Phagocytosis of Mycoplasma pneumoniae and Acholeplasma laidlawii measured as inhibition of [3H]uridine uptake by macrophages.

Many studies of the interaction between phagocytes and mycoplasmas have given controversial results. This is probably due both to the small size of the microorganisms and their ability to attach to the cell membrane, making it difficult to distinguish between adsorption and ingestion. To overcome these difficulties we took advantage of a phenomenon we noted occurring concomitantly with phase-contrast microscope-monitored phagocytosis of heat-killed C. albicans, i.e., a reduction of [3H]uridine uptake by macrophages from culture medium. This approach allowed us to measure the ability of mouse peritoneal macrophages and the macrophage-like P 388 D 1 continuous cell line to phagocytose Mycoplasma pneumoniae and Acholeplasma laidlawii. Live, UV-killed and specific antiserum-opsonized mycoplasmas were tested. A. laidlawii was ingested under all the conditions mentioned above, while live M. pneumoniae was not phagocytosed unless UV-killed. Phagocytosis of UV-killed M. pneumoniae was directly verified by transmission electron microscopy studies. Data obtained with opsonized M. pneumoniae indicated no ingestion by mouse peritoneal macrophages and incomplete phagocytosis with P388 D 1 macrophages, suggesting that different responses by different types of phagocytes can be observed. In spite of a lack of information concerning the biological meaning of the inhibition of macrophage RNA metabolism during phagocytosis, our data suggest that this phenomenon may be used to study the phagocytosis of microorganisms which are difficult to visualize.

Detection and characterization of Helicobacter pylori from patients with gastroduodenal diseases.

Polymerase chain reaction and cytotoxin assays were performed to identify as Helicobacter pylori type I (cagA+/tox+) or type II (cagA-/tox-) 56 (59.6%) strains from 94 patients. Of these patients 64 were affected by nonulcer dyspepsia (NUD), 10 by gastric ulcer (GU), 19 by duodenal ulcer (DU), and 1 by both GU and DU. H. pylori strains were tested for cagA using two sets of primers; target sequences were detected in 40-42/56 (71.4-75%) depending on the set of primers used, while cytotoxin-producing strains (tox +) were 26/56 (46.4%). Tox+ strains were isolated in 13/32 (40.6%), 2/7 (28.6%), and 11/17 (64.7%) in NUD, GU, and DU patients, respectively. However, the different percentage between cagA+ strains from NUD patients (13/32; 40.6%) and patients with ulcerative diseases (13/23; 54.2%) is not statistically significant (p = 0.462). Because the two sets of primers employed for amplification of cagA target sequences give different results, we concluded that cagA alone could not be taken as predictive factor for severity of gastroduodenal disease. It has been found that H. pylori type I is associated with duodenal ulcer disease.

Influence of yeasts and of their constituents on nucleoside uptake in peritoneal murine macrophages.

A marked reduction of [3H]-uridine uptake was observed when mouse peritoneal macrophages (pM phi) were exposed to heat-killed Candida albicans or Saccharomyces cerevisiae. By contrast, an increased nucleoside uptake was promoted by yeast products such as zymosan, laminarin, or yeast cell-wall extracts, which are mainly composed of beta-glucans and alpha-mannans. In a search for the active fungal component(s), the uptake process was shown to be differently affected by monosaccharides and polysaccharides. These findings support the view that a specific recognition of a pM phi membrane receptor is mediating the effect of the various substances.

The effect of Mycoplasma hominis and Ureaplasma urealyticum on hamster egg in vitro penetration by human spermatozoa.

The effects of some genital mycoplasmas on the in vitro penetration of human spermatozoa into the master egg were studied. Ureaplasma urealyticum serotypes 4, 8, and 6 showed high interfering activity: 6.3% (P less than 0.01), 12.3%, and 14.5%, respectively, against the 55.6% penetration rate of untreated sperm. Neither a cytotoxic effect of mycoplasmas on gametes nor a masking of the binding sites on the egg surface were demonstrated. In experiments carried out with U. urealyticum serotype 4, the production of diffusible relatively heat-labile factor(s) responsible for the inhibition of sperm penetration was postulated.

Mycoplasmic localization patterns on spermatozoa from infertile men.

Two mycoplasmas have been observed with increasing frequency in patients with genitourinary disorders: Mycoplasma hominis and Ureaplasma urealyticum. Mycoplasma cells of both these species have been demonstrated to be capable of attaching to human spermatozoa of infertile patients. The mechanisms for the association of infertility and mycoplasma infection have not been established. The main objective of this article was to explain the significance of some morphologic features of spermatozoa of patients with unexplained infertility using light and electron microscopy. These studies and quantitative analysis of ureaplasmas in the semen indicate that at least two patterns can be seen. Frequently, sphere-shaped particles adhering mainly to the midpiece of spermatozoa were detected. In a second, more complex pattern ureaplasmas were seen inside a swollen zone on the midpiece, which suggests that the infection does not occur in the urethra, but at another unknown site. Furthermore, the sphere-shaped particles cannot be associated with ureaplasmas because their titers in the semen of infertile patients were much lower than those expected.

The enzyme-linked immunosorbent assay for detection of the antispermatozoal antibodies.

An immune-enzymatic method was developed for the determination of antispermatozoal antibodies. Sera of 94 infertile patients were studied with the enzyme-linked immunosorbent assay (ELISA). Twelve patients were positive for antispermatozoal antibodies. Forty-six of these patients were studied also with the gelatin agglutination test (GAT). Eight sera were positive in the ELISA and nine in the GAT. With ELISA, immunoglobulin classes can be demonstrated; in fact, nine of our patients were positive for IgG and three for IgM. In all patients the IgA titer was less than 1:16. In addition, 61 seminal fluid specimens were studied by ELISA, and 7 were positive. The serum and seminal fluid of 12 patients were simultaneously studied. Seminal fluid was positive in only three patients, serum was positive in four, whereas serum and seminal fluid were both negative in five. This study illustrates that ELISA is apparently less sensitive than GAT; however, it is certainly more practical and an easier method for antibody research in sera and in seminal fluid.

Detection of Mycoplasma genitalium and Chlamydia trachomatis DNAs in male patients with urethritis using the polymerase chain reaction.

The use of PCR assays as a fast and reliable method is constantly improving and easing microbiological diagnosis. We used a polymerase chain reaction (PCR) assay designed to detect Mycoplasma genitalium and Chlamydia trachomatis in urethral swab samples of 56 males with urethritis and 44 asymptomatic patients as a control group. The PCR assay provides an amplification of target sequence within MgPa (M. genitalium protein attachment) gene. Results indicated that M. genitalium was present in 6 (10.7%) patients with urethritis and none in the control group. Eleven of 56 (17.8%) patients were positive for Chlamydia trachomatis when tested by an outer membrane protein primer-based PCR. The amplified DNA fragments were homogeneous as shown by restriction enzyme analysis and found to be consistent with the published sequences. The PCR assay employed was as reliable as the cultural method in detecting C. trachomatis in the urethral swabs of patients with urethritis (100% of sensitivity when compared with the cultural method) and it has been revealed as an essential method for detection of M. genitalium.

Effects of bacillus of Calmette-Guerin, Candida albicans and human recombinant interferon-gamma on RNA metabolism and on in vitro release of tumor necrosis factor-alpha by human monocytes/macrophages.

Bacillus of Calmette-Guerin (BCG) was found to be effective in the therapy of superficial bladder cancer, although the mechanisms by which this occurs have not yet been clarified. One hypothesis is related to the ability of monocytes/macrophages (MN/M phi) to release tumor necrosis factor-alpha (TNF-alpha), a monokine with cytotoxic and cytostatic effects against certain tumor cell lines. The present study demonstrates that BCG and C. albicans are both very efficient inducers of TNF-alpha, while they inhibit uridine uptake and incorporation into human MN/M phi RNA. However, unlike C. albicans, BCG is cytotoxic for MN/M phi, as determined by release of labelled leucine from target cells.

In vitro activity of antibiotics against Ureaplasma urealyticum and Chlamydia trachomatis strains from patients with nongonococcal urethritis.

The activity of minocycline, doxycycline, tetracycline, erythromycin, mepartricin and lincomycin against 35 freshly isolated Ureaplasma urealyticum strains was tested. Doxycycline was the most active. Twelve strains were resistant to minocycline and four of these were sensitive to erythromycin. Mepartricin showed no activity against the organisms at a concentration of 10 micrograms/ml. The susceptibility of 30 low-laboratory-passage Chlamydia trachomatis strains against tetracycline and erythromycin was tested. A variable degree of sensitivity to tetracycline and erythromycin was found, the median MIC values being 0.13 micrograms/ml and 0.025 micrograms/ml respectively. No resistant Chlamydia trachomatis strain was found.

Do mycoplasmas inhibit the human sperm fertilizing ability in vitro?

Although clinical observations have suggested that Ureaplasma urealyticum may be associated with reproductive failure, the role of ureaplasmas in human infertility remains controversial. However, the mechanism whereby ureaplasmas can interfere with the fertilization process is not known. To study possible mechanisms, we used the human sperm-hamster egg fertilization test. Eggs were exposed to the spermatozoa preincubated with Mycoplasma hominis or U. urealyticum (serotype 1-8). U. urealyticum serotype 4 showed greater interference activity on the penetration rate (6.6%) than did the other mycoplasmas, as compared with the control (55%). Furthermore, our data suggest that the inhibition of penetration was not related to a masking of sperm membrane sites, since the mean of adsorbed spermatozoa/egg after preincubation of spermatozoa with mycoplasmas was not significantly different from that of the control. In addition, eggs preincubated with U. urealyticum serotype 4 gave a reduction of the penetration rate by untreated spermatozoa.

A simplified method using enzyme-linked immunosorbent assay for titration of antisperm antibodies.

We selected sera from 44 patients (36 males and 8 females) that were positive for antisperm antibodies using ELISA (titer ranged from 1:32 to 1:512) for the evaluation of a simplified method for determination of antisperm antibodies. This method uses a correlation between a single absorbant value and the endpoint titer of the same serum. This simplified procedure increases the number of the sera that can be tested on each plate, resulting in considerable saving of time, reagent costs, and materials. A standard curve allows the direct determination of endpoint titer using the absorbance value found at a single dilution. This modification improves the utility of the assay for the epidemiological screening of antisperm antibodies in patients who may have an immunological cause of infertility.

Serodiagnosis of M. pneumoniae infections by enzyme-linked immunosorbent assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) has been used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA and its sensitivity compared with other serological methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and radioimmunoprecipitation (RIP) are reported. ELISA and MC showed greater sensitivity than CF and MI, while RIP showed serum titer two- to 16-fold higher. ELISA was specific as determined using other human mycoplasma. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution has been proposed. ELISA presented several advantages: sensitivity, rapidity, and low cost and, if adequately standardized, could become a reliable method for the serodiagnosis of M. pneumoniae infection.

Comparative evaluation of some methods for detection of M. pneumoniae antibodies.

Various methods of detection are available for measuring antibodies specific to M. pneumoniae. We examined over 600 sera of patients with clinical diagnosis of M. pneumoniae infection using complement fixation (CF) test. 91 gave positive reaction and were therefore selected for a comparative analysis with other methods such as metabolic inhibition (MI), mycoplasmocidal (MC) and radioimmunoprecipitation (RIP) methods. MC and RIP test gave antibody titers comparatively higher than MI and CF. Comparative studies on the sensitivity of the various methods considered were also carried out on 6 paired sera collected from the same patients at 10 days intervals. The increase in antibody titers were much higher when MC and RIP tests were considered. When RIP and MC methods were compared, the first appeared more sensitive in all cases studied.

Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann has been used for the detection of antibodies against Mycoplasma pneumoniae. These organisms, grown in flat-bottom wells of microtiter plates, were used as antigen. Preliminary results suggest that ELISA is specific and that its sensitivity is somewhere in between those of the metabolic inhibition and radioimmunoprecipitation methods. Unlike other serological methods, such as metabolic inhibition and mycoplasmacidal tests, in which the presence of antibiotics in sera may give false results, ELISA provided results that were not influenced by antibiotics. Furthermore, ELISA offers the possiblity of measuring not only immunoglobulin M, but also complement-independent antibodies, especially those of the immunoglobulin A class. Theoretical aspects concerning the different sensitivities of some serological reactions are discussed.

Nucleoside uptake in macrophages from various murine strains: a short-time and a two-step stimulation model.

Kinetics of [3H]-uridine uptake by murine peritoneal macrophages (pM phi) is early altered after exposure to a variety of stimuli. Alterations caused by Candida albicans, lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-gamma) were similar in SAVO, C57BL/6, C3H/HeN and C3H/HeJ mice, and were not correlated with an activation process as shown by the amount of tumor necrosis factor-alpha (TNF-alpha) being released. Short-time exposure to all stimuli resulted in an increased nucleoside uptake by SAVO pM phi, suggesting that the tumoricidal function of this cell either depends from the type of stimulus or the time when the specific interaction with the cell receptor is taking place. Experiments with priming and triggering signals confirmed the above findings, indicating that the increase or the decrease of nucleoside uptake into the cell depends essentially on the chemical nature of the priming stimulus. The triggering stimulus, on the other hand, is only able to amplify the primary response.

Solid-phase binding of clinical isolates of Escherichia coli expressing different piliation phenotypes. Effect of glycosaminoglycans.

Pili (or fimbriae) are frequently present on most Escherichia coli strains and they mediate binding to specific receptors. In the present work we used type 1 and P-fimbriated E. coli strains isolated from patients with urinary tract infections to study the antiadhesive effect of some synthetic commercial glucosaminoglycans (GAGs). Quantitative determinations of tritiated bacteria associated with specific receptor-activated resins indicated that displacement by GAGs was more effective in the presence of nonspecific binding. Glucuronilglucosaminoglycan sulfate (Glu-g) and esosaminoglycan sulfate (Eso-g) at a concentration of 10 mg/ml were the most active substances against P-fimbriated E. coli, while galactosaminoglucuronglycan sulfate (Gal-g) behaved as an anti-adhesive agent on type 1 fimbriated E. coli also at a lower concentration. The possible use of GAGs for the prophylaxis or treatment of chronic urinary tract infections implies a previous characterization of the piliation phenotype of the clinical isolates.

Enzyme-linked immunosorbent assay for serodiagnosis of Mycoplasma pneumoniae infections.

Enzyme-linked immunosorbent assay (ELISA) was used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA were compared with those of other serological methods, such as complement fixation, metabolic inhibition, mycoplasmacidal test, and radioimmunoprecipitation. ELISA and mycoplasmacidal test showed greater sensitivity than did complement fixation and metabolic inhibition. Radioimmunoprecipitation showed serum titers 2- to 16-fold higher. Specificity of ELISA was determined by testing other human mycoplasmas. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution is proposed.

In vitro antimycoplasmal activity of mepartricin.

The in vitro antimycoplasmal activity of mepartricin was evaluated on several mycoplasma strains. The results demonstrate that this polyene antibiotic possesses a high efficacy against these microorganisms.

Colony morphology, ultrastructure and morphogenesis in Mycoplasma hominis, Acholeplasma laidlawii and Ureaplasma urealyticum.

Colonies of Mycoplasma hominis, Acholeplasma laidlawii (three strains) and Ureaplasma urealyticum were examined by light and electron microscopy and their characteristic morphology, ultrastructure and morphogenesis are described. Mycoplasma hominis and A. laidlawii, PG8 and oral strains, developed typical 'fried-egg' colonies which were remarkably heterogeneous in size. The colonies of A. laidlawii strain NCTC 10116 were more homogeneous and grew mainly on the surface of the agar showing a fine granular appearance. Ureaplasma urealyticum produced smaller, granular colonies which grew deeply embedded in the agar and generally without much surface growth. The cellular ultrastructure in these colonies was also examined. The results indicate that several aspects of colony morphogenesis and ultrastructure varied for each of the three species examined.