction in a murine model using non-viable bacterial components of A. actinomycetemcomitans. Periodontitis is an infectious disease. Although hundreds of bacterial species, serotypes and strains are found in dental plaque, there are clearly differences between the healthy and diseased sites. A. actinomycetemcomitans, P. gingivalis and B. forsythus have been found associated with diseased sites and have been implicated as causative pathogens in 40 periodontitis. This study investigates if there is a predictable inflammatory reaction produced with non- -viable bacterial components of the bacterium A. actinomycetemcomitans. 3. To evaluate the inflammatory reaction (virulence) of 3 different strains (2 serotypes) of non-viable A. actinomycetemcomitans. Studies have demonstrated that the levels of virulence capabilities of specific bacteria can explain approximately 9 to 16% of the variability in disease expression. This has led to the concept that with live bacteria, different serotypes of the same bacteria display different pathogenic properties. We hypothesized that an inflammatory model using non-viable bacteria of different serotypes of A. actinomycetemcomitans will demonstrate the same varied virulence capabilities as live bacteria. 41 C H A P T E R T W O - M A T E R I A L A N D M E T H O D S 2.1 Animals Healthy male C D - I mice (Animal Care Facility, University of British Columbia), weighing approximately 35 gm, age 4-6 weeks, were used in all of the experiments. The mice were caged in groups of four and were maintained on standard lab chow (Purina) and water ad libitum. Approval for the study was granted by the Animal Care Committee of the University of British Columbia. 2.2 Bacteria Three strains of A. actinomycetemcomitans were used in the various experiments: JP2 (serotype b), Y 4 ( A T C C - American Type Culture Collection 43718) (serotype b) and I D H (Institute of Dentistry, Helsinki) 1705 (serotype e). The three A. actinomycetemcomitans strain were grown on Trypticase soy-serum-bacitracin-vancomycin ( T S B V ) agar plates (Slots 1982), incubated in 5% C 0 2 in air at 37° C for 2-3 days. The bacteria showed typical colony morphology, positive catalase reaction, and negative lactose fermentation (Asikainen et al. 1986). The bacteria were collected into PBS and stored at -20° C until used. The initial optical density (OD6oo) of JP2 was 0.2; Y 4 was 1.2, and I D H 1705 was 0.73. Cel l concentrations were established using the spectrophotometer (Varian) so that each undiluted strain had the same optical density (ODeoo)Of 0.2 with equivalent cell counts of approximately 5 X 10 9 bacterial equivalents/ml. The calibrations were done using sterile phosphate buffered 42 saline (PBS). Each undiluted sample of bacteria was kept frozen until use. After thawing, the aliquots were sonicated at output position 2 (Branson Sonifier 250) for 30-sec and dilutions of 0.2, 0.1, or 0.01 were made using sterile PBS (concentration 0.1M), as needed per experiment. The types and concentration of inoculae were as follows: 1. Sterile phosphate buffered saline 0 .1M as vehicle control 2. Immunopathological testing JP2- strain b, IDH-1705- strain e, A T C C 43718- strain b, at concentrations of 5.0 X 10 9 bacterial equivalents/ml (undilute), 1.0 X 10 9 bacterial 0.05 X 10 9 bacterial equivalents/ml (0.01 dilution). The various dilutions were made using the OD<5oo equivalent for each stock strain and mixing sterile phosphate buffered saline (0.1M) to the appropriate dilution/concentration. 2.3 Injections The inoculae to be used were prepared in sterile tubes, and mixed thoroughly. 0.05 cc of solution was drawn into 1.0 cc tuberculin syringes. The syringes were capped with 26 gauge needles. Recipient animals were placed into the anesthesia chamber and administered equal amounts of halothane and oxygen until unconscious. Hair was removed from either the right or left ear and/or abdomen (depending on the experimental design) and the site swabbed with 70% alcohol. Figure 1 demonstrates the subcutaneous injections of the mice in the ear and abdomen with 0.05 cc of the appropriate A. actinomycetemcomitans strain. The mice were then placed in their cages. They appeared well immediately after the injections and throughout the experimental period. 43 Figure 1(a) Ear Injection. Mice were injected subcutaneously (s.e.) at base of Ear Figure 1(b) Abdomen Injection. Mice were injected s.e. in ventral side of Abdomen 44 Figure 1(a) Injection of JP2 - Ea r 1(b) Injection of JP2 - Abdomen 2.4 Time Intervals The time intervals for each experiment were 5 days, 15 days and 25 days, chosen to correspond to histological phases of inflammation. The 5-day period represented an early acute cellular response, populated by polymorphonuclear leukocytes (PMNs) such as neutrophils. The 15-day time interval represented a late acute-early chronic response with the presence of increased PMNs, phagocytic cells and macrophages and 25 days was chosen to represent the late chronic response based on an infiltration of lymphocytes and cells of the monocyte-macrophage lineage (Newman & Nisengard, 1988). 2.5 M u r i n e Virulence Model/Studies Experimental Groups- the animals were randomly separated into 6 experiments for the virulence studies: 1. To establish Control vs JP2 Reaction- mice were injected s.e. ear and abdomen with phosphate buffered saline as control compared to a 5.0 X 109, 0.5 X 109 and 0.05 X 109 bacterial equivalents/ml inoculae of JP2 (serotype b) and sacrificed after 15 days to determine if there was an inflammatory reaction compared to the saline control. 2. To determine the Time-Course of Reaction- mice were injected s.e. ear and abdomen with JP2 (serotype b) undiluted 5.0 X l O 9 and 1.0 X 109 bacterial equivalents/ml and evaluated after 5, 15 and 25 days to determine the comparable extent of the inflammatory reaction. 3. To compare the Time-Kinetics of the 3 different strains of Aa- mice were injected s.e. ear and abdomen with undilute 5 X 109 bacterial equivalents/ ml ATCC JP2, Y4 46 ( A T C C 43718), I D H 1705 strains of A. actinomycetemcomitans and evaluated after 5, 15, 25 days to compare inflammatory reactions of the three strains over time. 4. To determine the Titratability of the 3 strains of A. actinomycetemcomitans - mice were injected s.e. abdomen only with 4 dilutions of each A. actinomycetemcomitans strain and sacrificed after 15 days to determine and compare the inflammatory reactions caused by the different concentrations of each strain. 5. To study the Immunohistologic Cell Response of the 3 strains of A. actinomycetemcomitans, tissue samples from mice in group 3 were processed for immunohistologic staining. Days 5 and 15 after injection were chosen to ensure a reaction that was more acute and detectable. 2.6 Histological Preparation and Protocol At the appropriate time intervals, the selected mice were euthanized using CO2 asphyxiation; hair was removed from the injection site and the area swabbed with alcohol. The injected tissue sites were removed with a scalpel and/or scissors. The specific tissues were put into labeled Histoprep tissue capsules (Fisher Scientific, B .C. ) and placed in a glyoxol, ethanol and buffer solution (Prefer, Anatek Ltd, MI) for up to 24 hours i f being prepared for Hematoloxylin and Eosin staining. The samples to be studied immunohistologically were placed into Tris Zn Buffer (QLT Pharmaceuticals, B.C. ) . The samples were immediately washed or placed in P B S overnight and washed thoroughly with tap water the next day, then placed into the Autotechnicron (Technicon Co. N . Y . ) for dehydration. 47 Dehydration of Samples The tissue samples were placed into the tissue receptacles of the Autotechnicon and the baskets rotated from one reagent beaker to another through the various processing stages. During emersion into the beakers, the samples are gently oscillated vertically to ensure free flow of the beaker reagent. The sequence and duration of immersion are timed automatically by a timing disc, which permits a definite sequence of varying time intervals to be preset. The first five reagent beakers are intended to dehydrate the tissues with varying concentrations of alcohol, while the last three beakers contain a clearing agent (Hemo D , Fisher Scientific, B;C) . The timing sequence was as follows: Dehydrating - 70% ethanol -1 hr - 95% ethanol - 2 hr -100%ethanol-2hr (X3) Clearing -Hemo-D-1 hr (X3) At the completion of the cycle, the tissue samples were tapped gently to remove as much Hemo-D as possible and then put into ceramic pots for waxing. Waxing Embedding Paraffin waxing pellets (Paraplast X-tra, Fisher Scientific, B .C. ) were placed into the waxing oven overnight at 56° C (2-4 degrees above the melting point of the wax). When the samples were removed from the Autotechnicron, they were placed into ceramic pots and covered with the melted paraffin, then placed into the vacuum chamber with a heat setting of 56° C for Vacuum Impregnation. The samples were subjected to 15 pounds of pressure for 1 48 hour, then removed to add fresh melted paraffin to the pot. The samples were placed in the vacuum chamber for 30 minutes at 15 pounds pressure two more times, and new melted wax added each time. When each wax impregnated sample was removed from the vacuum for the last time, it was quickly put into a prepared histological block for Embedding. Embedding After thorough impregnation, each tissue sample was placed in an appropriately sized mold. The bottom of the mold was covered with paraffin and the tissue oriented. With the proper sized embedding ring in place, paraffin was poured in to make a block about 1 cm. thicker than the piece of tissue. The molds were cooled to solidify the wax. When the blocks were solid, the waxed molds were removed and kept at 4°C until ready to section. Trimming and cutting of Tissue Samples After embedding, surplus wax was removed to prepare for sectioning with a Spencer Microtome 820. Sections 6u thick were cut into ribbons and separated for mounting on slides. Mounting Sections on Slides The ribbons were divided into sections and placed onto frosted slides (Fisher, B.C. ) which were flooded with water to initially stretch the sections. They were then poured off the slide into water in a thermostatically controlled water bath (46° C) and floated for a few minutes to get rid of creases. A clean slide was lowered into the water and withdrawn at an angle, bringing the section with it. The section was then oriented on the slide and placed into the drying oven set at 37° C overnight to dry completely. Prior to staining, the slides were placed into the waxing oven for 15-30 minutes. 49 Staining and Mounting for Histological Study Haematoxylin (Harris's Modified Haematoxylin, Fisher, B.C. ) and Eosin (Fisher, B .C) , ( H & E ) staining was done by hand in small batches. Both H and E were filtered prior to use. Running tap water was used for washing. The staining procedures, completed under the fume hood, consisted of placing the slides into beakers of the following reagents: Dewaxing - 5 minutes each: - Xylene ( X 2) - Absolute ethanol - 95% ethanol, - 70% ethanol Wash in tap water -3-4 dips Staining - Haematoxylin 4-5 minutes- wash in running water 20 minutes - 1.5% N a H C 0 3 or saturated L I 2 C 0 3 , wash 30 sec. - Eosin 1-3 minutes - Ethanol 100% ( X 3) for 1 minute each - Xylene ( X 2) for 5 minutes. Coverslip - Coverslips were placed on the slides using Entallan (Merck, Ont) and then left to dry. 50 2.7 Immunological Methods and Preparation 2.7.1 Antibodies The immunological staining performed in this study was done using the MicroProbe Staining System, a component of the FisherBiotech MicroProbe System, Fisher Scientific, Pittsburgh, PA . (capillary gap technology). Three antibodies were used; purified anti-mouse macrophage (F4/80) monoclonal antibody (Cedarlane), rat anti-mouse neutrophil monoclonal antibody (Serotec) and D A K O rabbit anti-human T cell C D 3 . The F4/80 antigen is found on most macrophages and on macrophage precursors. It is found in low levels on activated macrophages and eosinophils. Anti-mouse macrophage (F4/80) monoclonal antibody reacts with the mouse macrophage F4/80 antigen. The rat anti-mouse neutrophil monoclonal antibody is specific for mouse neutrophils. The C D 3 antigen is a highly specific marker for T cells, and the D A K O Rabbit anti-human T cell, C D 3 antibody reacts with the intracytoplasmic portion of the C D 3 antigen expressed by human and mouse T cells. The tissue samples slides were placed in a 37° C oven overnight, then deparaffinized and rehydrated in the following sequence: Xylene 5 minutes (X3) - 100% Alcohol 2 minutes (X2) - 95% Alcohol 2 minutes ( X I ) - 70% Alcohol 2 minutes ( X I ) The slides were then placed in fresh Automation buffer for 5 minutes. 51 The Reagents used in the immunological staining were as follows: 1. I X Automation buffer - Dilute 10 ml. of 10X Automation buffer to a final volume of 100 ml. with distilled water. 2. I X Phosphate Enhancer - Dilute 10 ml. of 10X Phosphate Enhancer to a final volume of 100 ml. with distilled water. 3. Primary antibody - Rabbit Anti-human T cell ,CD3 (1:100); Anti-mouse macrophage F4/80 (1:50); rat Anti-mouse neutrophil (1:100). 4. Reagent 1A - Biotinylated second antibody mix (supplied) 5. Reagent 2 - Avidine-alkaline phosphatase complex (supplied). 6. Reagent 3 - Working chromogen reagent: A) Transfer 5 ml. of distilled water to bottle labeled 3. B) Add 2 drops of concentrated buffer p H 8.2. M i x . C) Add one tablet of fast red T R tablets. Wait one minute. D) Add one drop of Naphthol A S - M X phosphate. M i x . Control slides were processed (subjected to immunological staining) without the addition of the primary antibodies. 2.7.2 Staining Method The Staining method for the Immunohistologic study involved the addition of the primary antibodies, (F4/80, C D 3 , or neutrophil) to the slides and incubating at 37°C for 60 minutes. After rinsing with I X Automation buffer (6 times), the biotinylated second antibody mixture (Reagent 1A) was added to the slides. The slides were then incubated at 37°C for 15 minutes, after which they were rinsed with I X Automation buffer (6 times). Reagent 2, the avidin-alkaline phosphatase complex was then added, and the slides incubated at 37°C for a further 20 minutes. 52 They were then dipped into Phosphate Enhancer for 1 minute, after which time the working chromogen reagent, Reagent 3 was applied. After incubating at room temperature for 10 minutes, the slides were rinsed twice with distilled water. Counterstaining was done with 1:5 dilution of Hematoxylin, then the slides were rinsed three times with distilled water. After being dipped into I X Automation buffer for 2 minutes, the slides were dipped twice into distilled water. They were then removed from their holders and Crystal mount applied. 2.8 Statistical Analysis The statistics used were the nonparametric Kolmogorov-Schmirnov test of Significance. Standard Error was calculated for the reproducibility data, presented in Figure 10. 53 CHAPTER THREE - RESULTS 3.1 Methodology for the Evaluation of Histological and Immunohistological Score The histological and immunohistologic evaluation of inflammatory responses in the literature is very subjective and relies on observational criteria only. The main objective of this part of the study was to try to standardize the methodology for grading the histopathological responses caused by non-viable A. actinomycetemcomitans. 3.1.1 Histological Score In this inflammatory model, two parameters were selected for grading of the histological slides. The first parameter of the grading system used here was the percentage of the microscopic field that demonstrated an inflammatory infiltrate. This was determined by examining the slide at X l O power and grading the area of cellular infiltrate on a 0-4 scale (0 = 0-9% of field, 1 = 10-14% of field, 2 = 15-19% of field, 3 = 20-24% of field and 4 = >25% of field). The second parameter was the cellular density of the infiltrate. This was determined by counting the number of inflammatory cells per optical grid square (each 0.49 sq. mm) at X40 power and grading the density on a 0-4 scale: 0 = 0 cells/grid square, 1 = 1-4 cells/grid square, 2 = 5-9 cells/grid square, 3 = 10-14 cells/grid square, and 4 = >15 cells/grid square. The overall histological score was determined by the addition of the two above parameters and assigning a score as follows: 54 Percent of Field + Density Histological Score (Grade) 0 0 1-2 (1+0 or 1+1) 1 3-4 (2+1 or 2+2) 2 5-6 (3+2 or 3+3) 3 7-8 (4+3 or 4+4) 4 Figure 2 represents examples of the percentage of field demonstrating an inflammatory infiltrate and cellular density of infiltration calculated to determine the Histological Score. 55 Figures 2(a), (b), (c) (d) Histologic Score (Grade) as calculated by addition of area and density. 2(a) Grade 1 - I D H 1705 at day 5 - abdomen at magnification X 10 and X 40. A ) fat B) blood vessel C) muscle layer D) P M N infiltrate 2(b) Grade 2 - Y 4 at day 15 - abdomen at magnification X 10 and X 40. 2(c) Grade 3 - Y 4 at day 5 - abdomen at magnification X l O and X 40. A ) fat B) muscle layer C) P M N infiltrate 2(d) Grade 4 - JP2 at day 5 - abdomen at magnification X l O and X 40. The photo shown at 10X magnification demonstrates an example of the area of the slide that contains inflammatory cells. The photo taken at 4 0 X magnification represents the most densely populated area for cell counting. 56 Figure 2(a) Histological Score - Grade 1 - magnification X l O and X40 . Figure 2(b) Histological Score - Grade 2 - magnification X l O and X 4 0 Figure 2(d) Histological Score - Grade 4 - magnification X l O and X40 . 3.1.2 Immunohistological Score The parameters used for grading o f the immunohistological slides were more difficult due to the non-specific binding seen on many of the slides and the more diffuse nature of the cellular response. The 0-4 scale was based predominantly on the number of cells (density) counted at X 4 0 power using the optical grid. Score 0 = 0 cells/grid square, 1=1-5 cells/grid square, 2 = 6-10 cells/grid square, 3 = 11-15 cells/grid square and 4 = > 15 cells/grid square. The immunological reaction for the three antibodies were graded the same. Figure 3 represents examples of the density of field with inflammatory infiltrate used to determine the Immunologic Score. 61 Figures 3(a), 3(b), 3(c) and 3(d) Immunohistological Score as determined by density of the field with an inflammatory infiltrate. 3(a) Grade 1 - I D H 1705 - C D 3 at day 15 - Abdomen at magnification X l O and X40 A ) +ve staining - T-cells B) -ve nonspecific binding 3(b) Grade 2 - Y 4 -Neutrophil at day 5 - Abdomen at magnification X l O and X 4 0 A) +ve staining - neutrophils B) -ve nonspecific staining 3(c) Grade 3 - JP2 - Neutrophil at day 15 - Abdomen at magnification X l O and X 4 0 A) +ve staining - neutrophils B) -ve nonspecific staining 3(d) Grade 4 - Y 4 - F4/80 at day 5 - Abdomen at magnification X l O and X 4 0 A ) +ve staining - monocyte/macrophages B) -ve nonspecific staining 62 Figure 3(a) Immunohistological Score - Grade 1 - magnification X l O and X 4 0 Figure 3(b) Immunohistological Score - Grade 2 - magnification X lO and X40 Figure 3(c) Immunohistological Score - Grade 3 - magnification X l O and X40 Figure 3(d) Immunohistological Score - Grade 4 - magnification X l O and X40. 3.2 Comparison of Subcutaneous Injections of Ear and Abdomen for Elicitation of the Inflammatory Response. In this study two sites were chosen for investigation; the ear and abdomen of CD1 mice. The ear (at the base of the skull) was chosen as it had a unique anatomical feature, the internal cartilage core, which could be used as a histological marker from which the degrees of inflammation could be measured. Moreover, as the ear is heavily vasculated, erythema could be easily assessed and the contralateral ear could be used as a control. Finally the degree of swelling after injection could be measured by micrometer. A negative feature of this site was the difficulty in performing a subcutaneous injection, due to the risk of perforation of the epithelium on the opposite side o f the ear. As well, when harvesting the ear, some of the inflamed tissue may have been destroyed or severed on removal. The abdomen was chosen as the second site for investigation as it had an external marker, the hair follicles from which the degrees of inflammation could be measured. Moreover, it was a relatively easy site to perform subcutaneous injections. The injection site could be easily localized and the degree of induration could be measured. In the first experiment, A. actinomycetemcomitans (Aa) bacteria, JP2 (strain b) was chosen for testing as this strain has been shown to be highly associated with L J P (Zambon, Slots & Genco, 1983). Three mice were prepared as described for injection with 0.05 ml JP2 at concentrations of 5 X 10 9 bacterial equivalents/ml (undilute), 0.5 X 10 9 bacterial equivalents/ml (0.1) and 0.05 X 10 9 bacterial equivalents/ml (0.01) and a PBS control (0. IM) . In the right ear and abdomen, each mouse received one of the concentrations of inoculate of bacteria, while on the contralateral side, the animal received 0.05 ml of phosphate buffered saline (0.1 M ) as a control. 67 The animals were sacrificed at 15 days and the tissues processed as indicated in the protocols. The entire wax blocks were cut, sections mounted, stained and microscopically evaluated to find the sections with the strongest inflammatory reaction present within the entire tissue sample. The most severe reaction was graded and recorded according to the proposed inflammatory grading model. The tissues injected with the saline control demonstrated a normal histological appearance with no inflammatory infiltrate as a result of the saline or the needle itself. This was true for both the ear and the abdomen samples. In the ear samples injected with varying dilutions of strain b JP2 and harvested on day 15, an inflammatory reaction was evident by the staining of the P M N infiltration, which was scored according to the Histologic Score criteria. The inflammatory reaction demonstrated in the abdomen samples injected with varying dilutions of strain b JP2 and harvested on day 15, demonstrated more intense staining and a heavier infiltration of P M N leukocytes than the ear tissues. This appeared true for all dilutions. 68 Figure 4(a) The top photograph (XlO) demonstrates the mouse ear injected with the saline control, devoid of P M N s , macrophages or inflammatory cells. In the bottom photograph (XlO) on day 15, after injection with undilute JP2, the inflammatory reaction is quite localized and intensely populated with P M N s . Many inflammatory cells are also evident in the muscle and fat layers. A) epithelium B) fat C) muscle D) P M N infiltrate 4(b) The top photograph (XlO) demonstrates the abdomen injected with the saline control. The bottom photograph (XlO) demonstrates the P M N reaction in the abdomen injected with undilute JP2, and harvested on day 15. There is a large area of inflammatory cells, densely populated with P M N s visible in both muscle and fat layers. A ) epithelium B) fat C) muscle D) P M N infiltrate E) hair follicle 69 Figure 4(a) Inflammatory Response in Ea r - P B S control and J P 2 at day 15 ( X l O ) Figure 4(b) Inflammatory Response in Abdomen - P B S control and J P 2 at day 15 ( X l O ) 3.3 Dose Dependency of Inflammatory Response in Ear and Abdomen (JP2) at Day 15. A s can be seen in figures 5 to 7, the mflammatory response to the varying dilutions of JP2 injected into both Ear and Abdomen sites demonstrate a higher response with the more concentrated A. actinomycetemcomitans. As the samples were graded for Histologic Score involving area and density of inflammatory cells, the abdomen site demonstrated a greater inflammatory response than the ear for all dilutions. However, the 1 X l O 9 bacterial equivalents/ml dilution - Ear, demonstrated a higher histologic score at 15 days than the undilute sample (5 X l O 9 bacterial equivalents/ml). In the Abdomen samples, these two dilutions produced equally high scores (Grade 4). 72 Figure 5 Graph of the inflammatory Dose Responses in the Ear and Abdomen using 5.0 XlO9,1.0 XlO 9, 0.5 XlO 9 and 0.05 XlO 9 bacterial equivalents/ml of JP2 at day 15. 73 Figure 5 Inflammatory Response of varying dilutions of JP2atdayl5 - Ear and Abdomen 74 Figure 6 Inflammatory Response of the varying dilutions of JP2 at day 15 - Ear (XlO) 6(a) 0.05 XlO 9 bacterial equivalents/ml. 6(b) 0.5 XlO 9 bacterial equivalents/ml. 6(c) 1.0 XlO 9 bacterial equivalents/ml. 6(d) 5.0 XlO 9 bacterial equivalents/ml. 7 5 igure 6(a) Inflammatory Response of 0.05 X l O 9 bacterial equivalents/ml of J P 2 - Day 15 Ear (XlO) 6(b) Inflammatory Response of 0.5 X l O 9 bacterial equivalents/ml of JP2 - Day 15 -Ea r (XlO) 76 Figure 6(c) Inflammatory Responses of 1.0 X l O 9 bacterial equivalents/ml of JP2 - Day 15 -E a r (XlO) 6(d) Inflammatory Response of 5.0 X l O 9 bacterial equivalents/ml of JP2 - Day 15 -Ea r (XlO) Figure 7 Inflammatory Response of the (XlO) 7(a) 0.05 XlO 9 bacterial equivalents/ml. 7(b) 0.5 XlO 9 bacterial equivalents/ml. 7(c) 1.0 X 109 bacterial equivalents/ml 7(d) 5.0 XlO 9 bacterial equivalents/ml. ing Dilutions of JP2 at day 15 - Abdomen 78 Figure 7(a) Inflammatory Response of 0.05 X l O 9 bacterial equivalents/ml - Day 15 Abdomen (XlO) 7(b) Inflammatory Response of 0.5 X l O 9 bacterial equivalents/ml - Day 1 5 -Abdomen (XlO) 79 Figure 7(c) Inflammatory Response of 1.0 X l O 9 bacterial equivalents/ml - Day 15 -Abdomen (XlO) 7(d) Inflammatory Response of 5.0 X l O 9 bacterial equivalents/ml - Day 1 5 -Abdomen (XlO) 3.3 Kinetic Analysis of Inflammatory Response of Three Strains of A. actinomycetemcomitans. We next determined whether there was a difference in the inflammatory response between the three different strains of A. actinomycetemcomitans at time periods: 5, 15, and 25 days. It has been demonstrated that Serotype b, found in increased proportions in L J P (Zambon, Slots & Genco, 1983; Asikainen et al, 1991), produced more leukotoxin (up tolO-20 times more) than serotypes a or c and may be particularly virulent in destroying periodontal tissues. Saarela et al. (1992) determined that serotype e was much less pathogenic, as it was found less frequently in periodontitis than serotypes a, b, c or d. Even within serotypes, leukotoxin production and virulence are not equivalent (Poulsen et al, 1994; Brogan et al, 1994). This study looks at the kinetic analysis of the three different strains to determine i f the inflammatory reaction seen in the mice would be consistent with reported findings. Three groups of mice were randomly caged in groups of four. In each group, 0.05 ml of 5 X l O 9 bacterial equivalents/ml (undiluted) of strains JP2, Y 4 , and I D H 1705 were injected s.e ear and abdomen. The animals were sacrificed on day 5 (early acute response), day 15 (acute), and day 25 (chronic response). Tissues were fixed in Zn-Tris buffer and sectioned for H and E and Immunohistological staining. The histologic specimens were evaluated to find the most pronounced inflammatory reaction in the tissues, which were then graded as described. As can be seen in Figure 8, the Ear samples of the undilute JP2 injections demonstrate a very acute, heavily infiltrated, localized P M N response on Day 5. The P M N inflammatory reaction of the Y 4 and I D H 1705 undilute strains indicate a reduced (less virulent) response which remains consistent over day 5 and 15. Histological examination revealed a chronic, diffuse inflammatory response for all strains at day 25, in which P M N s and macrophages could be seen. 81 In the Abdomen samples, JP2 demonstrates a strong P M N response both day 5 and day 15, becoming less intense, more diffuse (chronic) by day 25. Strain Y 4 produced a less intense inflammatory response day 5 and 15 than JP2, decreasing by day 25. The inflammatory response on day 5 of I D H 1705 produced a minimal staining of P M N s , macrophages or monocytes. The most acute reaction for I D H 1705 can be seen at day 15. The response at day 25 appears more intense than the initial response at day 5. The responses were quite equal for all strains by day 25. 82 Figure 8(a) Graph of the Kinetic Response of undilute strains - JP2, Y4, IDH 1705 (Ear) -Day 5,15 and 25 8(b) Graph of the Kinetic Response of undilute strains - JP2, Y4, IDH 1705 (Abdomen) - Day 5,15, and 25 8(c) Inflammatory Response of strains JP2, Y4 and IDH 1705 - Day 5,15,25 - Ear (X40) 8(d) Inflammatory Response of strains JP2, Y4 and IDH 1705 - Day 5,15, 25 -Abdomen (X40) 83 Figure 8(a) Kine t i c Response of J P 2 , Y 4 and I D H 1705 - E a r .5 O 4 W q o o "5) o 2 o % 1 if 0 0 mi 15 Day • JP2 • Y4 • 1705 25 Figure 8(b) Kine t ic Response of J P 2 , Y 4 and I D H 1705 - Abdomen co 5 -8 4 CO o 3 Ui _ o 2 o % 1 £ 0 r i 0 5 1 Day 5 25 • JP2 • Y4 • 1705 84 Figure 8(c) Inflammatory Response of JP2 , Y4 , I D H 1705 - Day 5, 15, 25 - Ea r (X40) 85 Figure 8(d) Inflammatory Response of JP2 , Y4 , I D H 1705 - Day 5,15, 25 - Abdomen 86 3.5 Dose Dependency of Inflammatory Response of Three Strains of A. actinomycetemcomitans In order to determine whether there was a difference in the inflammatory response mediated by the 3 strains of A. actinomycetemcomitans, mice were injected s.e. abdomen with 0.05 cc of four dilutions of the 3 strains of Aa. A l l of the mice were sacrificed at 15 days as described and the tissues were prepared for H and E staining. The samples with the most mflammatory response were graded and compared. Figure 9 illustrates a graph of the Dose Response at day 15 of the three different strains of A. actinomycetemcomitans at dilutions 0.05 X l O 9 bacterial equivalents/ml, 0.5 X 1 0 9 bacterial equivalents/ml, 1.0 X l O 9 bacterial equivalents/ml and 5.0 XlO 9 bacter ia l equivalents/ml. In this experiment, strain JP2 demonstrated the strongest inflammatory response at 1.0 X l O 9 and 5.0 X 1 0 9 bacterial equivalents/ml as determined by the Histologic Scores. Y 4 demonstrated a greater response than the other two strains at 0.5 X l O 9 bacterial equivalents/ml at day 15. At the lowest dilution of 0.05 X 10 9 bacterial equivalents/ml, a l l three strains produced an equal response. Strain I D H 1705 demonstrates the least variable and the lowest inflammatory response of the three strains. 87 Figure 9 Inflammatory Response of Varying Dilutions of strains JP2, Y4 and IDH 1705 - Day 15 - Abdomen (XlO) 88 Figure 9 G r a p h of Inflammatory Response of varying dilutions of strains J P 2 , Y 4 and I D H 1705 - Day 15 - Abdomen Histologic Score i i i i i i Histologic Score i i i i i i 1 0 0.05 Cell Eqn i 0.5 ivalents i 1 X10(9) 1-5 • JP2 • Y4 • 1705 89 3.6 Reproducibi l i ty of the Kine t i c Response. In order to detenriine the reproducibility of the kinetic response, a final experiment was done using 1:10 dilution (0.5X10 9 bacterial equivalents/ml) of the three strains of Aa for day 5, 15, 25. Four mice were used in each grouping for a total of 36. The Histologic Scores from all animals that were tested at 0.5 X l O 9 bacterial equivalents in all experiments were used to calculate the mean of the histologic Scores. Figure 10 demonstrates the mean of the histological scores of the 3 strains at 0.5 X 10 9 bacterial components/ml at day 5, 15, 25. As can be seen from the graph, the mean Histologic Score for JP2 was highest of the three species day 5 and 15, representing the acute phase of the inflammatory response. B y day 25, the chronic inflammatory phase, the JP2 response was consistently reduced, as revealed by the lower Histologic Score. Strain Y 4 demonstrated a mean Histologic Score that increased with time (not statistically significant), but overall, the inflammatory response was lower than the JP2 P M N response. As seen in the former experiments, strain I D H 1705 demonstrates low Histologic Scores, (particularly by day 25), which are not significantly different from strain Y 4 . There were extreme inconsistencies with one animal in both of the strain groups (Y4 and I D H 1705) that may have affected the mean values. 90 Figure 10 Graph of Mean Histologic Scores and Standard Errors of JP2, Y4 and IDH 1705 (dilution 0.5 X 109 bacterial equivalents/ml) - Day 15. 91 Figure 10 M e a n Histologic Scores and Standard Er ro r s of JP2 , Y 4 , and I D H 1705 (0.5 X l O 9 bacterial equivalents/ml) - Day 15. 92 3.7 Immunohistological Analysis of the Cellular infiltrate Mediated by Three Strains of A. actinomycetemcomitans. Day 5 and 15, representing the early and acute inflammatory phases, were chosen to investigate the immunohistologic response in this murine model. Using the antibody F4/80, neutrophil and C D 3 T-cell, the Immunohistologic Score for undilute strains JP2, Y 4 and I D H 1705 was determined as described earlier. The Immunohistological Score for the three antibodies is displayed in Table 1. As can be seen in Table land Figure 11, tissue sections from JP2- and Y4-injected animals demonstrate a very strong and equal response to F4/80, for both 5 and 15 days. I D H 1705 induced a strong F4/80 monocyte/macrophage response day 15, equal to the other two antibodies. The neutrophil response is the highest for JP2 at 5 days and less at 15 days. Y 4 shows a moderate neutrophil response day 5, but I D H 1705 demonstrates an equal neutrophil infiltration at day 15. The T-cell response induced by al l strains is similar for both days, with a very strong response detected day 15 for JP2. The weakest is demonstrated on day 15 by I D H 1705. 93 Table 1 Immunohistologic Score of F4/80, neutrophil and CD3 for 3 undilute strains of A. actinomycetemcomitans at day 5 and day 15. Figure 11 The Immunohistologic Response of 3 antibodies at day 5 and 15 for undilute JP2. 11(a) F4/80 - day 5, day 15 11(b) Neutrophil - day 5, day 15 11(c) CD3 - day 5, day 15 94 Table 1 Immunohistologic Score of the 3 Antibodies for undilute JP2, Y4 and IDH 1705 -Day 5 and 15 Aa Time (days) F4/80 Neutrophil CD3 T-cell JP2 5 4 4 2 15 4 3 4 Y4 5 4 3 2 15 4 2 2 1705 5 3 2 2 15 4 3 1 95 Figure 11(a) Immunohistologic Response of Antibody F4/80 for JP2 at day 5 and 15 (XlO) Figure 11(b) Immunohistologic Response of Neutrophil Ant ibody for J P 2 at day 5 and Figure 11(c) Immunohistologic Response of Antibody CD3 for JP2 at day 5 and 15 (XlO) CHAPTER FOUR - DISCUSSION 4.1 Inflammatory Response of Viable Bacteria Compared to Non-viable Bacteria in Mice Robert Koch, in 1800, first demonstrated the technique of creating abscesses subcutaneously in mice. Since that time, hundreds of investigators have used and modified this technique. In models involving mice, researchers have been able to study in vivo, the etiology, control, treatment and ultimately prevention of disease in man and animals (Madden and Fujiwara, 1982). In abscess models the pathogenic activities of periodontal pathogens injected s.e. into tissues other than the periodontium have attempted to provide controlled and interpretable results for assessing selected host-parasite interactions and examining pathology and virulence potential associated with soft tissue destruction. One of the first reported techniques for producing subcutaneous encapsulated abscesses was first developed by introducing cultures of B. frigalis or S. aureus homogenized with autoclaved cecal contents by subcutaneous inoculation in mice (Wilkins et al, 1977; Dickerman et al, 1980 and Joiner et al, 1980). Since then, various suspending media and bacteria or combinations of bacteria have been used in the study of pathogenesis and virulence. These studies utilize such bacteria as A. actinomycetemcomitans (Taubman et al, 1984; Chen et al, 1991; Kurita-Ochiai et al, 1992; Ebersole etal, 1995), C. rectus (Ebersole etal, 1995), and F. nucleatum (Baumgartner et al., 1992; Ebersole et al, 1995; Feuille et al, 1996). Other species used for abscess models include P. anaerobius and V. parvula (Baumgartner et al, 1992), T. denticola (Ebersole et al, 1995), P. gingivalis (Van Steenbergen et al, 1982, 1986; Chen et al, 1987; Genco C A et al, 1991; Baumgartner et al, 1992; Kesavalu et al, 1992, 1995; Schifferle et al, 1993; Ebersole et al, 1995; Eke et al, 1996; Feuille et al, 1996; Gemmell et al, 1997), P. intermedia (Chen et al, 99 1987; Baumgartner et al., 1992), P. melaninogenica (Van Steenbergen et al, 1981; Baumgartner et al, 1992) and W. rectus (Kesavalu et al, 1991; Chen et al, 1991; Ebersole et al, 1995). The subcutaneous infection in the M A M is characterized by localized abscess formation (with gangrenous necrosis of skin and subcutaneous layers and/or dry gravity abscesses with no pus) at the injection site within 3 days. This may occur with or with out a secondary spreading, phlegmonous abscess containing pus, blood and exudate. These secondary spreading abscesses often involve subcutaneous tissues and muscle layers in the dorsal, flank and ventral side of the abdomen, base of the tail or thoracic region. Live bacteria can be cultivated from the secondary abscesses, depending on the species or combinations of species. Lesion size remains stable through 7-9 days, with resolution occurring 12-15 days. Wi th virulent organisms, the mice display severe cachexia, ruffled hair, hunched bodies, weight loss and ultimately death (Kesavalu etal, 1991; Chenet al, 1991; Genco etal, 1991; Schifferle et al, 1993; Ebersole et al, 1995; Eke etal, 1996). Subcutaneously injected heat-killed F. nucleatum caused the formation of local lesions only, with no spreading abscess formation at distant sites (Feuille et al, 1996). Investigations of heat-killed P. gingivalis demonstrated no lesions or abscess development when injected subcutaneously (Genco C A et al, 1991; Kesavalu et al, 1992, 1995; Eke et al, 1996; Feuille et al, 1996;). Genco, on examination of lesions caused by non-viable P. gingivalis, reported seeing a few P M N s on histologic examination on day 1, but no P M N s after day 3. Joiner et al. (1980) reported no lesion development with heat-killed B. fragilis or S. aureus. Non-viable C. rectus did not elicit a localized abscess when injected s.e. (Ebersole etal, 1995). 100 No dilutions of the three strains of non-viable A. actinomycetemcomitans used in these experiments led to observable abscess formation and none of the animals appeared cachectic or died as a result of injection of the bacteria. This is in agreement with other investigators using non-viable bacteria as reported. 4.2 Non-viable Bacterial Murine Model compared to Murine Abscess Model The murine abscess models use subcutaneously injected live bacteria, at concentrations of 1 X l O 7 to 5 X l O 1 1 bacterial ml, depending on the bacterial strain used. The non-viable bacterial model involves injections of sonicated killed bacteria (three strains of A. actinomycetemcomitans), at concentrations of 0.05 X l O 9 to 5.0 X l O 9 bacterial equivalents/ml to study the virulence ability of each of the strains by a quantitative analysis of the histologic and immunohistologic response. The virulence of A. actinomycetemcomitans is largely dependent on its surface ultrastructure that includes fimbriae, vesicles of lipopolysaccharide, and extracellular amorphous material (important for adhesion). Although the A. actinomycetemcomitans strains were non-viable, their outer membrane virulence factors were still able to cause a tissue inflammatory response, without causing death to the mice. 101 4.2.1 Histologic Response in the M A M and Non-viable Bacterial Model. Most of the abscess models provided descriptions of the mice or the encapsulated abscesses produced by injection of live bacteria. Virulence was determined by lesion size and shape (Chen et al, 1991; Kesavalu et al, 1990; Ebersole et al, 1995; Feuille et al, 1996) or lesion area and volume (Joiner et al, 1979; Kesavalu et al, 1992, 1995; Schifferle et al, 1993) produced by the various strains and dilutions of the bacteria. Other indicators of virulence were the spreading capability of the abscesses to distant sites (Van Steenbergen et al, 1986; Chen et al, 1987; Eke et al, 1996) or death of the animal (Wilkins et al, 1977; Chen et al, 1987; Baumgartner et al., 1992; Ebersole et al, 1995). Histology was not reported in most of the studies. In the murine abscess models that did look at the histological response, either descriptively or quantitatively, a P M N infiltrate and inflammatory exudate were apparent in the early abscess lesions 1-5 days postinoculation (Joiner et al, 1979; Kesavalu et al, 1990; Genco et al, 1991; Ebersole et al, 1995). In a virulence study by Kesavalu et al (1990), tissue sections of the skin lesions produced by Wolinella recta revealed coagulation necrosis of the epidermis, as well as underlying cutaneous muscle. Marked exudation of serum proteins and neutrophils were seen beneath the necrotic area. Joiner et al. (1980) described the abscess produced by the injection of B. fragilis or S. aureus as having a distinct band of white blood cells, 95% of which were P M N s around the periphery of the abscess by 24 hours. Garant (1976) demonstrated primary inflammatory cells consisting of P M N s , macrophages, lymphocytes and monocytes in the gingival tissues of germ-free rats 65 days after infection with A. naeslundii. As is characteristic of the P M N host defense mechanism to periodontopathogens in the gingival sulcus of humans (Schroeder & Graf-deBeer, 1976; Van Dyke & Hoop, 1990), P M N s also responded to the nonviable bacterial components injected into the tissues of the mice in these experiments. 102 The current model of A. actinomycetemcomitans -induced inflammation demonstrates a nonencapsulated inflammatory response to all A a strains and dilutions used in the study. JP2 demonstrates the most intense P M N reaction day 5 and 15 (Figure 2(d), 8(a), 8(b), 10). A T C C 43718 produces a strong acute P M N reaction similar to JP2 (Figure 3(d), 9) and the mean Histologic Score demonstrates the most intense P M N reaction at day 25 compared to the other two strains (Table 1). The inflammatory infiltration is much less intense with strain I D H 1705 (Figure 10), which is considered as a non-periodontopathogen (Saarela et al., 1992). These findings are in agreement with Ebersole et al. (1995) who showed a comparison of the virulence capacity of various A. actinomycetemcomitans strains and serotypes in a M A M , indicating a progression of virulence (lesion size), with JP2 >_Y4 >_serotype c. Virulence differences within bacterial strains (other than A.a) were also demonstrated by Van Steenbergen et al, 1982; Kesavalu etal, 1990; Genco etal, 1991 and Ebersole etal, 1995). 4.2.1.1 Time/Dose Effects on the Histological Response. The Histological Scores of the varying doses of A. actinomycetemcomitans demonstrate a weaker cellular response with higher dilution of the bacteria. Figure 5 demonstrates the dose responses (abdomen) of varying dilutions of JP2 on day 15. The ear sample however, produced a more severe reaction with the 1.0 X 1 0 9 bacterial equivalents/ml on dayl5 than the undilute JP2. The ear site provided a less predictable and less severe inflammatory reaction and eventually the abdomen site alone was used in future experiments. Figure 9 illustrates the histologic response to the three strains of varying doses on day 15. JP2 demonstrates the highest histologic scores for dilutions 5.0 X l O 9 and 1.0 X l O 9 bacterial equivalents/ml, with strain Y 4 showing an equally intense inflammatory reaction at 0.5 X 1 0 9 bacterial equivalents/ml. I D H 1705 demonstrated the 103 weakest reaction at higher concentrations but was equal to the other strains at the lowest concentrations. A l l o f the strains demonstrated a minimal P M N , macrophage response at the lowest dilution, 0.05 X l O 9 bacterial equivalents/ml. The inflammatory response to A. actinomycetemcomitans at this low dose does not appear to be related to the virulence of the strain. In many M A M virulence studies, lesion size and death of the mice were shown to be dose dependent. Genco etal. (1991) demonstrated the pathological course of P. gingivalis using many strains and dilutions, and found that at higher doses severe abscesses or death resulted, where lower doses demonstrated smaller localized lesions or no pathology at all. These results are in agreement with Ebersole et al. (1995) who demonstrated dose dependency for P. gingivalis, W. recta, and F. nucleatum. Although the mice in the non-viable bacterial model did not die, virulence variation was shown to be dose dependent for A. actinomycetemcomitans as indicated by the Histologic Score. In the M A M studies, most of the animals developed severe abscesses by 24-48 hour, with septicemia or death by day 4-5. If the mice did not die, the lesion size remained stable until day 7- 9. Resolution would occur by approximately 12-15 days. The acute responses for the live bacterial model occurred within hours. Day 5 represents the acute time period in the non-viable bacteria study for strain JP2 (Figure 2(d), 8(a) and (b)); day 15 demonstrates a similar or less severe reaction for JP2, the P M N response being less localized and more diffuse. Y 4 is also time dependent, but with less variation in inflammatory response between day 5 and day 15. B y day 25, the inflammatory response was similar for all three strains. 104 Non-viable P. gingivalis elicited only a slight P M N response on day 1. No P M N s were seen after day 3 (Van Steenbergen et al, 1982; Genco et al, 1991). This is markedly different from the inflammatory response detected using non-viable A. actinomycetemcomitans in this study. Further investigation is needed to draw conclusions about the comparative virulence of non-viable P. gingivalis and non-viable A. actinomycetemcomitans. 4.2.2 Immunohistological Response in the M A M and Non-Viable Bacterial Model In this study, antibodies F4/80, anti-neutrophil and anti-CD3, were used to study specific cells recruited into the lesions during the inflammatory response. The Immunohistologic Scores are presented in Table 1 for the three antibodies after injection with undilute JP2, Y 4 and I D H 1705 strains on A. actinomycetemcomitans. A l l three antibodies identified specific leukocyte subpopulations at day 5 and day 15. 4.2.2.1 Time Effects on the Immunohistologic Response JP2 and Y 4 (both strain b), demonstrate a very strong F4/80 response (monocyte and macrophage) at day 5 and 15. I D H 1705 did not demonstrate quite as strong a response on day 5, but was equal to the other two strains by day 15. This was closely matched by the neutrophil antibody response, with JP2 showing the highest Immunohistologic Scores and I D H 1705 again 105 showing a stronger reaction at day 15 than day 5. As expected, the F4/80 and neutrophil responses were high at days 5 and 15, representing the early acute and late acute phases of inflammation in this study. P M N s and macrophages are the most important phagocytic cells in the defense of the host against acute bacterial infections (Van Dyke & Hoop, 1990) and this was demonstrated in this study for both P M N s and macrophages. In a similar quantitative study of P. gingivalis performed by Gemmell (1997), H and E staining demonstrated a P M N , macrophage inflammatory response from day 1 in their murine abscess model study. They also detected and counted C D 4 and C D 8 T cells, noting their presence in the lesions from day 1, with increasing numbers during the time course of the experiment. This is in agreement with the results of this study for JP2, where the C D 3 response is greater at day 15 than day 5. For Y 4 , both days 5 and 15 demonstrate an equal response while I D H 1705 produced a low Immunohistologic Score, especially at day 15. While the Immunohistologic Scores are relatively low, these results suggest that in this A. actinomycetemcomitans-induced inflammatory mouse model, there is a protective immune response to non-viable bacteria, depending on the virulence characteristic of the strain of A. actinomycetemcomitans. JP2 (serotype b), mounts a greater T cell response than Y 4 (serotype b), which in turn, demonstrates a greater response than I D H 1705 (serotype e). The late or chronic response at 25 days was not looked at in this study, so no conclusions can be made for the later immunologic response of the antibodies at that time. 4.3 Strengths and Weaknesses of the A. actinomycetemcomitans-induced Model of Inflammation The previously discussed abscess models of inflammation have many limitations, the first of which is its imprecise quantitation. Lesion size is variable, making assessment difficult, and 106 animal death often results. Histologic analysis is not always available. Quantitative assessment of the dynamics of the infection and resulting pathology is an important component in elucidating the pathogenesis of bacterial infections. The MAM is not capable of assessing these dynamics quantitatively. The application of this A. actinomycetemconitans-induced model of inflammation allows the use of the virulence components of A.a, which results in localized pathology, but does not kill the mice. Acute lesions, chronic infection, and resolution can be studied in this animal model. This model can localize or concentrate the bacteria, to study the PMN migration and phagocytosis during the critical stages of inflammation. The quantitative measurements take into consideration the number of cells seen in the inflammatory response, as well as the percentage of spread of the inflammation. The relationship between these cells relating to time and dose of bacteria can also be determined. Further studies could be designed to determine what effect particular drugs would have on cellular and immunological responses, or determine what mechanisms could enhance/inhibit the destructive host responses to periodontopathogens. This animal model could be used to grade virulence factors such as fimbriae or membrane proteins if assessed individually. The results of this study however must not be used to directly compare the mouse inflammatory and immunohistological mechanisms to the human subject, nor can we concluded that all bacteria will produce the same degree of inflammatory response as various strains of A. actinomycetemcomitans. The vast majority of the virulence studies to date have been done with Porphyromonas gingivalis strains and this study took the liberty to compare results as much as possible to the mechanisms of the response, rather than to the actual bacterial responses of P. gingivalis. 107 The reproducibility of the inflammatory response to various strains and serotypes of A. actinomycetemcomitans demonstrated a strong trend, rather than a statistically significant quantitative difference in virulence between strains of A. actinomycetemcomitans in the non-viable mouse model. Further studies with a greater number of mice would be needed to statistically prove the trends shown in this work. As only one observer did the entire experiment, intraobserver variation may have been inevitable. A repeat of the study with several observers and lab technicians could determine the validity of these results. However, this study did demonstrate a higher Histologic and Immunologic Score with strain JP2 than either Y 4 or I D H 1705 throughout the experiments. Strain IDH1705, considered the least virulent strain (Saarela et al, 1992), demonstrated the lowest Histologic Scores and the least Immunohistologic response overall. 108 CONCLUSIONS The objectives of this study were to develop a quantifiable, reproducible model of A. actinomycetemcomitans-mduced inflammation, that would demonstrate virulence differences between strains. This mouse model of inflammation, using non-viable A. actinomycetemcomitans bacteria, was able to identify and grade the virulence differences between several strains by the intensity of the induced inflammatory response. Inflammatory responses for various dilutions of bacteria were also determined and graded at selected time intervals, and found to be time dependent. It was demonstrated that non-viable A. actinomycetemcomitans induced both a histologic and immunohistologic response in a mouse model. The results suggest that the non-viable A. actinomycetemcomitans-induced lesions in C D - I mice are consistent with a strong innate immune response involving the recruitment of neutrophils. This is followed by the infiltration of phagocytic macrophages and T-cells. There are currently available no other animal models that study inflammation in a quantifiable, reproducible fashion. There are implications for future uses of this model to determine the quantitative bacteriology involved in virulence and immunological responses, treatment trials (antibiotics, anti-inflammatory agents, steroids), and ultimate healing of induced lesions in animal models. 109 R E F E R E N C E S Alexander M B , Damoulis PD. 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