Testing of viscous anti-HIV microbicides using Lactobacillus.

The development of topical microbicides for intravaginal use to prevent HIV infection requires that the drugs and formulated products be nontoxic to the endogenous vaginal Lactobacillus. In 30min exposure tests we found dapivirine, tenofovir and UC781 (reverse transcriptase inhibitor anti-HIV drugs) as pure drugs or formulated as film or gel products were not deleterious to Lactobacillus species; however, PSC-RANTES (a synthetic CCR5 antagonist) killed 2 strains of Lactobacillus jensenii. To demonstrate the toxicity of formulated products a new assay was developed for use with viscous and non-viscous samples that we have termed the Lactobacillus toxicity test. We found that the vortex mixing of vaginal Lactobacillus species can lead to reductions in bacterial viability. Lactobacillus can survive briefly, about 2s, but viability declines with increased vortex mixing. The addition of heat inactivated serum or bovine serum albumin, but not glycerol, prevented the decrease in bacterial viability. Bacillus atrophaeus spores also demonstrated loss of viability upon extended mixing. We observed that many of the excipients used in film formulation and the films themselves also afford protection from the killing during vortex mixing. This method is of relevance for toxicity for cidal activities of viscous products.

Development of a liposome microbicide formulation for vaginal delivery of octylglycerol for HIV prevention.

The feasibility of using a liposome drug delivery system to formulate octylglycerol (OG) as a vaginal microbicide product was explored. A liposome formulation was developed containing 1% OG and phosphatidyl choline in a ratio that demonstrated in vitro activity against Neisseria gonorrhoeae, HSV-1, HSV-2 and HIV-1 while sparing the innate vaginal flora, Lactobacillus. Two conventional gel formulations were prepared for comparison. The OG liposome formulation with the appropriate OG/lipid ratio and dosing level had greater efficacy than either conventional gel formulation and maintained this efficacy for at least 2 months. No toxicity was observed for the liposome formulation in ex vivo testing in a human ectocervical tissue model or in vivo testing in the macaque safety model. Furthermore, minimal toxicity was observed to lactobacilli in vitro or in vivo safety testing. The OG liposome formulation offers a promising microbicide product with efficacy against HSV, HIV and N. gonorrhoeae.

Killing of Neisseria gonorrhoeae, Streptococcus agalactiae (group B streptococcus), Haemophilus ducreyi, and vaginal Lactobacillus by 3-O-octyl-sn-glycerol.

The microbicide candidate octylglycerol inactivates sexually transmitted bacterial pathogens at concentrations which spare normal vaginal flora (lactobacillus). Standard minimum microbicidal concentration assays and time-kill assays revealed the drug concentrations and times required for inactivation. Octylglycerol concentrations must exceed the binding capacity of any human serum albumin to be effective.

Preclinical safety assessments of UC781 anti-human immunodeficiency virus topical microbicide formulations.

The nonnucleoside reverse transcriptase inhibitor UC781 is under development as a potential microbicide to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). Two gel formulations of UC781 (0.1% and 1.0%) were evaluated in a range of preclinical safety assessments, including systemic absorption analysis following topical application in the pig-tailed macaque models for vaginally and rectally applied topical microbicides. High-sensitivity high-performance liquid chromatography analysis of serum samples showed that no systemic absorption of UC781 was detected after repeated vaginal or rectal application of either product. However, high levels of UC781 were detectable in the cervicovaginal lavage samples up to 6 h after product exposure. Both formulations were safe to the vaginal microenvironment, even with repeated daily use, as evidenced by colposcopy, cytokine analysis, and lack of impact on vaginal microflora. By contrast, rectal application of the 1.0% UC781 formulation caused an increased expression of numerous cytokines not observed after rectal application of the 0.1% UC781 formulation. These results provide additional support for the continued development of UC781 formulations as anti-HIV microbicides.

Response of guinea pigs to a vaccine containing a new adjuvant (SAF) and gram-negative bacteria.

Porphyromonas gingivalis is a gram-negative pathogen associated with severe periodontitis in man and other animals. A vaccine against P. gingivalis infection may improve resistance to such infection and disease progression in susceptible individuals. A vaccine composed of formalin-killed P. gingivalis and Syntex adjuvant formulation or saline was tested in guinea pigs. Blood was drawn before and at 2, 4, 6, 8, 24, and 27 weeks after immunization. There was no morbidity or mortality as a result of vaccination, and necropsy revealed no organ abnormalities or residual injection site granulomas. Serum immunoglobulin G (IgG) antibody titer and avidity were measured by enzyme-linked immunosorbent assay, and Western blots were done to determine the immunodominant antigens. The IgG titer increased more rapidly and reached higher values in the animals receiving SAF plus P. gingivalis vaccine than in those receiving saline plus P. gingivalis. Antibody titer decreased by 27 weeks, but avidity was twofold greater at 27 than at 8 weeks. Western blots indicated that protein and carbohydrate antigens are recognized.

Direct detection of Porphyromonas gingivalis in Macaca fascicularis dental plaque samples using an oligonucleotide probe.

Oligonucleotide probes complementary to the hypervariable regions of the 16S rRNA of Porphyromonas gingivalis, and previously shown to specifically identify human P. gingivalis strains to the species level, were tested for their ability to recognize P. gingivalis from nonhuman primates (Macaca fascicularis), either as distinct isolates or in subgingival dental plaque. The 32P-labeled probes hybridized with all 147 monkey isolates identified as P. gingivalis by morphology and biochemistry, but did not hybridize with any of the 331 isolates representing 17 genera of bacteria unrelated to P. gingivalis, or to the more closely related P. endodontalis and P. asaccharolytica. This corresponds to sensitivities and specificities of 100%. Of 76 M. fascicularis plaque samples, P. gingivalis was detected by probe and culture in 67. Of 26 human plaque samples taken from separate individuals free of periodontal disease, 23 failed to demonstrate P. gingivalis by probe or culture. The results of the combined 102 monkey and human plaque samples indicate that, when compared to culture as the "gold standard," the P. gingivalis probe had a sensitivity of 96%, a specificity of 87%, and an overall agreement with culture of 93%. These results reveal that the oligonucleotide probes used to identify P. gingivalis are specific for this organism, and give results comparable to culture methods for detecting the presence of P. gingivalis in M. fascicularis dental plaque.

Macaca fascicularis as a model in which to assess the safety and efficacy of a vaccine for periodontitis.

We have assessed Macaca fascicularis as a potential model in which to test the efficacy and safety of a vaccine for periodontitis. Twenty-eight animals were surveyed and 20 studied in more detail. Clinical periodontal status was assessed, the subgingival microflora analyzed especially for the presence and proportions of Porphyromonas gingivalis and titers and avidities of serum antibodies reactive with P. gingivalis measured. Probing depths ranged from 0.90 mm to 3.80 mm, Gingival Index scores from 0.00 to 4.00 and Plaque Index scores from 0.00 to 3.00. About 40% of sites bled on probing. The animals manifested a subgingival flora characteristic of the anaerobic gram-negative bacteria found in human periodontal pockets, including Actinobacillus actinomycetemcomitans, P. gingivalis, Bacteroides forsythus, Campylobacter rectus, Prevotella intermedia and Fusobacterium nucleatum. P. gingivalis was detected in 70 of 80 samples studied, ranging from 0.01% to 20% of the total flora. Serum antibody reactive with antigens of P. gingivalis was observed in all animals, with titers ranging from 1.0 enzyme-linked immunosorbent assay (ELISA) unit to 25 ELISA units and avidities from 0.10 M to 2.20 M. Antibody titer and maximum percentage of P. gingivalis were inversely correlated, indicating that a humoral immune response may be effective in reducing P. gingivalis overgrowth. M. fascicularis appears to be an excellent model for use in vaccine development.

Macaca nemestrina: a non-human primate model for studies of periodontal disease.

The non-human primate Macaca nemestrina was evaluated for use as a potential model in periodontal research by study of 16 animals. Using one incisor, premolar, and molar per quadrant, we measured supragingival plaque, severity of gingival inflammation, and pocket depth, and analyzed the subgingival flora. Serum IgG titers and avidities to antigens of Porphyromonas gingivalis (Pg) and Actinobacillus actinomycetemcomitans (Aa) were also assessed. Ten animals were between 13 and 24 years old, and six were between 4 and 5 years old. While mean gingival inflammation scores were significantly higher for older than for younger animals (2.2 vs 1.8, p < 0.05), mean plaque index scores and mean probing depths did not differ significantly. The animals harbored a subgingival microflora considered to be pathogenic for humans including Aa, Pg, Bacteroides forsythus, Prevotella intermedia I and II, Campylobacter recta and Fusobacterium nucleatum. Aa, however, was found only in the younger animals. All of the animals had serum IgG antibodies reactive with antigens of Pg and Aa, and titers for Pg but not for Aa were significantly higher in the older relative to the younger animals (t test p < 0.02). In contrast, antibody avidity did not significantly differ between the two groups. A combined clinical assessment index based on maximum probing depth, gingival index score, and tooth loss was used to assess the overall disease severity. Titers were positively associated with disease severity (Spearman's rank correlation 0.57, p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of antibiotic treatment on clinical conditions and bacterial growth with guided tissue regeneration.

Mucogingival flaps were reflected over pairs of mandibular molar teeth with Class II furcation invasions. The dimensions of the furcations were measured. The teeth were debrided and an expanded polytetrafluoroethylene (e-PTFE) membrane was placed and retained over one furcation of each pair (test site) for 4 weeks. The second site served as a control. Eight patients (group 1) with 12 e-PTFE sites received no antibiotic. Seven patients (group 2) with 12 e-PTFE sites were administered amoxicillin/clavulanate potassium for 10 days. Paper-points were used to collect bacterial samples and clinical indices were recorded at baseline and weekly for 4 weeks. Paper-point samples and the e-PTFE collected at week 4 were sonicated and analyzed by DNA probes for seven putative pathogens. At baseline no parameter showed statistical differences between groups or sites. At week 1 significantly greater levels of Prevotella intermedia type I (P < 0.05) and Fusobacterium nucleatum (P < 0.01) were found in group 1. At week 4, paper-point samples from test sites (P < 0.05) and e-PTFE materials (P < 0.001) showed significantly higher presence of Bacteroides forsythus in group 1. No significant microbial changes were found for control sites over time or between groups. The total bacterial load at test sites over time increased similarly for patients administered or not administered the antibiotic. Clinical signs of inflammation were significantly greater in group 1 and associated with the presence of B. forsythus (P < 0.01).

Effects of treatment on antibody titer to Porphyromonas gingivalis in gingival crevicular fluid of patients with rapidly progressive periodontitis.

Twenty-eight patients diagnosed as having rapidly progressive periodontitis (RPP) were enrolled in a study in which samples of subgingival microflora were harvested from test teeth and assayed for the presence of Porphyromonas gingivalis, and GCF collected and analyzed by ELISA for specific antibody for P. gingivalis. Clinical conditions were measured and recorded, and treatment by scaling and root planing provided at baseline and at 3, 6, 9, and 12 months. Reduction in pocket depth, stabilization of attachment level, and resolution of inflammation were comparable to previously reported values. By 3 months, mean and median specific antibody concentration had decreased, and continued to decrease through 12 months. The proportion of samples in which specific antibody was not detectable increased from 27% at baseline to 73% at month 12. GCF samples from sites at which P. gingivalis was present had greater than 2-fold higher median specific antibody than samples from P. gingivalis-negative sites. At baseline, specific antibody titer of 30-second GCF samples positively correlated with pocket depth, and GCF volume significantly correlated with antibody titer and concentration, and with pocket depth. In addition, change in specific antibody titer of 30-second samples from baseline to both 6 and 12 months correlated positively with pocket depths. Thus sites infected by P. gingivalis manifested high levels of specific antibody, and levels were related to clinical status. Following treatment, antibody levels decreased significantly as pocket depths decreased, attachment levels stabilized, and inflammation resolved.

Titer and subclass distribution of serum IgG antibody reactive with Actinobacillus actinomycetemcomitans in localized juvenile periodontitis.

Most patients with localized juvenile periodontitis (LJP) manifest serum IgG antibodies specifically reactive with antigens of Actinobacillus actinomycetemcomitans serotype b (Aa-b). Whether these antibodies are protective, destructive, or irrelevant to the progress of the disease remains unclear. We report results of studies aimed at assessing the subclass IgG responses in 35 LJP patients and 35 periodontally normal control subjects using well-characterized monoclonal antibody subclass reagents in an enzyme-linked immunosorbent assay. Our data show that the mean value for total IgG reactive with antigens of Aa-b was more than sevenfold higher for patients than for normal control sera (2349.6 micrograms/ml for patients vs 332.2 micrograms/ml for controls). Individual patients and control subjects were classified as high- or low-titer, using twice the median value for total anti-Aa-b IgG in control sera as the cutoff. Of 35 patients, 26 (74%) were high-titer, and 9 (26%) were low-titer. This compares to 5 normal control subjects (14%) high-titer and 30 (86%) low-titer. IgG2 accounted for the major quantitative response in both patients and control subjects. Indeed, the mean IgG2 values for both concentration and percentage of total specific IgG were greater than the combined values for specific anti-Aa-b IgG1, IgG3, and IgG4. Of the 26 high-titer sera, IgG2 predominated in 24, with IgG1 and IgG3 predominating in 1 each; IgG2 predominated in only 2 of the low-titer sera.

Reduction in adherence of Actinomyces viscosus after exposure to low-frequency acoustic energy.

The ability of low-frequency (200 Hz) acoustic energy to reduce the adherence of Actinomyces viscosus T14V to saliva-treated hydroxyapatite (SHA) disks was studied. An acoustic pressure range between 0 and 65 kPa and exposure durations between 0 and 8 min were used to study the levels necessary to significantly alter adherence. The effects of acoustic exposure on both bacteria in liquid and bacteria already adhering to SHA disks were studied. A modified enzyme-linked immunosorbent assay was used to assess bacterial adherence. For bacterial suspensions exposed prior to addition to SHA disks, it was found that reductions in adherence were greater for lower bacterial concentrations. Exposure of bacteria already adhering to SHA disks resulted in a decrease in adherence that was independent of the bacterial concentration and linearly related to the logarithm of the exposure duration. In addition to affecting adherence, acoustic energy also dispersed bacterial aggregates. Our results support the concept that low-frequency sonic energy applied orally may be of therapeutic value in reducing adherence and colonization of teeth by plaque bacteria.

Rapid presumptive identification and further characterization of Bacteroides forsythus.

Bacteroides forsythus is a fastidious anaerobic gram-negative organism associated with various forms of periodontal disease. It is dependent on N-acetylmuramic acid for growth. A method for rapid presumptive identification of human-derived strains of B. forsythus is presented, based on the following eight criteria: (i) positive activity for alpha-glucosidase, (ii) positive activity for beta-glucosidase, (iii) positive activity for sialidase, (iv) positive activity for trypsinlike enzyme, (v) negative indole production, (vi) requirement for N-acetylmuramic acid, (vii) colonial morphology, and (viii) gram stain morphology from blood agar medium deficient in N-acetylmuramic acid. Enzymes were assayed with rapid filter paper spot tests based on fluorogenic substrates (4-methylumbelliferone derivatives and N alpha-carbobenzoxy-L-arginine-7-amino-4-methylcoumarin hydrochloride). Gas-liquid chromatography analysis of the metabolic products of B. forsythus grown in peptone yeast extract broth supplemented with N-acetylmuramic acid and heat-inactivated horse serum revealed predominant amounts of acetate, propionate, butyrate, isovalerate, and phenyl acetate, with minor amounts of isobutyrate and succinate. The described presumptive identification scheme facilitated recognition of four strains of B. forsythus which were isolated from subgingival plaque samples from monkeys (Macaca fascicularis). With the exception of indole production, these organisms were essentially identical to the human strains of B. forsythus for all phenotypic and genotypic characteristics examined.

Sialidases (neuraminidases) in bacterial vaginosis and bacterial vaginosis-associated microflora.

Bacterial vaginosis, Prevotella species, and Bacteroides species have been associated with prematurity and upper genital tract infection. Prevotella (Bacteroides) species and Bacteroides fragilis have also been associated with preterm birth. However, the mechanism by which lower genital tract infection causes upper genital tract disease remains poorly understood. Sialidases (neuraminidases) are enzymes which enhance the ability of microorganisms to invade and destroy tissue. Elevated levels of sialidase activity were detected in 42 (84%) of 50 vaginal fluid specimens from women with bacterial vaginosis and none of 19 vaginal fluids from women without bacterial vaginosis (P less than 0.001). Vaginal fluid from women with bacterial vaginosis had a median specific activity of 9.8 U compared to 2.5 U of sialidase in women without bacterial vaginosis (P less than 0.001). In order to determine the probable source of sialidases in vaginal fluid, the microorganisms recovered from women with bacterial vaginosis before and after treatment were assayed. Of 28 specimens from women with bacterial vaginosis, 27 (96%) yielded sialidase-positive bacteria, at a median concentration of 10(6.5) CFU/ml of vaginal fluid. Prevotella and Bacteroides species accounted for the sialidase activity in 26 of the vaginal fluids, and Gardnerella vaginalis accounted for the sialidase activity in the remaining fluid. After treatment, sialidase was detected in the vaginal fluid of 1 (5%) of 22 women who responded to therapy and in all of 6 women for whom therapy failed. These data suggest that vaginal fluid sialidase is highly correlated with bacterial vaginosis and that the probable sources for this enzyme activity are the Bacteroides and Prevotella species present in the vagina.

Characterization of unusual tetracycline-resistant gram-positive bacteria.

Tetracycline-resistant Tet M-negative isolates of Actinomyces viscosus, Eubacterium lentum, Mobiluncus curtisii, and Mobiluncus mulieris were screened with the Tet K, Tet L, and Tet O DNA probes. Ten (71%) of the resistant Mobiluncus strains hybridized with the Tet O probe, two of the three E. lentum strains hybridized with the Tet K probe, and the A. viscosus isolate hybridized with the Tet L probe.

Humoral immune responses to Porphyromonas gingivalis before and following therapy in rapidly progressive periodontitis patients.

We have performed studies aimed at elucidating the nature of the humoral immune response in rapidly progressive periodontitis (RPP). We analyzed the sera of 36 periodontally normal subjects and 36 RPP patients for titers and avidities of IgG antibodies reactive with the antigens of Porphyromonas gingivalis using ELISA, prior to and following treatment. We used whole-cell sonicate, purified lipopolysaccharide (LPS), and total extractable protein as plate antigens. Twelve of the patients had antibody titers at least 2-fold greater than the median of the controls and were designated as seropositive. The remaining 24 patients had titers that did not exceed twice the median titer of the controls and were designated as seronegative. For both patient groups, antibody titers were highest when whole-cell antigen was used, intermediate for LPS, and lowest for the protein fraction. Following treatment, median titer for seropositive patients decreased from pretreatment values of 241.7 to 76.5, while median titer for seronegative patients increased from 39.5 to 80.1. Avidities of pretreatment sera from both patient groups for all 3 antigen preparations were lower than the median avidities of the control sera. Avidity significantly increased following treatment to levels greater than those for control sera in both patient groups. Thus, some young adults with severe periodontitis mount a humoral immune response and produce high levels of serum IgG antibodies reactive with antigens of P. gingivalis, while others do not. The antibodies produced are of relatively low avidity, and may therefore be relatively ineffective biologically. Therapy, which greatly reduces antigen load, appears to stimulate production of higher avidity IgG antibodies in both patient groups; in the seropositive group, low avidity antibodies appear to be replaced by antibodies of higher avidity. Both the purified LPS and protein fractions contain reactive antigen(s), although LPS binds more antibody. Our data are consistent with the idea that many RPP patients do not produce protective levels of biologically functional antibody during the course of their natural infection, but they may be stimulated to do so by treatment.

Use of synthetic oligonucleotide DNA probes for identification and direct detection of Bacteroides forsythus in plaque samples.

Oligonucleotide DNA probes complementary to the hypervariable region of the 16S rRNA of Bacteroides forsythus were tested for their specificity and sensitivity against reference and clinical isolates of 73 different species of bacteria. The probes were used to detect the organism directly from nucleic acid extracts of subgingival plaque samples, and the results were compared with results of detection by culture methods. The data demonstrate that the probes are very specific (100%) and sensitive (100% when they are compared with those obtained by the culture method) and could reliably detect the organism in plaque samples. When a probe to a conserved region of the 16S rRNA (UP9A) was used to estimate the quantity of bacteria present in plaque samples, it gave results comparable to those of culture methods. The data suggest that total microbial load may be a parameter in periodontal disease.

The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.

Rapid presumptive identification of black-pigmented gram-negative anaerobic bacteria by using 4-methylumbelliferone derivatives.

A rapid method for presumptive identification of black-pigmented gram-negative anaerobic rods was developed. Using filter paper spot tests for indole production, sialidase, alpha-glucosidase, beta-glucosidase, alpha-fucosidase, and trypsinlike enzyme activities, 100% of Porphyromonas gingivalis, Prevotella intermedia, and Bacteroides levii and 89% of Prevotella corporis isolates were correctly identified to the species level. Porphyromonas asaccharolytica and Porphyromonas endodontalis could not be differentiated from each other but could be distinguished from all other species tested. Similarly, Prevotella denticola, Prevotella loescheii, and Prevotella melaninogenica could not be differentiated from each other. The methods described are based on 4-methylumbelliferone derivatives of the various substrates and are simple to perform, rapid (less than 15 min), and applicable to difficult-to-cultivate anaerobic rods.