bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of double‐stranded replicative forms of HCV RNA in liver tissue from chronically infected patients
Authors: Blight K. , Trowbridge R. , Gowans E. J.
Published: December 2007

SUMMARY. The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double‐stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV‐infected individuals, HCV‐specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT‐PCR). Initially, a strand‐specific RT‐PCR resulting from chemical modification of the 3′ end of the RNA was established using in vitro transcribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV‐infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2m LiCl (designed to precipitate single‐stranded and partially double‐stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single‐stranded RNA only). Amplification of positive sense HCV RNA from the LiCl‐insoluble fraction, but not from the LiCl‐soluble fraction nor in the RNase A‐digested sample, was consistent with the interpretation that single‐strand, but not double‐stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double‐stranded RNA species is formed during the HCV replication cycle.
Reactogenicity and immunogenicity of a new recombinant hepatitis B vaccine containing Pre S antigens: a preliminary report
Authors: Hourvitz A. , Mosseri R. , Solomon A. , Yehezkelli Y. , Atsmon J. , Danon Y. L. , Koren R. , Shouval D.
Published: December 2007

SUMMARY. A new vaccine against hepatitis B virus (HBV) infection, produced in mammalian Chinese hamster ovary (CHO) cells, contains the small (s), middle (Pre S2) and large (Pre SI) surface proteins of HBV. Three injections of a 5‐μg or 10‐μg dose were administered intramuscularly (i.m.) at 0, 1 and 6 months to a group of 105 young adults, who were monitored for a period of 6 months after the third injection. Seroconversion rates were 100% after the second injection of the 5‐μg or 10‐μg dose. Geometric mean litres of HBsAb at 1 month after the third injection were 12156 mIU ml‐1 and 13 482 mIU ml‐1 in those receiving the 5‐μg and 10‐μg dose respectively. The vaccine was well tolerated with no significant adverse events. These preliminary results suggest that the Pre S‐s recombinant vaccine, produced in mammalian cells, is highly immunogenic, leading to 100% seroconversion in the population tested after injection of only two doses of 5 μg.
2‘,3’‐dideoxy‐3‘‐fluoroguanosine inhibits duck hepatitis B virus in vivo
Authors: Lofgren B. , Vickery K. , Zhang Y. Y. , Nordenfelt E.
Published: December 2007

SUMMARY. Duck hepatitis B virus (DHBV) belongs to the same virus family as the human hepatitis B virus (HBV). Domestic ducks infected with DHBV can be used as an animal model for chronic hepatitis B virus infection in therapeutic trials. In this study the antiviral effect of the guanosine analogue 2′,3′‐dideoxy‐3′‐fluoroguanosine (FLG) was tried in vivo on chronically DHBV‐infected ducks. The ducks were either congenitally infected, or inoculated with DHBV immediately post‐hatch. FLG was given as intraperitoneal injections twice daily, at different dosages. Serum DHBV levels were determined by DNA dot‐blot hybridization. A strong inhibition of serum DHBV DNA was observed with FLG doses down to 1 mg kg‐1 day‐1. given for 7 to 10 days. With the corresponding thymidine analogue, 2′,3′‐dideoxy‐3′‐fluorothymidine; however, no inhibition was obtained. This difference may be due to different phosphorylation mechanisms. Independently of FLG dose, serum DHBV DNA returned to pretreatment levels within a few days after cessation of therapy. After a long‐term trial (FLG, 5mg kg‐1 day‐1 for 33 days), the same relapse of DHBV production was seen. Thus, FLG is an efficient inhibitor of DHBV replication, and is a candidate for treatment of HBV infections. However, the effect is transient, and therefore combination with other types of anti‐HBV drugs should be considered.
The expression of hepatitis B virus polymerase in hepatocytes during chronic HBV infection
Authors: McGarvey M. J. , Goldin R. D. , Karayiannis P. , Thomas H. C.
Published: December 2007

SUMMARY. A recombinant protein corresponding to part of the amino‐terminal domain of hepatitis B virus (HBV) polymerase was expressed in Escherichia coli. Antisera raised against this protein stained hepatocytes, from human liver biopsies, predominantly in the nucleus but some cytoplasmic staining was also observed. No staining was observed in hepatocytes from uninfected patients. Liver biopsies, taken from patients who were infected with human immunodeficiency virus (HIV) as well as HBV showed more intense staining with these antisera than that seen in patients who were infected with HBV alone. The staining pattern suggests that either the whole HBV polymerase protein, or a portion encoding the amino‐terminal domain, is translocated to the nucleus. This event may be an important early step in replication of the HBV genome.
Genotypes and multiple infections with hepatitis C virus in patients with haemophilia A in Japan
Authors: Fujimura Y. , Ishimoto S. , Shimoyama T. , Narita N. , Kuze Y. , Yoshioka A. , Fukui H. , Tanaka T. , Tsuda F. , Okamoto H. , Miyakawa Y. , Mayumi M.
Published: December 2007

SUMMARY. Hepatitis C virus (HCV) RNA was tested for, and HCV genotypes determined, in 96 patients with haemophilia A in Japan. Of 88 patients aged ≥ 10 years, 74 (84%) were positive for HCV RNA at a frequency higher than that in patients aged less than 10 years (one of eight, 13%P<0.001). Genotype I/1a was detected in 30 (40%), II/1b in 12 (16%), III/2a in eight (11%), IV/2b in five (7%) and V/3a in 12 (16%); mixed infection with HCV of two different genotypes was identified in the remaining nine (12%). This distribution was markedly different from that in 767 Japanese HCV carriers without haemophilia, in whom II/1b accounted for the majority (68.7%), I/1a was rare (0.5%), V/3a was absent, and mixed infection was observed rarely (1.3%). Mixed infection was transient in all of the seven haemophilic patients who were followed for 1 to 7 years. One of them was infected with genotype II/ 1b and an unclassifiable genotype, which showed nucleotide sequence similarity to genotype 4c from Zaire (82% homology in the El gene) and to 4a from Egypt (91% homology in a part of the NS5b region). In this patient, HCV of genotype II/1b disappeared while that of group 4 survived during a 4‐year observation period. These results indicate different epidemiology of HCV genotypes in Japanese haemophiliacs, attributable to HCV contaminating factor VIII imported in the past, and an increased opportunity in haemophiliacs for mixed infection with HCV of different genotypes.
Stimulation of human lymphocyte proliferation and CD40 antigen expression by phosphorothioate oligonucleotides complementary to hepatitis B virus genome
Authors: Chen C. , Zhou Y. , Yao Z. , Zhang Y. , Feng Z.
Published: December 2007

SUMMARY. We have studied the proliferation and CD40 antigen expression of lymphocytes, and the cytotoxicity to monocytes, of antisense phosphorothioate oligodeoxynucleotides complementary to the SP II promoter of HBV mRNA (sequence I) and the X gene (sequence II) in patients with chronic hepatitis B. The oligo sequence I stimulated proliferation of both T and, to a lesser extent, B cells. The percentage of cells expressing CD40 in T and B cell co‐cultures increased from 4.2% to 13.8% after oligo stimulation in patients, while it increased from 4.7% to 48.6% in healthy controls. The sense sequence (sequence III) of the X gene also enhanced the expression of CD40 antigen in patients with hepatitis B. The proportion of CD40 cells (26%) in a resting B‐cell preparation from hepatitis B patients decreased to zero after a 5‐day culture with sequence I, but IgG levels in the culture supernatant increased. The cytotoxic properties of monocytes were not influenced by the oligos. These findings indicate that antisense oligos against hepatitis B virus (HBV) have mitogenic effects on the proliferation of human lymphocytes in a non‐specific manner and may activate T cells to express CD40 antigen.
Hepatitis E virus infection in north‐east Italy: serological study in the open population and groups at risk
Authors: Gessoni G. , Manoni F.
Published: December 2007

SUMMARY. Developed western countries are considered to be relatively free from endemic foci of hepatitis E virus (HEV) infections. The aim of this study was to assess the seroepidemiology of HEV in north‐east Italy. Of the 2361 individuals studied 1889 were representative of the general population and 472 were from groups at high risk for viral infections: 279 drug users and 193 patients on chronic haemodialysis. All sera were tested for hepatitis C virus antibody (HCVAb), human immunodeficiency virus antibody (HIVAb) and for hepatitis B virus (HBV) serology. Two solid‐phase enzyme‐linked immunosorbent assays (ELISA) were used to study the seroepidemiology of HEV IgG, the first (using recombinant antigens) for screening, the second (using synthetic peptides) for confirmation of initially reactive samples. The prevalence of circulating hepatitis E virus antibody (HEVAb) was 2.6% in the open population, 5.4% among drug users and 9.3% among patients on chronic haemodialysis. In the open population a positive relationship between age and prevalence of HEVAb was observed. A relationship between presence of HEVAb and serological evidence of previous HBV or HCV infections was also observed in this study. It was concluded that HEV infections are present in north‐east Italy and are more frequent among subjects at risk for blood‐borne viral infections. The positive correlation, observed in the open population, between age and prevalence of HEVAb suggests the presence of a cohort effect.
Histopathological aspects of viral hepatitis
Authors: Scheuer P. J. , Davies S. E. , Dhillon A. P.
Published: December 2007

SUMMARY. The main method of classification of chronic viral hepatitis is now by cause, and the old histology‐based classification is no longer considered appropriate. However, liver biopsy remains an important part of patient assessment and, in the context of clinical trials, biopsy findings are often scored in a semiquantitative manner. The concepts of grading and staging, borrowed from tumour pathology, have been introduced, representing the severity of the necroinflammatory lesion and the extent of its structural consequences respectively. The pathology of the individual forms of viral hepatitis A to G shows more similarities than differences. However, some pathological features are commonly associated with specific viruses. The combination of portal lymphoid follicles, bile duct damage, lobular activity and steatosis give chronic hepatitis C a characteristic histological profile. Very similar appearances have been noted in the limited number of biopsies so far reported from patients with known combined hepatitis C and G virus infection.
Cultural methods for the identification and quantitation of Streptocococcus mutans and lactobacilli in oral samples
Authors: Jordan Harold V.
Published: December 2007

Literature on cultural techniques that have been used for monitoring populations of acidogenic and aciduric bacteria in oral samples was reviewed. Development of selective culture media for specific identification and quantitation of S. mutans and lactobacilli was discussed and evaluated. Adaptation of these selective cultural methods into simplified diagnostic tests for use in the dental clinic was described. Additional considerations were presented for future development of cultural procedures for assessment of microbiological risk factors in caries.
DNA probe detection of periodontal pathogens
Authors: French C. K. , Savitt E. D. , Simon S. L. , Eklund S. M. , Chen M. C. , Klotz L. C. , Vaccaro K. K.
Published: December 2007

The technology for hybridization of nucleic acids using isotopically labeled whole genomic or species‐specific cloned deoxyribonucleic acid (DNA) probes for Actinobacillus actinomycetemcomitans, Bacteroides intermedius and Bacleroides gingivulis can be used to detect as few as 102 cells of these periodontal pathogens. The sensitivity and specificity of the test is not affected by the presence of 5x 10s unrelated bacteria in constructed mixed culture samples. Current feasibility tests with non‐isotopic reagents also give reliable and rapid detection of these oral pathogens in pure or mixed cultures. DNA probes should prove useful in a rapid, specific, and sensitive assay for these periodontal pathogens.
Rapid method for detection of lactose fermenting oral microorganisms
Authors: Alcoforado Gil A. P. , McKay Terry L. , Slots Jørgen
Published: December 2007

Abstract A rapid inexpensive assay for lactose fermentation which can be performed on individual colonies in situ and which does not negate subcultivation is described. The assay is based on the ability of bacterial β‐galactosidase to hydrolyze 4‐methylumbelliferyl‐β‐D‐galactoside (MUG) and form 4‐methylumbelliferone, a compound which is brightly fluorescent under long‐wave ultraviolet light. 1% MUG in dimethyl sulfoxide was stable at room temperature for at least 21 days. Maximal MUG fluorescence reactions required viable bacterial cultures. The MUG test represents a convenient means to distinguish the lactose‐negative Bacteroides intermedius and Haemophilus actinomycetemcomitans, both suspected major periodontopathogens, from the lactose‐positive Bacteroides melaninogenicus group and Haemophilus aphrophilus, organisms of little or no periodontopathic significance. The MUG test may also greatly help in distinguishing lactose from nonlactose fermenting organisms of nonoral origin.
Longitudinal clinical and microbiological study on the relationship between infection with Streptococcus mutans and the development of caries in humans
Authors: Lang Niklaus P. , Hotz Peter R. , Gusberti Francesco A. , Joss Andreas
Published: December 2007

This 24 months prospective study was undertaken to test the principle that infection with S. mutans on human fissure and smooth tooth surfaces at risk for caries will lead to decalcification and/or cavitation lesions. Forty‐eight elementary school students aged 7‐8 years were recruited and enrolled in the study in an overlapping fashion. The children were examined annually for plaque, gingivitis and dental caries which was scored both clinically and radiographically. In addition, 4‐6 microbiological samples were obtained every 3 months from each participant on predetermined sites of mesial aspects or fissures of first molars using sterile floss or hypodermic needles. The test sample represented 258 tooth surfaces at risk producing a total of 1528 microbiological samples. The samples were processed and identified on selective and non‐selective media using anaerobic techniques. The total colony forming units (CFU/ml) and % of anaerobic and facultative organisms and S. mutans were enumerated. During the entire duration of the study 40 initial (DI) and 6 carious lesions with cavitation (DC) developed in 36 subjects. Eleven established initial lesions (EI) and 12 remineralized surfaces (R) were followed. The control sample consisted of three groups of sites which remained caries‐free throughout the study: 34 sites in control group (CA) of caries‐free children (DFS = 0), 35 sites in children with some filled teeth (CB) (DFS > 5) and 36 sites in children with only one initial lesion (CC) (DFS= 1 or 2) were followed microbiologically for 1‐2 years. DI and DC showed significantly higher levels of S. mutans than controls 9 and 6 months prior to the clinical diagnosis, while R surfaces yielded significantly lower S. mutans levels 9, 6 and 3 months prior to the diagnosis of the reversal. However, in one single DC lesion, S. mutans could not be documented at any observation period prior to the diagnosis of the cavity. Thus, the results confirmed the role of S. mutans as an important, but not exclusive initiator of dental caries. Furthermore, a temporal relationship between infection with S. mutans and clinical diagnosis could be demonstrated.
Comparative studies on the biology of Actinobacillus actinomycetemcomitans leukotoxin in primates
Authors: Taichman Norton S. , Simpson David L. , Sakurada Shigeyo , Cranfield Michael , DiRienzo Joseph , Slots Jørgen
Published: December 2007

These studies concern the biological properties of the Actinobacillus actinomycetemcomitans leukotoxin in different primates. We compared the sensitivity of primate polymorphonuclear leukocytes (PMNs) to the leukotoxin and determined whether animals had toxin‐neutralizing antibodies in serum. In addition, subgingival plaques from representative non‐human primates were analysed for leukotoxin‐producing A. actinomycetemcomitans. PMNs from different primates varied significantly in sensitivity to leukotoxic extracts of A. actinomycetemcomitans. Neutrophils from humans, the Great Apes and most Old World Monkeys were destroyed upon in vitro exposure to the toxin. By contrast, PMNs from Prosimii, New World Monkeys and the Lesser Apes were not sensitive to the leukotoxin. Human PMNs were more sensitive when compared to PMNs of non‐human primates. Chimpanzee PMNs were closest to human PMNs in terms of sensitivity to the toxin. The data indicate that the leukotoxin has unique specificites for primate PMNs and is especially potent against human leukocytes. Sera from the vast majority of adult primates were capable of neutralizing the A. actinomycetemcomitans leukotoxin, including animals whose PMNs were resistant to the toxin. This suggests that most non‐human primates have been exposed to toxin‐producing strains of A. actinomycetemcomitans, and this was confirmed in representative species where leukotoxic A. actinomycetemcomitans could be isolated from subgingival plaque samples. By contrast, less than 20% of sera from normal humans neutralized the leukotoxin whereas over 90% of sera from juvenile periodontitis patients had relatively high toxin‐neutralizing antibody liters. It appears that the majority of adult primates harbor A. actinomycetemcomitans and produce antibodies against the leukotoxin of this organism. In humans, on the other hand, these phenomena are usually associated with destructive periodontal disease. Differences between man and non‐human primates in terms of their sensitivity to and/ or in the dynamics of the immune response against the leukotoxin, may be important determinants in explaining why this organism seems to belong to the normal indigenous oral microflora in some primates but acts as a periodontal pathogen in man.
Effects of extracts from Bacteroides gingivalis, Bacteroides intermedius, and Bacteroides asaccharolyticus on the growth of fibroblast lines obtained from healthy and inflamed human gingiva
Authors: Larjava Hannu , Uitto Veli‐Jukka
Published: December 2007

The effect of sonicates from Bacteroides gingivalis, Bacteroides intermedius, and Bacteroides asaccharolyticus on the uptake of (3H)‐thymidine by different gingival fibroblast cell lines was studied. Extracts from B. intermedius stimulated thymidine uptake by as much as 150% over that of controls. This effect varied according to both the fibroblast line and the concentration of the bacterial extract. B. asaccharolyticus extracts inhibited the thymidine uptake of all fibroblast cell lines at high concentrations, but in low doses the thymidine uptake was stimulated in some fibroblast lines by up to 200%. B. gingivalis extract caused a strong, dose‐dependent inhibition of thymidine uptake in all of the fibroblast lines tested. Low concentrations of B. gingivalis extract (10 μg protein/ml) inhibited DNA‐synthesis by 12‐60%, and high concentrations (100 μg protein/ml) by 70‐90%. The results suggest that the response of periodontal fibroblasts to oral Bacteroides varies considerably depending on the Bacteroides species, as well as on the individual characteristics of the cell lines being tested.
Penicillin‐degrading enzymes in sites associated with adult periodontitis
Authors: Walker C. B. , Tyler K. Z. , Low S. B. , King C. J.
Published: December 2007

The incidence and enzymatic activity of penicillin‐degrading enzymes (beta‐lactamases) in gingival crevicular fluid were determined for 406 sites in 52 patients with adult periodonititis. Beta‐lactamase activity was detected in 64% of the patients and in 24% of the sites sampled. The enzyme was present in 13% of 206 sites and 36% of 200 sites having probing depths of ≤3 mm and >3 mm, respectively. The incidence of the enzyme was positively correlated with increases in pocket depth up to 8 mm. However, there was no correlation between probing depths and the enzyme activity present. The range of activity detected was equivalent to 0.001 to 0.569 enzyme units per ml of gingival fluid. Approximately 60% of the sites in which beta‐lactamase was detected, or 19% of all sites sampled with probing depth > 3 mm, contained enzymatic activity sufficent to inactivate penicillins at the concentrations available in gingival fluid.
Bacteroides forsythus in adult gingivitis and periodontitis
Authors: Lai C.‐H. , Listgarten M. A. , Shirakawa M. , Slots J.
Published: December 2007

Abstract Bacteroides forsythus, a newly named species from the human mouth, possesses a distinct cell wall ultrastructure and a unique set of cell surface antigens. To determine the distribution of B. forsythus in the periodontal region, supragingival plaque was collected from 16 healthy adults and from 11 adults with mild gingivitis, 12 with severe gingivitis and 17 with periodontitis. Subgingival samples from 27 diseased sites were examined as well. B. forsythus was located in plaque smears by indirect immunofluorescence microscopy. Rabbit antibody to B. forsythus strain FDC 335 constituted the primary antibody and fluoresceinlabelled goat‐antirabbit antibody the secondary antibody. There was a significant difference (p<0.05) between the proportions of B. forsythus in supragingival samples of healthy subjects (0.2%) vs. individuals with mild gingivitis (1.3%), severe gingivitis (1.0%) and adult periodontitis (0.9%). A significant difference (p< 0.001) in B. forsythus proportions was also detected between supragingival (0.9%) and subgingival (15.3%) samples from adult periodontitis patients. In a separate study, patients previously treated for moderate to severe adult periodontitis were monitored over a 12‐month period for evidence of disease recurrence. A significant increase in the proportions of B. forsythus was found at sites with breakdown as compared to stable sites. Breakdown was defined as loss of attachment of 2 mm or more from base line, as measured from a reference stent, or a probing depth increase of 3 mm or more. These results support the assertion that B. forsythus is associated with advanced periodontitis as well as recurrent periodontitis. Monitoring the subgingival proportions of B. forsythus may be of diagnostic value in patients on maintenance therapy. Whether B. forsythus causes periodontal disease or is a secondary colonizer of periodontal lesions remains to be determined.
Applicability of monoclonal antibodies to quantitatively monitor subgingival plaque for specific bacteria
Authors: Gmür Rudolf
Published: December 2007

The aim of the present investigation was to assess the applicability of monoclonal antibodies For the quantitative determination of specific bacteria in subgingival plaque. Periodonlal pocket samples from sites with recurrent periodontal breakdown were investigated in parallel by culture analysis, by direct immunofluorescence with species‐specific polyclonal antiscra to Bacteroides gingivalis and Bacteroides intermedius, and by indirect immunofluorescence with one monoclonal antibody specific for Bacteroides forsythus, 3 monoclonal antibodies directed against B. gingivalis and 3 monoclonal antibodies recognizing B. intermedius, B. forsythus was consistently detected in high numbers by immunofluorescence, but never by cultural analysis. For B. gingivalis and B. intermedius culture scores were similar or lower than those obtained with the serological technique. Monoclonal and polyclonal antibodies yielded quantitatively very similar results. Neither evidence for a significant interference of host antibodies with the labelling of target bacteria by single epitope‐specific monoclonal reagents, nor for unspecific staining due to the labelling of crossreactive antigens was observed. The results demonstrate that, following careful selection and characterization, monoclonal antibodies can indeed by successfully applied for a rapid, quantitative and highly sensitive species ‐ or serotype‐specific screening of subgingival plaque.
Yeasts, enteric rods and pseudomonads in the subgingival flora of severe adult periodontitis
Authors: Slots Jørgen , Rams Thomas E. , Listgarten Max A.
Published: December 2007

The subgingival occurrence of yeasts and species of Enterobacteriacae and Pseudomonas was studied in 500 adults with severe periodontitis. All subjects had sites non‐responding or “refractory” to conventional periodontal therapy. Most subjects had received one or more courses of broad‐spectrum systemic antibiotics. Subgingival microbial samples were collected with paper points and transported in VMGA III. The bacterial samples were plated on enriched brucella blood agar and incubated anaerobically, and on TSBV, TBC, and Sabouraud agar, which were incubated in 10% CO2. Yeasts were speciated using the API 20C micro‐method system and the germ tube test (for Candida albicans). Enteric organism's and pseudomonads were speciated with the API 20E kit system. Yeasts, enteric rods and pseudomonads were subjected to antibiotic susceptibility testing. The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedium was also determined in the patients studied. In the 500 periodontitis patients, yeasts were detected in 84 (16.8%), enteric rods or pseudo‐monads in 51 (10.2%), and both yeasts and enterics or pseudomonads in 6 (1.2%). Candida albicans comprised 83.3% of the isolated yeasts. Enterobacteriaceae averaged 21–39% of the cultivable flora in culture‐positive cases, with Enterobacter cloacae, Enterobacter agglomerans, Proteus mirabilis, Klebsietta pneumonias, and Klebsiella oxytoca being the most frequent species. Pseudomonas aeruginosa was isolated from 10 patients, averaging 23.4% of the cultivable subgingival flora. All Candida isolates, and 86–95% of the enteric rods and pseudomonads, were resistant to 1 μg/ml of tetracycline, penicillin G, and erythromycin. In patients positive for yeasts, enteric rods or pseudomonads, A. actinomycetemcomitans was isolated from about one‐fifth, B. intermedius from about one‐third, and B. gingivalis from 5% of the individuals. The present findings suggest that yeasts or enteric rods or pseudomonads occur in the subgingival flora of about one‐third of “refractory” adult periodontitis patients. We caution against using antibiotics without prior microbiological screening in treating this patient group.
Actinobacillus actinomycetemcomitans fimbriae
Authors: Rosan Burton , Slots Jørgen , Lamont Richard J. , Listgarten Max A. , Nelson Genevieve M.
Published: December 2007

Freshly isolated Actinobacillus actinomycetemcomitans strains were found to possess fimbriae. These appendages appeared to be irreversibly lost after 30 to 40 subcultures in the laboratory. The fimbriated strains were associated with a specific type of colonial morphology designated SP (Star Positive); the non‐fimbriated variant colony was designated SN (Star Negative). The fimbriae were approximately 5 nm wide and could be several μm in length. The fimbriated cells tended to aggregate readily whereas the non‐fimbriated cells formed uniform suspensions. Three stains of fimbriated A. actinomycetemcomitans isolated from different patients were compared to their non‐fimbriated variants for attachment to hydroxyapatite (HA) and saliva coated hydroxyapatite (SHA). Two of the fimbriated variants exhibited a 3‐ to 4‐fold enhancement of attachment to HA and SHA over their non‐fimbriated variant. However, there did not appear to be any specificity for SHA over HA. One strain (Pag) showed no major differences in attachment of the fimbriated or non‐fimbriated variant, albeit the fimbriae were indistinguishable from those of the other A. actinomycetemcomitans strains. Thus, there may be molecular differences among fimbriae of various A. actinomycetemcomitans strains which determine both their tissue specificity and their affinity for particular substrates.
Fimbriation of Actinobacillus actinomycetemcomitans
Authors: Preus Hans R. , Namork Ellen , Olsen Ingar
Published: December 2007

Presence of fimbriae on a bacterium may promote its initial colonization, survival and ability of tissue penetration. Thus, electron microscopy was performed on negatively stained periodontal Actinobacullus actinomycetemcomitans from a patient with Papillon‐Lefevre syndrome. The isolates were, in contrast to reference strains found to carry a considerable amount of fimbriae (5 nm in diameter). The presence of fimbriae may support the hypothesis of the invasive ability of this bacterium.
A simple and rapid plaque‐sampling assay for monitoring the number of cariogenic organisms
Authors: Wyatt C. C. L. , MacEntee M. I. , McBride B. C.
Published: December 2007

Specimens of debris and plaque adhering to the facial surface of teeth and samples of saliva from children and young adults were compared as a source of cariogenic bacteria. The specimens were easily obtained and offered an estimate of bacterial colonization that was comparable to the saliva. Material adhering to the facial surface of teeth provides a convenient source of bacteria and may be useful to assess caries risk in subjects who cannot supply a sample of saliva.
Growth and metabolic properties of Bacteroides intermedius in anaerobic continuous culture
Authors: Hamilton I. R. , McKee A. S. , Bowden G. H.
Published: December 2007

Two strains of Bacteroides intermedius BH20/30 and BH18/23, have been grown in anaerobic continuous culture under various conditions for periods up to 54 days. Strain BH20/30 grew over a relatively wide pH range from 5–8 with a maximum at pH 7.0 at a dilution rate (D) of 0.1 h‐1 with a glucose limitation, while strain BH 18/23 had an optimum between 5.8 and 7.3 and would not grow above and below this range. The maximum growth rate (μmax) for the latter strain was shown to be 0.23 h‐1, or a doubling time of 3.0 at the upper limit of pH 7.3. The yield values (Yglucose) for strain BH18/23 reached 187–177 g cells (dry weight) per mole of glucose in the optimum pH range (6.0–7.0) and amino acid analysis of the spent medium indicated that these high values were the result of the combined use of glucose and amino acids; the cultures also exhibited proteolytic activity. The major acid end‐products in the same pH range were formate and succinate with lesser concentrations of acetate, isovalerate and fumarate; small amounts of lactate appeared as the cells were stressed at pH values above 7.5 when the culture was‘washing out’of the chemostat. Glucose metabolism appeared to function through the glycolytic pathway in B. intermedius BH 18/23 since the glycolytic inhibitors, sodium fluoride and sodium iodoacetate, completely inhibited glucose utilization as did the proton ionophore, gramicidin, and the ATPase inhibitor, N,N1‐dicyclohexylcarbodiimide (DCCD). Inhibition by these latter compounds indicated that the saccharolytic Bacteroides utilize proton gradients generated by proton‐extruding ATPase (H+/ATPase) to conserve energy. B. intermedius BH 18/23 was shown to possess activity in membrane preparations for enzyme II (glucose) of the P‐enolpyruvate phosphotransferase system (PTS) indicating that sugar transport by the organism occurs, at least in part, by this group translocation process.
The capacity of subgingival microbiotas to produce volatile sulfur compounds in human serum
Authors: Persson Sten , Claesson Rolf , Carlsson Jan
Published: December 2007

Hydrogen sulfide is formed by the subgingival microbiotas of periodontal pockets. The capacity of these microbiotas to form various volatile sulfur compounds in human serum was studied. Bacterial samples from nine deep periodontal pockets were incubated for 7 days in human serum and the amounts of volatile sulfur compounds and the degradation of serum proteins were determined. Hydrogen sulfide was the predominant volatile sulfur compound, but also methyl mercaptan was formed in significant amounts. Only traces of dimethyl sulfide and dimethyl disulfide were detected. There was an extensive degradation of the serum proteins. In most of the reaction mixtures hydrogen sulfide reached highly toxic levels.
A comparison of antibody levels to Bacteroides gingivalis in serum and crevicular fluid from patients with untreated periodontitis
Authors: Baranowska H. I. , Palmer R. M. , Wilson R. F.
Published: December 2007

The levels of IgG antibody to Bacteroides gingivalis were measured in serum and sequential samples of crevicular fluid from healthy and diseased sites in patients with untreated periodontitis using ELISA. All subjects had detectable serum litres but there was a wide variation in litre between subjects. Moderate to strong correlations were found between serum and crevicular fluid levels of IgG. A statistically significant difference was observed between sequential samples of crevicular fluid. There was no difference in the level of specific IgG lo B. gingivalis in crevicular fluid between healthy and diseased sites within the same individual.
The distribution of trypsin‐like enzyme activity in cultures of a virulent and an a virulent strain of Bacteroides gingivalis W50
Authors: Smalley John W , Birss Andrew J , Kay Helen M , McKee Ailsa S , Marsh. Phillip D
Published: December 2007

The distribution of trypsin‐like enzyme activity was studied in 48‐ and 72‐h batch cultures of Bacteroides gingivalis W50 and an avirulent variant (W50/BE1) of the parent strain. Activity was measured at pH 7.4 in cells, the extracellular vesicle (ECV) and soluble extracellular protein (EP) fractions recovered by ammonium sulphate precipitation from spent growth medium. Both organisms produced cell surface and extracellular vesicles, but whilst strain W50 produced more ECV, W50/ BE1 yielded more of the EP fraction by weight. Whole cultures of W50 displayed a three‐fold greater trypsin activity than those of W50/BE1. However, 90% of the total enzyme activity of W50 cultures was associated with the particulate fraction (cells and ECV totalled), whereas this fraction accounted for only 10‐30% of the total for W50/BE1. Unlike W50/BE1, the specific activities of W50 cells and ECV rose in 72‐h cultures. Conversely, cultures of W50/BE1 displayed an increase in the yield and specific activity of the EP fraction.
Immunomodulatory effects of Bacteroides products on in vitro human lymphocyte functions
Authors: Shenker Bruce J. , Slots Jorgon
Published: December 2007

Bacteroides spp. have been implicated in the pathogenesis of several diseases, including periodontal diseases. In this study sonic extracts of 6 Bacteroides spp. were examined for their abilities to alter human lymphocyte function. We found that soluble extracts from Bacteroides intermedius, Bacteroides endodontalis, Bacteroides asaccharolylicus, Bacteroides melaninogenicus, and to a lesser degree Bacteroides loescheii, caused dose‐dependent inhibition of human lymphocyte responsiveness to both mitogens and antigens. Suppression involved altered DNA, RNA and protein synthesis as well as immunoglobulin production. In contrast, Bacteroides gingivalis did not suppress these responses; instead, it stimulated lymphocyte proliferation and enhanced immunoglobulin production. It has been proposed that impaired host defense may play a pivotal role in the pathogenesis of many infections. The data presented in this paper suggest that microbial mediated immunosuppression may conceivably alter the nature and consequences of host‐parasite interactions in periodontal disease.
Staphylococci in human periodontal diseases
Authors: Rams Thomas E. , Feik Diane , Slots Jörgen
Published: December 2007

The occurence of subgingival staphylococci was determined in 506 individuals with advanced adult periodontitis, 108 with early‐onset periodontitis, 13 with localized juvenile periodontitis, 18 with gingivitis, and 13 with 20 failing osseointegrated titanium dental implants. Subgingival samples were collected with paper points and transported in VMGA III. The bacterial samples were plated on Staphylococcus 110 medium which was incubated in 10% CO2, and on enriched brucella blood agar, which was incubated anaerobically. Staphylococcal isolates from 94 adult periodontitis subjects were speciated using the API STAPH Trac micromethod kit system and the Bacto Staph latex agglutination test for coagulase activity. Staphylococcus epidermidis comprised 45.8% and Staphylococcus aureus 22.3% of total staphylococcal isolates. At 1 μg/ml, in vitro resistance by staphylococci was found to tetracycline (14.4% of isolates), penicillin (4.9%), erythromycin (12.1%), and metronidazole (31.9%). Subgingival staphylococci were isolated from approximately 50% of gingivitis and periodontitis patients. No statistically significant differences were found between these patient groups in the prevalence or mean proportions of Staphylococci recovered. “Periimplantitis” lesions exhibited significantly higher proportions of Staphylococci (15.1%) than gingivitis (0.06%) or periodontitis (1.2%) lesions. Staphylococci may play a role in some failing osseointegrated dental implants.
Some aspects of protease production by a strain of Streptococcus sanguis
Authors: Rogers A. H. , Zilm P. S. , Pfennig A. L. , Gully N. J.
Published: December 2007

Our previous studies indicated that Arginine (Arg) plays a key nutritional role in Streptococcus sanguis P4A7 and that this organism can grow on whole casein as the sole nitrogen source. Its protease activities were therefore studied after glucose‐limited continuous culture in a chemically‐defined medium with either free amino acids or casein as the nitrogen source. Both culture supernatant and cell‐associated endopeptidase (EP) and exopeptidase (amino‐AP and carboxy‐CP) activities were determined. Growth rate (μ) had little effect on EP, 75% of which was consistently in culture supernatants; AP and CP both decreased as μ was increased and both were predominantly cell‐associated. At high growth pH, EP was substantially increased while AP and CP activities were optimal at pH 7. The most striking nutritional effect occurred under nitrogen limitation (glucose excess) when EP and AP were greatly increased and CP greatly decreased. It was concluded that S. sanguis is well equipped to scavenge its environment for Arg under a wide range of growth conditions.
Improved detection of oral spirochetes with an anaerobic culture method
Authors: Umeda M. , Ishikawa I. , Benno Y. , Mitsuoka T.
Published: December 2007

A method for routine culturing of oral spirochetes was studied in periodontitis patients and subjects with healthy gingivae. Subgingival plaque bacteria, including oral spirochetes were cultured anaerobically using the steel wool jar method, (he glove‐box procedure and the plate‐in‐bottle method. They were also observed directly with a phase‐contrast microscope. The number of colonies of recovered spirochetes was significantly higher with the plate‐in‐bottle method when Medium 10 (M10) or M10 supplemented with 10% rumen fluid was used. In almost all cases, spirochetes were detected with this culture method. The ratios of spirochetes from patient samples, cultured by the plate‐in‐bottle method, and of other microorganisms, cultured in Eggerth‐Gagnon (EG) medium by the steel wool jar method, were almost identical of those observed by phase‐contrast microscopy. These results indicate that it can be just as possible to culture certain oral spirochetes as other anaerobic bacteria.
Humoral immunity in root caries in an elderly population. I.
Authors: Butler John E. , Spradling Jill E. , Peterman Jeffrey H. , Joshi Kalpana S. , Satam Mangesh , Challacombe S. J.
Published: December 2007

IgA, IgG and IgM antibody activity (ELISA Units/ml) to Streptococcus mutans, Actinomyces viscous and Escherichia coli CF8 in serum, parotid saliva and whole saliva was measured using the amplified ELISA (a‐ELISA) while the concentration (μg/ml) of each isotype of immunoglobulin as well as albumin and lactoferrin, was determined using sandwich ELISAs. Selection of suitable reagents from those commercially available was based on specificity tests using purified human immunoglobulin; most polyclonal reagents required further absorption to attain class specificity. Cross‐absorption studies indicated the absence of patient antibodies that were cross‐reactive among the bacteria studied, except for IgM in some cases. Expression of response in ELISA Units (E.U.) per μg of immunoglobulin, i.e. specific activity, revealed that IgG specific activity was significantly higher in parotid saliva than in either whole saliva or serum for all bacteria studied; serum and whole saliva did not differ except for the higher specific activity in whole saliva to E. coli. The value of one E.U. was determined using the Comparative Antibody‐immunoglobulin Capture Assay (CACA). Using this novel method, we estimated that about 0.05 percent of serum IgA was specific for Streptococcus mutans, 0.008 for Actinomyces viscosus and 0.004 for Escherichia coli CF8. The percentage of specific IgM antibodies was higher than for IgA and IgG. The concentration of IgA anti‐Streptococcus mutans, Actinomyces viscosus and Escherichia coli levels are approximately 92 ng/ml, 25 ng/ml and 16 ng/ml in whole saliva and 46 ng/ml, 9.4 ng/ml and 6.3 ng/ml in parotid saliva. We calculated that normal, elderly adults secrete circa 25 ng of IgA anti‐Streptococcus mutans per min into both whole and stimulated parotid saliva. The ratio of salivary antibodies to the locally produced protein, lactoferrin (LF) and the serum‐derived proteins (albumin) together with simultaneous monitoring of salivary flow rates, provide additional insight into saliva dynamics. Among randomly‐selected patients, IgA and LF concentrations are inversely correlated with flow rate and > 70% of the IgG in whole saliva can be calculated to be of local origin; the latter is also consistent with the higher specific activity of the IgG in whole saliva than in serum. Using albumin:IgG ratios, the small amount of IgG in parotid saliva can be accounted for by transudation from serum.
Killing of oral, gram‐negative, facultative bacteria by the rabbit defensin, NP‐1
Authors: Miyasaki Kenneth T. , Bodeau Amy L. , Selsted Michael E. , Ganz Tomas , Lehrer Robert I.
Published: December 2007

Oral, gram‐negative, facultative bacteria, including Actinobacillus actinomycetemcomitans, Eikenella corrodens, and Capnocytophaga spp. have been associated with destructive periodontal infection. Neutrophils play a critical role in defending the periodontium against destructive infection. Defensins are antimicrobial peptides that have been isolated in human, rabbit, guinea pig, and rat leukocytes that may constitute an important nonoxidative mechanism of killing. The purpose of this study was to examine the sensitivity of a battery of oral, gram‐negative, facultative bacteria to the bactericidal effects of the isolated rabbit peptide NP‐1. All species tested were killed by NP‐1; however, there was strain‐to‐strain variation in sensitivity. The bactericidal effect was not dependent on net bacterial growth, although metabolic activity was evident as assessed by bacterial oxygen consumption. We conclude that bacteria are sensitive to the cidal mechanism involved in defensin‐mediated bacterial killing and that the conditions of this assay system support the killing of bacteria by the defensin peptides.
Black‐pigmented, asaccharolytic Bacteroides species resembling Porphyromonas gingivalis (Bacteroides gingivalis) from beagle dogs
Authors: Yamasaki T. , Nagata A. , Kiyoshige T. , Sato M. , Nakamura R.
Published: December 2007

Black‐pigmented, asaccharolytic Bacteroides strains, which positively reacted with anti‐Bacteroides gingivalis (now reclassified as Porphyromonas gingivalis) serum, were isolated from beagle dogs, and their characteristics were studied and compared with those of P. gingivalis. The strains from dogs were different from P. gingivalis in their catalase activity, nutritional requirements and oxygen tolerance, and double‐immunodiffusion tests showed serological dissimilarity between the strains from dogs and P. gingivalis. However, the strains from dogs had guanine‐plus‐cytosine contents of 49.0 to 49.3 mol %, which were very similar to P. gingivalis, and they showed strong DNA‐DNA hybridization with P. gingivalis. These results indicated that beagle dogs had Porphyromonas species analogous to P. gingivalis in their oral cavities.
The association of selected bacteria with the lesions of root surface caries
Authors: Bowden G. H. , Ekstrand J. , McNaughton B. , Challacombe S. J.
Published: December 2007

Plaque from the root surfaces of 165 subjects (mean age 65.5 years, 22–26 teeth/subject) was analysed for specific bacteria. Five subject groups were defined: A (DMFS 16.4), B (DMFS 55.9), C1 (DMFS 55.6), C2 (DMFS 57.0) and C3 (DMFS 48.1). Groups C1 and C2 had unrestored root surface lesions: Groups A, B and C3 were free of unrestored root caries and differed in their coronal caries experience. Streptococcus mutans was isolated more frequently from the root lesions in Groups C1 and C2 than from intact root surfaces in Group A, Streptococcus oralis, Streptococcus mitis 1 and Streptococcus sanguis were isolated more frequently from Group A. The percentage contribution that S. mutans made to plaque from lesions in Groups C1 and C2 was higher than that from plaque in Group A and Actinomyces viscosus serovar 2 contributed more to plaque in Group C1 than in samples from Group A. The percentage counts of Lactobacillus in plaque from lesions in Groups C1 and C2 were higher than those from intact roots in Groups A, B, and C3. Subjects were also grouped on the presence of Lactobacillus and S. mutans in plaque samples. Samples with both organisms (n= 17) showed significantly higher isolation frequencies of specific strains of S. mitis 1 and also A. viscosus serovar 2 compared with samples of plaque containing S. mutans or Lactobacillus. Actinomvces naeslundii serovar 1 was not isolated from samples containing both S. mutans and Lactobacillus. The results confirm an association of S. mutans and Lactobacillus with root surface lesions and suggest a relationship between lesions and A. viscosus serovar 2. Selection of samples based on the presence of S. mutans and Lactobacillus showed that specific and different communities may be associated with root surface lesions. These communities reflect the local environment and as such could be valuable indicators of the state of the disease or risk of progression of lesions.