Comparative study of seminal parameters between samples collected in 1992 and samples collected in 2010.

OBJECTIVE: The aim of this study was to conduct a comparative study of semen quality in two large populations; one evaluated in 1992 and another in 2010, in order to evaluate any possible decline in male fertility due, at least in part, to environmental factors. MATERIAL AND METHODS: A total of 701 subjects in 1992 (TOTAL group 1992) and a total of 626 subjects in 2010 (TOTAL group 2010) were enrolled in our Andrology Unit. Each group was subdivided into 3 subgroups: Subfertile, Pathology and Control. Standard semen analysis was performed using the Superimposed Image Analysis System, according to WHO guidelines 1987 (for TOTAL group 1992) and WHO guidelines 1999 (for TOTAL group 2010). RESULTS: The mean values of sperm number (concentration/ml as well as the total ejaculate) and progressive motility were significantly higher in TOTAL group 2010 than TOTAL group 1992. Atypical forms in TOTAL group 1992 semen samples were significantly lower than TOTAL group 2010. The mean age of TOTAL Group 2010 was significantly higher compared with TOTAL Group 1992. In particular, the mean age gap was more evident in Subfertile subjects. CONCLUSIONS: In conclusion, environmental factors have not determined a significant decline in seminal parameters in the past 18 years.

Mepacrine antagonises tumour cell growth induced by natural polyamines.

BACKGROUND: Mepacrine is an antiproliferative agent, characterised by an aliphatic chain similar to that of natural polyamines whose activation is closely associated with cell proliferation and may lead to malignant transformation and neurodegenerative diseases. This study aims to investigate a possible antagonism between mepacrine and polyamines in tumour proliferation. MATERIALS AND METHODS: MCF-7 and Vero cells were cultured in Eagle's minimum essential medium and then subjected to graded concentrations of putrescine, spermine and spermidine alone and in combination with mepacrine. Methyl thiazole tetrazolium test and Western-blotting were performed. RESULTS: Putrescine and spermidine at 0.5 mg/l significantly stimulated cell growth, whereas mepacrine treatment confirmed the enhanced p21 expression previously reported by a recent study and growth inhibition. When used in combination, mepacrine antagonized MCF-7 growth induced by polyamines. CONCLUSION: Our results suggest that mepacrine may represent a choice in the treatment of tumours induced by the modified concentration of polyamines.

New neurocutaneous syndrome with defect in cell trafficking and melanosome pathway: the future challenge.

OBJECTIVE: Case study of a CNS impairment lacking in presumptive cause; case presents with a clinical phenotype encompassing multiple differently expressed and combined symptoms, as well as a subtle skin defect. MATERIALS AND METHODS: A 6-year-old male with apparently isolated mental delay, speech delay, attention deficit/hyperactivity disorder, epilepsy, and subtle and insignificant skin dyschromias. The patient underwent a systematic evaluation, including clinical history; medical, neurological and ophthalmologic examinations. Skin, teeth, nails, hair and sudation were examined for defects. Routine laboratory tests for blood, urine, were performed. The proband had thyroid function tests, electrocardiography, genitourinary system and abdominal examinations. Special examinations pertaining to mental performance, biochemistry, chromosome studies, imaging and electrodiagnostic studies, and skin biopsy were also performed. RESULTS: Investigators ruled out genetic syndromes, congenital infections, fetal deprivation, perinatal insults, intrauterine exposure to drug abuse, and postnatal events such as CNS infections as possible common causes of brain impairment. Being all further test negative, the patient exhibited an ultrastructural defect of the skin, identical to that previously described [Buoni S, Zannolli R, de Santi MM, Macucci F, Hayek J, Orsi A et al. Neurocutaneous syndrome with mental delay, autism, blockage in intracellular vesicular trafficking and melanosome defects. Eur J Neurol 2006;13:842-51], suggesting that some cell compartments, such as rough endoplasmic reticulum, lysosomes, Golgi apparatus, and the vesicular zone (racket) of Birbeck granules, sharing similar components, can be altered, resulting in a common defect in cell trafficking, associated to melanosome defects. CONCLUSIONS: This new devasting, ultrastructural phenotype accompanied by apparently unspecific and mixed neurological symptoms should represent a future challenge to finally discover the pathogenesis of many childhood CNS symptoms, that currently seem to lack any apparent cause.

Toxicity and gut associated lymphoid tissue translocation of polymyxin B orally administered by alginate/chitosan microparticles in rats.

Fluorescent calcium alginate/chitosan microparticles, prepared using a spray-drying technique followed by crosslinking reactions with calcium ions and chitosan, were assayed in-vivo for polymyxin B (PMB) oral toxicity, uptake by Peyer's patches and PMB oral absorption. A single PMB dose (300 mg kg(-1)), loaded in microparticles or dissolved in water, was administered to rats by oral gavage under fasted and fed conditions. By monitoring incidence of mortality, animal behaviour, clinical signs and abnormality in several organs, PMB in water solution was found lethal at a dose lower than the LD50 (790 mg kg(-1)) in the fasted state and toxic for the gastrointestinal tract in the fed state. However, no signs of acute toxicity at the level of the gastrointestinal tract were observed when animals were administered PMB loaded in microparticles under fasted and fed conditions. A lower PMB dose (125 mg kg(-1)), loaded in microparticles or dissolved in water, was given to rats in a fed state to determine PMB levels in Peyer's patches, urine and serum as well as to detect the loaded microparticles inside Peyer's patches for three days after dosing. Abnormalities were observed at gut level only when PMB was dosed in a water solution. Detectable antibiotic levels in Peyer's patches and urine as well as more constant PMB serum concentrations were provided by dosing PMB loaded in microparticles. Therefore, the use of alginate/chitosan microparticles to target the lymphatic system could improve safety when administering PMB orally.

Effects of anti-malarial drugs on MCF-7 and Vero cell replication.

BACKGROUND: Previous in vivo studies performed in our laboratories demonstrated that anti-malarial drugs may or enhance or slow down Ehrlich's ascites tumour progression in infected mice. In the light of these observations, an in vitro study was undertaken to assess the response of human tumour cells to various anti-malarial drugs and consequently the safety of the anti-malarial therapy. MATERIALS AND METHODS: MCF-7 cells and Vero cells (control line) were cultured in Eagle's minimum Essential medium (EMEM) and then subjected to graded concentrations of different anti-malarial drugs. Trypan-blue exclusion, MTT and Western blotting tests were performed. RESULTS: The findings showed that pyrimethamine (12.5 mg/L), chloroquine (12.5 mg/L) and primaquine (1.56 mg/L) stimulated MCF-7 cell growth. The proliferative effect was inhibited by doxorubicin only in cultures treated with chloroquine and primaquine. These results might indicate that some anti-malarial drugs have a worrying tumour-promoting effect which should not be underestimated when undertaking anti-malarial prophylactic measures.

Antibacterial agent discovery using thymidylate synthase biolibrary screening.

Thymidylate synthase (TS, ThyA) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate to 2'-deoxythymidine 5'-monophosphate, an essential precursor for DNA synthesis. A specific inhibition of this enzyme induces bacterial cell death. As a second round lead optimization design, new 1,2-naphthalein derivatives have been synthesized and tested against a TS-based biolibrary, including human thymidylate synthase (hTS). Docking studies have been performed to rationalize the experimentally observed affinity profiles of 1,2-naphthalein compounds toward Lactobacillus casei TS and hTS. The best TS inhibitors have been tested against a number of clinical isolates of Gram-positive-resistant bacterial strains. Compound 3,3-bis(3,5-dibromo-4-hydroxyphenyl)-1H,3H-naphtho[1,2-c]furan-1-one (5) showed significant antibacterial activity, no in vitro toxicity, and dose-response effects against Staphylococcus epidermidis (MIC=0.5-2.5 microg/mL) clinical isolate strains, which are resistant to at least 17 of the best known antibacterial agents, including vancomycin. So far this compound can be regarded as a leading antibacterial agent.

Pharmacological and toxicological evaluation of a new series of thymidylate synthase inhibitors as anticancer agents.

Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3-bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR35) and 6-bromo-3,3-bis(3-chloro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [3H]-thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [3H]-thymidine test was the best in predicting toxicity against melanoma cells.

Effects of glycyrrhizin on UVB-irradiated melanoma cells.

It is known that liquorice root is rich in compounds which exert several pharmacological actions. In the present study, we evaluated the effect of glycyrrhizin (the main constituent of liquorice root) and of its metabolite aglycone, 18beta-glycyrrhetinic acid, on UVB-irradiated human melanoma cells: SKMEL-2 from metastatic tissue and SKMEL-28 from primary malignant melanoma. Tests performed (Trypan blue exclusion test, MTT and Western blot) showed that glycyrrhizin is not toxic for both types of cells. In SKMEL-28 cells, Bcl-2 expression was low after UVB irradiation, but it was increased when treated with glycyrrhizin. On the contrary, in the SKMEL-2 cell culture, Bcl-2 expression was not modified by the substances under study. The results show that glycyrrhizin treatment might offer protection from the damage induced in humans by UVB radiation, while it seems to be ineffective on metastatic cells. Further studies must be performed to understand the mechanism of the protective effect.

High frequency of (TG)mTn variant tracts in the cystic fibrosis transmembrane conductance regulator gene in men with high semen viscosity.

OBJECTIVE: To evaluate a possible correlation between abnormal semen consistency and cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and variant tracts. DESIGN: Study of CFTR mutations and variant tracts in men with high semen viscosity as compared with normospermic men. SETTING: University-based centers for andrology, clinical biochemistry, and cystic fibrosis. PATIENT(S): Forty-six male partners from infertile couples with sine causa high semen viscosity compared with 72 normospermic men. INTERVENTION(S): Semen sample collection. MAIN OUTCOME MEASURE(S): We obtained the (TG)mTn polymorphic tracts and a panel of 31 mutations of CFTR, semen viscosity, and semen variables. RESULT(S): The frequencies of the (TG)12 and T5 variant alleles were statistically significantly higher in men with high semen viscosity (17.4% and 7.6%, respectively) than in the normospermic control group (6.9% and 1.4%, respectively). The frequency of the genotypes carrying (TG)12 or T5 was statistically significantly higher in men with high semen viscosity (39.1%) than in the normospermic control group (16.7%). Four men with high semen viscosity showed the variant (TG)12T5 haplotype; one of these men presented variant tracts on both alleles. None of the normospermic controls showed a (TG)12T5 haplotype. CONCLUSION(S): Semen hyperviscosity could be considered a "minimal clinical expression" of cystic fibrosis; CFTR gene sequence variations may constitute the genetic basis for this disease.

Selectivity of action of glycyrrhizin derivatives on the growth of MCF-7 and HEP-2 cells.

Since 1986, there have been reports in the literature that glycyrrhizin (GL), 18 alpha-glycyrrhetinic acid (18 alpha-GA) and 18 beta-glycyrrhetinic acid (18 beta-GA) can inhibit the growth of some murine tumours. Recently, testing the activity of GL and 18 alpha-GA on human tumour cells, we observed that both substances are good anti-proliferative agents, especially on those cells whose replication rate is slow. In the present study, the MTT test was performed on MCF-7, Hep-2 and VERO cells treated with increasing concentrations of GL and of its derivatives. Results showed that the substances do not have a toxic effect on VERO cells and that their anti-proliferative effect is evident only on the cell line (MCF-7) that evolves slowly. Finally, the TUNEL test revealed the presence of apoptosis in MCF-7 cells treated with dosages of GL and 18 alpha-GA, thereby confirming that these natural phytoestrogens could be considered as interesting new agents for cancer therapy.

Inhaled Solid Lipid Microparticles to target alveolar macrophages for tuberculosis.

The goal of the work was to evaluate an anti-tubercular strategy based on breathable Solid Lipid Microparticles (SLM) to target alveolar macrophages and to increase the effectiveness of the conventional tuberculosis (TB) therapy. Rifampicin loaded SLM composed of stearic acid and sodium taurocholate were characterized for aerodynamic diameter, surface charge, physical state of the components, drug loading and release as well as drug biological activity on Bacillus subtilis strain. Moreover, SLM cytotoxicity and cell internalization ability were evaluated on murine macrophages J774 cell lines by MTT test, cytofluorimetry and confocal laser microscopy. SLM exhibited aerodynamic diameter proper to be transported up to the alveolar epithelium, negative charged surface able to promote uptake by the macrophages and preserved drug antimicrobial activity. The negligible in vitro release of rifampicin indicated the capacity of the microparticle matrix to entrap the drug preventing its spreading over the lung fluid. In vitro studies on J774 cell lines demonstrated SLM non-cytotoxicity and ability to be taken up by cell cytoplasm. The microparticulate carrier, showing features suitable for the inhaled therapy and for inducing endocytosis by alveolar macrophages, could be considered promising in a perspective of an efficacious TB inhaled therapy by means of a Dry Powder Inhaler device.

18beta-glycyrrhetinic acid and glabridin prevent oxidative DNA fragmentation in UVB-irradiated human keratinocyte cultures.

BACKGROUND: UVB radiation is the major etiological factor in the pathogenesis of skin aging and cancer development. New approaches to prevent and reverse UVB damage are needed to reduce sunlight-induced skin cancer. This study aimed to investigate a possible protective activity of liquorice root extracts glycyrrhizin (GL), 18ß-glycyrrhetinic acid (18ß-GA) and glabridin (GLB) against UVB radiation damage in human keratinocyte cultures. MATERIALS AND METHODS: The MTT test was performed to assess cell viability. DNA damage was evaluated by comet assay, whereas generation of intracellular reactive oxygen species (ROS) was measured by fluorescent 2'7'-dichlorodihydrofluorescein diacetate assay. In addition, the activation of p53, regulation of BCL-2 and PARP cleavage were analyzed by Western blot analysis. RESULTS: The treatment of human keratinocytes with 18ß-GA and GLB prevented direct and indirect DNA damage avoiding apoptosis activation. CONCLUSION: 18ß-glycyrrhetinic acid and glabridin are potent antioxidants that prevent oxidative DNA fragmentation and the activation of apoptosis-associated proteins in human keratinocytes.

Role of the pharmaceutical excipients in the tamoxifen activity on MCF-7 and vero cell cultures.

BACKGROUND: Microparticles are used for controlled drug delivery. With the aim of improving both bioavailability and tamoxifen selective toxicity, the activity of tamoxifen embedded in calcium alginate/chitosan microparticles was studied. MATERIALS AND METHODS: Tamoxifen alone and embedded in microparticles prepared with sodium alginate from Kelco (62% mannuronic acid and 38% guluronic acid) and from Fluka (30% mannuronic acid and 70% guluronic acid) was added to MCF-7 and Vero cultures and evaluated for antiproliferative activity by the MTT test. RESULTS: The use of Kelco or Fluka alginate resulted in different LD(50) values on Vero and MCF-7 cultures, showing a higher cytotoxicity toward Vero cells treated with tamoxifen embedded in Kelco microparticles (25 microM vs. 48 microM on MCF-7 cells) but a selective toxicity with Fluka microparticles (25 microM and 10 microM on Vero and MCF-7 cells respectively). CONCLUSION: Microparticle formulation may improve selective toxicity according to the alginate employed: differences in the chemical alginate composition can dramatically change both drug activity and toxicity.

Human semen hyperviscosity: prevalence, pathogenesis and therapeutic aspects.

The aims of this study were (a) to determine the prevalence of subjects with semen hyperviscosity (SHV) in a large population of male partners of subfertile couples; (b) to identify any correlation between SHV and infections or inflammation of the genital tract; (c) to assess the effects of therapeutic approaches for treating SHV; and (d) to assess sperm kinetic parameters after successful treatment of SHV. A retrospective study of 1 833 male partners of subfertile couples was conducted. Next, clinical, seminal, bacteriological and ultrasound studies involving 52 subjects suffering from SHV were performed, and the SHV was classified as being mild (length of thread > 2 cm and 4 cm and 6 cm). The prevalence of SHV was observed in 26.2% (480) of the subjects, with 13.2% suffering from mild, 6.6% from moderate and 6.4% from severe SHV. Treatment was completely successful in only 27 subjects (52.0%), primarily in those who had mild basal SHV with a positive semen culture. In these subjects, progressive motility percentage, straight line velocity and linearity were significantly higher than pre-treatment levels. SHV is often found in subjects with subfertility. Pathogenesis was strictly related to infective/inflammatory factors in only 48.0% of cases; therefore, it is possible that biochemical, enzymatic or genetic factors have a role in this condition.

Human semen refrigeration at + 4 degrees C: bio-kinetic characteristics.

The aim of our study was to evaluate the bio-kinetic characteristics of human semen refrigerated for different periods and to compare the effects of refrigeration at +4 degrees C against cryopreservation of human sperm at -196 degrees C. Semen was obtained from 30 male partners of infertile couples (infertile subjects) with the following semen profile: sperm count >or=10 x 10(6)/ml; progressive motility >or=20%; atypical forms <70% and white blood cells <1.0 x 10(6)/ml. Fifteen normospermic subjects were also selected as controls (control subjects). The following tests were carried out on basal, refrigerated and cryopreserved sperm: a) sperm kinetic properties (by Superimposed Image Analysis System); b) the Hypoosmotic Viability Test (HVT) (combined Hypoosmotic Swelling and Viability Test). The results of the study showed that the percentage recovery of kinetic properties and of HVT were optimum for up to 48 h. After refrigeration for 72 h, a drastic decrease in straight motility recovery was observed. No significant differences were observed between cryopreservation and refrigeration at +4 degrees C for 48 h for motility or HVT recoveries in samples from control subjects. However, in infertile subjects, a significant decrease in straight progressive motility and HVT recoveries was observed in cryopreserved samples compared to those refrigerated for 48 h. Neither refrigeration nor cryopreservation led to the growth of pathogenic bacteria in any of the cases studied. Based on the above results, refrigeration could represent a useful alternative to the cryopreservation method.

'In vitro' study of chemotherapeutic activity of sulphimidazole on some sensitive and metronidazole-resistant Trichomonas vaginalis strains.

Trichomonacidal treatment based on 5-nitroimidazoles is problematic both when Metronidazole, the drug of choice, is ineffective owing to the presence of resistant strains and when bacterial aerobic infections are present. Sulphimidazole (SIZ) possesses two distinct functional groups: one sulphonamide, the other 5-nitroimidazole. Since SIZ is active against aerobic and anaerobic bacteria, we set out to discover whether, in view of the presence of the 5-nitroimidazole group, it could also be effective against Trichomonas vaginalis. Twelve strains of T. vaginalis were cultured in Modified Thioglycate Medium in anaerobic conditions; subsequently, their growth was monitored in the presence of Metronidazole (MZ), SIZ, Sulphamethoxazole (SMX), Trimethoprim (TMP) and their associations. Eight strains proved to be sensitive to Metronidazole (minimum lethal concentration=0.5 microgml(-1)) and four to be resistant (minimum lethal concentration=40-60 microgml(-1)). SIZ was active against both the sensitive and the Metronidazole-resistant strains (minimum lethal concentrations=0.5-1 and 10 microgml(-1), respectively), thus showing that the chemotherapeutic activities of the two functional groups coexisting in SIZ remain unimpaired.

Interaction between saquinavir and antimycotic drugs on C. albicans and C. neoformans strains.

Candidiasis and cryptococcosis are the most common fungal diseases among patients suffering from HIV infection. In the present work we assess whether the combined therapies, proteinase inhibitors and antimycotic drugs, could modify the therapeutic effect of antimycotics. An in vitro study to evaluate the antifungal effect of saquinavir and antimycotic drugs combination on yeast growth was performed. Strains of C. albicans and C. neoformans from HIV-seropositive patients were used. Susceptibility tests of yeasts to amphotericin B, 5-fluorocytosine, miconazole and fluconazole, singly and in combination with saquinavir, were performed in two different media. In the combinations the antimycotic agents and saquinavir were tested at sub-inhibitory concentrations: 0.1-10 microg ml(-1) and 12.50 microg ml(-1), respectively. The fractionary inhibitory concentration (FIC) index was also calculated. The results show that the interaction between saquinavir and all the antimycotic drugs never resulted in antagonism. Fluconazole acts in more synergistic way, no matter which medium is used. The combined therapy miconazole/saquinavir results in synergism, especially in Sabouraud. The total absence of antagonism and the presence of synergism suggest that a combined therapy could be proposed in the treatment of HIV-seropositive patients to reduce side effects, thanks to the use of lower doses of antimycotic drugs.