Franco Busolo

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.

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.

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.

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.

A new ELISA method for the detection of serum bindable anti-platelet antibodies (SPBIG)

Thrombocytopenia is often associated with an immunological mechanism. That event was first demonstrated in idiopathic thrombocytopenic purpura with the identification in the immunoglobulin fraction of the factor responsible for the cytopenia [l]. These antibodies are primarily IgG [2] but IgM, C3 and immunocomplexes were also shown [3,4]. The immunospecificity of the antibody has been demonstrated and seems to be directed towards antigens that are associated with platelet glycoproteins ]5,6]. Conversely, circulating immunocomplexes would attach non-specifically to platelet Fc receptor [7]. Despite these studies, a reliable detection of platelet antibodies in vitro has remained controversial. Two approaches are usually employed to detect anti-platelet antibodies, namely the assay of platelet-associated Ig (PAIG) and of serum platelet-bindable Ig (SPBIG) [2]. PAIG assay shows a better correlation with platelet count and high sensitivity in ITP IS], but is impaired by a low specificity (91. On the other hand, the measurement of SPBIG, combines technical simplicity with a low sensitivity [2]. The aim of our study was to propose a practical application of ELISA for a reliable measurement of SPBIG in ITP according to the criteria of specificity and sensitivity.

Nucleoside transport in activated macrophages

The study of [3H]-uridine uptake by mouse peritoneal macrophages showed that this is an active, temperature- and protein synthesis-dependent phenomenon, which is early altered when are exposed to a variety of stimuli. Murine recombinant interferon-gamma, a stimulus able to activate macrophage and to induce the production of tumor necrosis factor-alpha, within few hours markedly increased [3H]-uridine uptake by mouse macrophage. Other stimuli devoid of activation capacity, such as inert phagocytable latex beads, did not affect this phenomenon, which appeared to be related to macrophage activation. The increase in [3H]-uridine uptake may be an useful phenomenon in studying the early biochemical events associated with macrophage activation.

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.

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.