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.

Serum antibody response to antigens of oral gram-negative bacteria by cats with plasma cell gingivitis-pharyngitis.

The etiology of a form of periodontal disease in domestic cats known as plasma cell gingivitis-pharyngitis is not understood. Actinobacillus actinomycetemcomitans and Bacteroides species have been strongly implicated as the cause of periodontitis in humans and other mammalian species, and most affected patients manifest serum antibodies reactive with the infecting bacteria. We and others have isolated Bacteroides species from the oral flora of cats. Using enzyme-linked immunosorbent assay (ELISA) and immunoblot procedures, we measured serum antibodies in affected and control cats reactive with human isolates of A. actinomycetemcomitans, B. gingivalis, and B. intermedius, and purified lipopolysaccharide (LPS) from these and other species, and Bacteroides of cat origin. Affected cats had serum antibody titers reactive with these Gram-negative anaerobic bacteria that were significantly elevated relative to those of normal control cats. The quantitatively major antigens recognized by cat serum antibodies are proteins; this contrasts sharply with serum antibodies from humans with juvenile periodontitis, where LPS is the quantitatively major antigen fraction. Our data support the idea that plasma cell gingivitis-pharyngitis in cats may have a bacterial etiology, and that Gram-negative anaerobes similar to those that cause periodontitis in humans and other mammals may be involved.

The use of whole-cell DNA probes for the identification of Bacteroides intermedius isolates in a dot blot assay.

Bacteroides intermedius includes two distinct groups of organisms that are phenotypically indistinguishable by conventional methods. These two groups are represented by the type strain of the species ATCC 25611T (B. intermedius type I) and by ATCC 33563 (B. intermedius type II). Members of each group can be distinguished from each other by analysis of the cellular protein composition by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by DNA-DNA homology studies, because they share less than 40% homology. The purpose of this study was to prepare specific DNA probes for the two groups of Bacteroides intermedius and to test them against field isolates. Whole-cell DNA probes were prepared from B. intermedius types I and II and tested against 253 field strains of Bacteroides which had been identified by conventional phenotypic tests as B. intermedius. Of these, 170 (67%) hybridized with the B. intermedius type I DNA probe, 28 (11%) with the type II, and 23 (9%) failed to react with the B. intermedius probes but did hybridize with either B. melaninogenicus, B. loescheii, or B. corporis whole-cell DNA probes. The 32 (13%) remaining isolates failed to hybridize with any of the five Bacteroides probes or with probes to B. asaccharolyticus, B. buccae, B. buccalis, B. denticola, B. gingivalis, B. oralis, or B. oris. These data demonstrate the usefulness of whole-cell DNA probes for the identification of phenotypically similar or identical field isolates.

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.

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).

Abnormal lymphocyte profiles and leukotriene B4 status in a patient with Crohn's disease and severe periodontitis.

THIS CASE REPORT DESCRIBES clinical and laboratory findings for a 60-year-old woman with recently diagnosed Crohn's disease and severe generalized periodontitis. Comparison of dental radiographs taken in 1975, in 1983, and at the time of our evaluation in 1986 revealed dramatic progression of alveolar bone loss over that period. Standard laboratory blood tests did not reveal any remarkable significant leukocyte abnormalities, but flow cytometric analysis of lymphocytes revealed a paucity of B cells stained with anti-light chain antibodies, and an increased proportion of T lymphocytes which were dully-stained with anti-CD5 monoclonal antibody. B cell function as determined by in vitro proliferative responsiveness to anti-IgM antibody was only about 50% of that observed with cells from two healthy normal subjects. Serum leukotriene B4 (LTB4) was elevated to 150% of normal values, in spite of the fact that the patient was taking a systemic anti-inflammatory drug which is known to reduce LTB4 levels. The microbial flora was highly mixed and included several putative periodontopathic bacteria. Therapy consisted of oral hygiene instruction, scaling and root planing, mucoperiosteal-flap curettage, extracoronal splinting, and selective extraction of three teeth. The periodontal status improved markedly with therapy. Possible relationships between the patient's immunological status, her Crohn's disease, and the severe periodontal breakdown are discussed.

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.

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.

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.

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.

Tetracycline resistance and TetM in oral anaerobic bacteria and Neisseria perflava-N. sicca.

Tetracycline-resistant organisms isolated from six patients with periodontal disease included Bacteroides spp., Eubacterium spp., Fusobacterium nucleatum, Neisseria perflava-N. sicca, Peptostreptococcus anaerobius, Veillonella parvula, and facultative streptococci. All but the Bacteroides spp. and Eubacterium spp. hybridized with the TetM determinant. An additional 417 bacterial strains were screened, and 4% of both the oral streptococci and the Fusobacterium spp. hybridized with the TetM probe.

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.

Detection of sialidase (neuraminidase) activity in Actinomyces species by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid in a filter paper spot test.

A rapid method for the detection of acetylneuraminyl hydrolase, EC 3.2.1.18 (sialidase or neuraminidase), was developed by using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid as substrate in a filter paper spot test. The method was compared to conventional assays that use 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid and bovine submaxillary mucin and was found to be in excellent agreement. Organisms with greater than 10 U of enzyme activity (in nanomoles per minute per milligram of cell protein) gave positive reactions, while those with 2.7 to 9.0 U gave only weak reactions. Isolates with less than 2.7 U of activity were detected upon prolonged incubation. Sialidase activity was detected in 79% of 71 clinical isolates representing five species of Actinomyces. The percentage of sialidase-producing isolates of each species varied considerably: Actinomyces israelii, 63%; A. meyeri, 73%; A. naeslundii, 85%; A. odontolyticus, 73%; and A. viscosus, 100%.

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.

Constitutive uptake and degradation of fatty acids by Yersinia pestis.

Yersinia pestis was found to utilize palmitic acid as a primary carbon and energy source. No inhibition of growth by palmitic acid was observed. Comparison of palmitic acid uptake by cells pregrown either with or without palmitic acid demonstrated that fatty acid uptake was constitutive. High basal levels of two enzymes of beta-oxidation, beta-hydroxyacyl-coenzyme A dehydrogenase and thiolase, and the two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were found in cells grown in defined medium with glucose. Elevated levels of all four enzymes were found when cells were grown with acetate as a primary carbon and energy source, and even higher levels were observed when palmitic acid was provided as a primary carbon and energy source. High-pressure liquid chromatography was used to demonstrate that, in the presence of glucose, uniformly labeled [14C]palmitic acid was converted to intermediates of the tricarboxylic acid cycle and glyoxylate shunt. Pregrowth with palmitic acid was not required for this conversion. Strains lacking the 6- or the 47-megadalton plasmid did not take up [3H]palmitic acid but did possess levels of enzyme activity comparable to those observed in the wild-type strain.

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.

Langmuir and scatchard parameters do not describe the binding of Actinomyces viscosus to saliva-treated hydroxyapatite.

The binding of Actinomyces viscosus T14V to saliva-treated spheroidal hydroxyapatite (SHA) beads was studied. The association constant (K) and the total number of binding sites (N) obtained from the Langmuir plots were in good agreement with those reported by other workers (approx. 3 X 10(-8) and 3 X 10(8), respectively). The values for N obtained from Scatchard plots differed from those obtained from Langmuir plots by factors of 10(6) or more. These results suggest that either these equations are inappropriate to describe binding or certain assumptions regarding this system are not being met. The use of these models requires, among other constraints, that the process be reversible and that measurements be taken at equilibrium. A method was developed which allowed a close examination of the equilibrium dynamics without perturbation of the system. The results suggest that the adsorption process is only poorly reversible. Adsorption to SHA was not at equilibrium after 1.5 h. Even when bacteria were allowed to adsorb for longer periods, and the system appeared to approach equilibrium, the increased time of adherence did not significantly alter the derived K or N values. Our results suggest that the use of Scatchard and Langmuir plots is inappropriate to describe binding of A. viscosus to SHA.

Sialidase (neuraminidase) activity among gram-negative anaerobic and capnophilic bacteria.

A filter paper spot test with 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid as a substrate was used to study the prevalence of sialidase activity among gram-negative anaerobic and capnophilic bacteria. A total of 567 isolates representing four genera of obligate anaerobes and four genera of capnophilic organisms was tested. Sialidase activity was detected in 94% of 66 isolates from the Bacteroides fragilis group, 98% of 66 B. bivius isolates, and all isolates of the following species (number of isolates follows species name): B. capillosus, 4; B. levii, 2; B. denticola, 22; B. loescheii, 23; B. melaninogenicus, 32; B. forsythus, 44; and B. buccalis, 2. However, sialidase activity was detected in only 29% of 7 B. buccae isolates, 79% of 14 B. disiens isolates, and 55% of 11 B. oralis isolates. Sialidase activity was not detected among any of 13 isolates of B. gracilis, 12 isolates of B. ureolyticus, 61 isolates of B. intermedius, or 26 isolates of B. corporis. Porphyromonas (Bacteroides) asaccharolytica (20 isolates) and P. endodontalis (8 isolates) did not demonstrate sialidase activity, while 25 isolates of P. gingivalis were sialidase positive. Sialidase activity was found in 10 (100%) of 10 isolates of Capnocytophaga ochracea of C. sputigena but not in any of 4 C. gingivalis isolates. Other gram-negative anaerobic or capnophilic bacteria, including the following, were negative for sialidase activity: Fusobacterium nucleatum, 39 isolates; Wolinella recta, 19 isolates; Eikenella corrodens, 17 isolates; Haemophilus aphrophilus, 10 isolates; and Actinobacillus actinomycetemcomitans, 10 isolates. These data demonstrate sialidase activity in several species of the genera Bacteroides and Porphyromonas and suggest that this characteristic may be useful for identification.