In vitro environmental regulation of Porphyromonas gingivalis growth and virulence.

Porphyromonas gingivalis appears to be a major contributor to periodontal disease, especially soft tissue destruction, which is reflected by the ability to cause invasive, spreading lesions, and tissue inflammation in a murine abscess model. This study investigated the role of hemin on the regulation of growth and virulence of P. gingivalis strains. P. gingivalis strains W50, A7A1-28, 3079, 381, W50/BEI, and NG4B19 were grown in broth and on blood agar plates. P. gingivalis cells grown under iron-depleted conditions for multiple passages showed significantly decreased lesion size in mice, in contrast to cells grown under iron-normal (5 microg/ml) and iron-elevated conditions. Statistically significant (P < 0.01) decreases in gingipain enzyme activity were found among the strains grown under iron-depleted conditions. P. gingivalis grown in the presence of blood induced significantly different lesion type, lesion size, lesion onset, and mortality. Elevated hemin resulted in increased cell-associated iron in P. gingivalis, which increased the capacity of the microorganism to survive at times of iron deprivation. These results indicate that hemin or iron availability regulates multiple aspects related to P. gingivalis virulence, including growth, survival, gingipain levels, and iron accumulation.

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5'-phosphate-dependent protein acting as a haemolytic enzyme.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts.

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.

Hemoxidation and binding of the 46-kDa cystalysin of Treponema denticola leads to a cysteine-dependent hemolysis of human erythrocytes.

Cystalysin, a 46-kDa protein isolated from the cytosol of Treponema denticola, was capable of both cysteine dependent hemoxidation and hemolysis of human and sheep red blood cells. The activities were characteristic of a cysteine desulfhydrase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting analysis of the interaction of cystalysin with the red blood cells revealed an interaction of the protein with the red blood cell membrane. Substrates for the enzyme (including L-cysteine and beta-chloroalanine) enhanced the interaction, which occurred with both whole red blood cells as well as with isolated and purified red blood cell ghosts. SDS-PAGE and western immunoblotting employing anti-hemoglobin serum revealed that, during the hemoxidative events, the hemoglobin molecule associated with the red blood cell membrane, forming putative Heinz bodies. Spectrophotometric analysis of the hemoxidative events (cystalysin + cysteine + red blood cells) revealed a chemical modification of the native hemoglobin to sulfhemoglobin and methemoglobin. Hemoxidation also resulted in the degradation of both the red blood cell alpha- and beta-spectrin. The results presented suggest that the interaction of cystalysin with the red blood cell membrane results in the chemical oxidation of the hemoglobin molecule as well as an alteration in the red blood cell membrane itself.

Sulfhemoglobin formation in human erythrocytes by cystalysin, an L-cysteine desulfhydrase from Treponema denticola.

Cystalysin, isolated from the oral pathogen Treponema denticola, is an L-cysteine desulfhydrase (producing pyruvate, ammonia and hydrogen sulfide from cysteine) that can modify hemoglobin and has hemolytic activity. Here, we show that enzymatic activity of recombinant cystalysin depends upon stochiometric pyridoxal phosphate. The enzyme was not functional as an L-alanine transaminase, and had a strong preference for L-cysteine over D-cysteine. Cystalysin preferred small alpha-L-amino acids as substrates or inhibitors and was far more active towards L-cysteine than towards the other standard amino acids that undergo pyridoxal phosphate-dependent beta-elimination reactions (serine, threonine, tryptophan and tyrosine). Cystalysin tolerated small modifications to the carboxylate of L-cysteine (i.e., the methyl and ethyl esters of L-cysteine were good substrates), but the smallest possible peptide with an N-terminal cysteine, L-cysteinylglycine, was a very poor substrate. These results, combined with the implicit requirement for a free amine for pyridoxal phosphate-dependent reactions, imply that cystalysin cannot catabolize cysteine residues located within peptides. Cystalysin has Michaelis-Menten kinetics towards L-cysteine, and there was little or no inhibition by ammonia, H2S, pyruvate and acetate. Human erythrocytes incubated with H2S or with cystalysin and cysteine primarily accumulated sulfhemoglobin and methemoglobin, along with minor amounts of choleglobin and protein aggregates. Erythrocytes retained the ability to reduce methemoglobin in the presence of H2S. Cystalysin could not modify hemoglobin when beta-chloroalanine was the substrate, indicating an absolute requirement for H2S production. Cystalysin appears to be an unregulated L-cysteine catabolizing enzyme, with the resulting H2S production being essential to the atypical hemolytic activity.

Studies on the binding of Treponema pectinovorum to HEp-2 epithelial cells.

We developed a radioassay to assess the adherence of the oral treponemes Treponema denticola and Treponema pectinovorum to live HEp-2 epithelial cells. T. pectinovorum bound firmly to the epithelial cell monolayer in a concentration-dependent manner. The results indicated that a subpopulation of T. pectinovorum appeared to bind and that the binding could be influenced by environmental factors. Increasing concentrations of fetal bovine serum inhibited binding, whereas T. pectinovorum membrane vesicles and co-incubation with T. denticola ATCC 35404 increased the number of cells bound to the monolayer. Treatment of T. pectinovorum with periodic acid, but not trypsin or proteinase K, decreased the binding suggesting that a cell surface carbohydrate, such as the O-antigenic component of the lipopolysaccharide, mediates attachment of the bacteria to the epithelial cells. Co-infection of the HEp-2 cells with both T. denticola and T. pectinovorum did not interfere with each other in attachment to the epithelial cell suggesting that they do not compete for the same cellular receptor on the host cell surface. This study demonstrates that T. pectinovorum is capable, in vitro, of forming a tight association with host cells and that this binding could represent an initial step in the pathogenesis of T. pectinovorum.

Environmental modulation of oral treponeme virulence in a murine model.

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.

Cystalysin, a 46-kDa L-cysteine desulfhydrase from Treponema denticola: biochemical and biophysical characterization.

A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.

Immunization with Porphyromonas gingivalis cysteine protease: effects on experimental gingivitis and ligature-induced periodontitis in Macaca fascicularis.

Targeting bacterial virulence factors such as proteases for immunization may hold the key to limiting or preventing loss of attachment and alveolar bone in periodontal disease. This study examined the clinical, microbiological, and immununological responses following active immunization with a purified Porphyromonas gingivalis cysteine protease (porphypain-2) in the nonhuman primate (Nhp) Macaca fascicularis. One group of Nhp was immunized with porphypain-2 antigen while control Nhp received placebo injections. All Nhp were subjected to experimental gingivitis followed by ligature-induced periodontitis in a split-mouth design. An enzyme-linked immunosorbent assay demonstrated that immunization elicited a significantly elevated and specific IgG antibody response to both whole cell P. gingivalis (36-fold) and to porphypain-2 (194-fold). Checkerboard hybridization DNA analysis of subgingival plaque from ligated sextants demonstrated that 25% more Gram-negative anaerobic species became significantly elevated from baseline and at earlier timepoints in the control group than in the immununized group. Immunization with this protease did not suppress the emergence of P. gingivalis. Clinical indices showed few changes related to immunization. Alveolar bone density changes demonstrated a highly significant loss in ligated sextants compared to non-ligated sextants within the control group (P < 0.001), and a smaller but significant difference within the immunized group (P = 0.043). Comparison of ligated sextants only demonstrated more bone loss in the control group versus the immunized group (-13.07+/-9.51 versus -9.41+/-6.18; computer-assisted densitometric image analysis units +/- SD); the difference approached, but did not reach, significance. The results suggest that porphypain-2 may contribute to the pathogenic potential of the subgingival plaque microbiota in the Nhp model of ligature-induced periodontitis, and that active immunization with porphypain-2 appeared capable of altering this pathogenic response.

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.

Trypsin-like protease activity of Porphyromonas gingivalis as a potential virulence factor in a murine lesion model.

Porphyromonas gingivalis possesses a large number of enzymatic activities which might be important in the virulence of this putative periodontopathogen. The purpose of this study was to examine these enzymatic activities in vivo in a murine model to assess their role in soft tissue destruction. Whole cells of P. gingivalis strains whether grown on blood agar plates or in broth exhibited high levels of alkaline phosphatase (ALPase), a trypsin-like protease (TLPase), acid phosphatase (ACPase), N-acetyl beta-glucosaminidase (Na beta-Gase) enzymes and collagenolytic activities. P. gingivalis W50 treated with 2 mM Na-P-tosyl-L-lysine chloromethyl ketone (TLCK)/phenylmethylsulfonyl fluoride (PMSF) prior to subcutaneous infection of mice failed to induce a phlegmonous abscess and lethality characteristic of animals challenged with untreated P. gingivalis. Comparison of wild type P. gingivalis strain 3079.03 with its protease-deficient (TLPase-negative) mutant NG4B19 revealed the mutant to be avirulent (no lesion and no death) in this model. P. gingivalis BEI and SW5 mutants (parent W50), which partially lacked TLPase enzyme activity produced only localized lesions, and no death. Thus, the TLPase enzyme appears to be correlated with the lesion type (spreading or localized), lesion size, and death in this mouse abscess model. Therefore, the enzymatic activities of P. gingivalis and specifically the TLPase enzyme could play an important role in periodontal disease by enhancing bacterial spread and degrading gingival tissues.

Effect of iron regulation on expression and hemin-binding function of outer-sheath proteins from Treponema denticola.

The effect of the iron-chelating compounds EDDA and BPD on polypeptide regulation in the putative oral pathogen Treponema denticola was studied. SDS-PAGE analysis of the T. denticola strains grown in the presence of EDDA or BPD, i.e. iron-limiting environmental conditions, revealed the expression of 44 and 43 kDa polypeptides in the outer sheath, a 73 kDa polypeptide in the cell membrane, and a 16 kDa polypeptide in the soluble cell fraction. The hemin-binding activity of purified outer sheaths from T. denticola TD-4 grown in the presence of 6.4 mM EDDA was significantly greater than that observed in control (absence of EDDA) outer sheaths. Both activities were inhibited by proteinase K. SDS-PAGE, LDS-PAGE and TMBZ staining revealed the 44 and 43 kDa outer-sheath polypeptides to be expressed by T. denticola strains GM-1. MS-25, ATCC 33520 and ATCC 33404 (TD-4), strains which possessed strong hemin-binding activity. The 44 kDa hemin-binding polypeptide was purified by 1% CHAPS solubilization, HPLC, and SDS-preparative electrophoresis. N'-terminal sequence analysis indicated the purified 44 kDa polypeptide to belong to a new, undescribed group of polypeptides possessing hemin-binding activity.

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli.

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola.

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40 kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lactoferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29-34 kDa protein.

Purification and characterization of a 45 kDa hemolysin from Treponema denticola ATCC 35404.

A 45 kDa polypeptide capable of erythrocyte (RBC) lysis and hemoglobin oxidation was isolated from Treponema denticola, ATCC 35404 (TD-4) after sequential ammonium sulfate (2.8-3.6 M) precipitation and preparative electrophoresis. The purified polypeptide produced a single protein band on PAGE at a relative molecular weight of 45 kDa in the presence and absence of SDS. The polypeptide was sensitive to proteinase K and pronase, and heating at 80 degrees C. The protease inhibitors, PMSF, TLCK and benzamidine had no inhibitory affect on activity. It was non heat-modifiable, and lost all hemolytic and hemoxidative function in SDS. Cysteine and other sulfhydryl-containing compounds were required for hemolytic and hemoxidative activities. The isoelectric point of the polypeptide was 5.3 and N'-terminal sequence analysis indicated it to belong to a new, so far undescribed group of peptides possessing hemoxidation and hemolytic activities. Functionally, it was capable of rapid hemoxidation of sheep and human erythrocytes (hemoglobin to methemoglobin) coupled to erythrocyte lysis, or hemolysis.

Characterization of hemolysis and hemoxidation activities by Treponema denticola.

This study determined the presence of two hemolytic activities in the oral treponeme, Treponema denticola, strains ATCC 35404 (TD-4), ATCC 33520, GM-1, and MS25. These activities, referred to as hemolytic and hemoxidative (HeA, HeO, respectively), were found to be both secreted into the extracellular environment, and cell associated. The extracellular activity was associated with small molecules with relative molecular weights of < 1000 Da, and its activity was cysteine independent; the cell-associated HeA and HeO activities were associated with a molecular weight fraction > 10 kDa, and were cysteine dependent. The HeO activity of the fractionated material observed was due to the oxidation of hemoglobin to methemoglobin, and preceded the HeA lysis of the RBCs by approximately 2 h. Heating at 80 degrees C and treatment with proteinase K resulted in the complete destruction of these activities in the fraction > 10 kDa, while lipase at high concentration (800 micrograms/ml) reduced the HeA and HeO activities in the extracellular fraction by approximately 50%. Proteinase inhibitors had a variable effect on HeA and HeO activities in both extracellular and cell-associated fractions. Scanning and transmission electron microscopy revealed a progressive destruction of the RBC membrane, with membrane protrusions formed early in the interaction, which progressed to irregular holes in the membrane, and the complete loss of membrane integrity.

Decidual cyst: endovaginal sonographic sign of ectopic pregnancy.

PURPOSE: To determine the prevalence of decidual cysts in patients with ectopic pregnancy and assess the value of endovaginal sonographic demonstration of decidual cysts in predicting ectopic pregnancy. MATERIALS AND METHODS: A series of 288 proved ectopic pregnancies was reviewed, and a series of 179 patients with pregnancies of less than 8 weeks menstrual age was prospectively examined. RESULTS: Decidual cysts were identified in 30 (14.4%) of 208 ectopic pregnancies. In 12 patients a decidual cyst was the first sonographic sign of ectopic pregnancy, and in six patients it was the only abnormal sonographic finding. Four of five patients with decidual cysts had an ectopic pregnancy. Decidual cysts had a sensitivity of 21%, specificity of 92%, positive predictive value of 80%, and negative predictive value of 42% in the diagnosis of ectopic pregnancy. CONCLUSION: Patients with decidual cysts are at high risk for ectopic pregnancy and should be monitored or treated appropriately, depending on the clinical findings.

Interstitial line: sonographic finding in interstitial (cornual) ectopic pregnancy.

PURPOSE: To evaluate the relationship of the endometrial canal and decidua vera to the interstitial gestational sac and to determine if this relationship can be used to increase the predictive value of ultrasound (US) in the diagnosis of interstitial ectopic pregnancy. MATERIALS AND METHODS: The US findings in 12 patients with interstitial ectopic pregnancy were reviewed. Radiologists also reviewed the cases of 40 patients with various diagnoses to assess the accuracy of the interstitial line sign. RESULTS: US showed a definite gestational sac in four of the 12 patients (33%); the rest had a heterogeneous mass in the cornual region. Thinning of the myometrial mantle was seen in these four patients. The gestational sac appeared eccentric in three of these but in only three of 12 (25%) overall. The endometrial canal or interstitial portion of the tube was identified in 11 of 12 patients (92%). The interstitial line had better sensitivity (80%) and specificity (98%) than eccentric gestational sac location (sensitivity, 40%; specificity, 88%) and myometrial thinning (sensitivity, 40%; specificity, 93%) for the diagnosis of interstitial ectopic pregnancy. CONCLUSION: The interstitial line sign is a useful diagnostic sign of interstitial ectopic pregnancy.

Properties of adhering and nonadhering populations of Mycoplasma genitalium.

The interaction between Mycoplasma genitalium and human lung fibroblasts (HLFs) was studied with the use of wild-type and hemadsorption-negative (HA-) mycoplasmas. [35S]-methionine-labeled M. genitalium adhered to HLFs by first-order kinetics, with maximal interaction occurring at approximately 2 hours. Electron microscopy of chemically fixed cells revealed an almost immediate association of mycoplasmas with the HLF plasma membrane that was mediated by the mycoplasma tip and a nap-like layer, which appeared to extend from the tip around much of the mycoplasmal unit membrane. Following cytadherence, M. genitalium appeared capable of invading the intracellular spaces of targeted HLF cells, possibly by receptor-mediated endocytosis. Spontaneously arising HA- variants of M. genitalium strain G37 failed to adhere to HLF cells and were distinguished on the basis of their protein profiles. SDS-PAGE analysis of the class I (lacking the 140-kd protein but containing a polypeptide doublet at approximately 140-kd) and class II (lacking the 140-kd protein and doublet) variants of M. genitalium revealed that class I variants contain a doublet protein in the 140-kd region, which reacted with a monoclonal antibody generated to the adhesin-implicated 140-kd protein (P140) of wild-type M. genitalium. Class II variants completely lacked the 140-kd protein or immunologically related peptides.