ReProComet: a new in vitro method to assess DNA damage in mammalian sperm.

The increasing request of chemical safety assessment demands for the validation of alternative methods to reduce the resort to animal experimentation. Methods that evaluate reproductive toxicity are among those requiring the largest use of animals. Presently, no validated in vitro alternative exists for the assessment of reproductive toxicity. Mammalian sperm are sensitive targets of DNA-reactive chemicals, which form premutagenic adducts. Here, we propose a new method based on comet assay to detect DNA damage induced by potential germ cell mutagens in bull sperm available from assisted reproduction practices. In somatic cells, chemical-induced adducts can be revealed by comet assay that detects DNA breaks produced during adduct repair. Mature sperm, however, are devoid of repair enzymes, and adducts are processed only after fertilization. For this reason, comet assay is not sensitive to detect DNA lesions induced in sperm by most chemicals. To overcome such limitation, we developed a modified comet assay based on the addition of a protein extract from HeLa cells to agarose-embedded sperm on microscopic slides. To test the method, sperm were treated in vitro with methyl methanesulfonate (MMS) or melphalan (MLP) and comet assay was conducted both with and without protein supplementation. No effect of MMS or MLP was detected without protein supplementation; on the contrary, a clear-cut dose-dependent effect was measured after addition of the cell extract. These results represent a proof of concept of a novel in vitro mutagenicity test on sperm that could offer a promising approach to complement previously validated in vivo germ cell genotoxicity assays.     

Asymmetric dimethyl-arginine (ADMA) response to inflammation in acute infections.

BACKGROUND AND METHODS: The endogenous inhibitor of nitric oxide synthase (NOs) asymmetrical dimethyl-arginine (ADMA) has been implicated as a possible modulator of inducible NOs during acute inflammation. We examined the evolution in the plasma concentration of ADMA measured at the clinical outset of acute inflammation and after its resolution in a series of 17 patients with acute bacterial infections. RESULTS: During the acute phase of inflammation/infection, patients displayed very high levels of C-reactive protein (CRP), interleukin-6 (IL-6), procalcitonin and nitrotyrosine. Simultaneous plasma ADMA concentration was similar to that in healthy subjects while symmetric dimethyl-arginine (SDMA) levels were substantially increased and directly related with creatinine. When infection resolved, ADMA rose from 0.62 +/- 0.23 to 0.80 +/- 0.18 micromol/l (+29%, P = 0.01) while SDMA remained unmodified. ADMA changes were independent on concomitant risk factor changes and inversely related with baseline systolic and diastolic pressure. Changes in the ADMA/SDMA ratio were compatible with the hypothesis that inflammatory cytokines activate ADMA degradation. CONCLUSIONS: Resolution of acute inflammation is characterized by an increase in the plasma concentration of ADMA. The results imply that ADMA suppression may actually serve to stimulate NO synthesis or that in this situation plasma ADMA levels may not reflect the inhibitory potential of this methylarginine at the cellular level.     

Prevalence, clinical characteristics, quality of life, and prognosis of patients with congestive heart failure and isolated left ventricular diastolic dysfunction.

Prevalence of isolated left ventricular (LV) diastolic dysfunction has been reported to be as high as one-third of all heart failure (HF) cases, with an increasing prevalence in the elderly population. However, there is a paucity of prospective data about the prevalence and prognosis of isolated LV diastolic dysfunction in an unselected population of patients hospitalized with HF. Therefore, we prospectively evaluated 179 consecutive patients discharged from our hospital with HF to assess the prevalence of systolic versus diastolic LV dysfunction among patients hospitalized with HF and to compare their demographics, clinical features, self-perceived quality of life (QOL), and 6-month readmission rate and mortality. Among them, 133 (59% men, median age 74 years) showed in sinus rhythm and had no significant primary valvular disease. LV diastolic dysfunction was diagnosed on the basis of the European Study Group on Diastolic HF echocardiographic criteria. QOL was assessed at hospital discharge and 6-month follow-up visit using the Minnesota Living with HF questionnaire. Survival of patients with HF was compared with that of age- and sex-matched general population. In all, 29 patients (22%) had isolated LV diastolic dysfunction and 102 (78%) had prevalent LV systolic dysfunction (ie, LV ejection fraction 相似文献   

Significance of cyclin D1 expression in meningiomas: a preliminary study.

Forty-four evaluable patients with intracranial meningiomas were assessed for the expression of the cell-cycle regulator cyclin D1 and of proteins involved in proliferation and apoptosis such as PCNA, MIB-1, p53 and bcl-2. Analyses were carried out by western blot and immunohistochemistry after immediate processing of fresh tumor specimens. By western blot, expression of cyclin D1 significantly correlated with p53 (p=0.02) and with proliferative activity, as assessed by PCNA expression (p=0.0009). By immunohistochemistry, a significant relationship between cyclin D1 and the proliferation marker MIB-1 was confirmed (p=0.05), whereas significance with bcl-2 expression was not found (p=0.01). Moreover, although the association with tumor grade appeared of borderline statistical significance (p=0.07), all the grade II/III meningiomas showed increased expression of cyclin D1 and high proliferative activity. In conclusion, data from this preliminary study seem to suggest a potential value of the combined expression of cyclin D1 and proliferation indicators in defining subgroups of meningiomas with a more aggressive biological behavior.     

Clinical epidemiology and survival of progressive multifocal leukoencephalopathy in the era of highly active antiretroviral therapy: Data from the Italian Registry Investigative Neuro AIDS (IRINA)

Human immunodeficiency virus (HIV)-associated progressive multifocal leukoencephalopathy (PML) remains a relevant clinical problem even in the era of highly active antiretroviral therapy (HAART). Aims of the study were to analyze clinical and treatment-related features and the survival probability of PML patients observed within the Italian Registry Investigative Neuro AIDS (IRINA) during a 29-month period of HAART. Intravenous drug use, the presence of focal signs, and the involvement of white matter at neuroradiology increased the risk of having PML. A reduced probability of PML was observed when meningeal signs were reported. Patients starting HAART at PML diagnosis and previously naïve for antiretrovirals showed significantly higher 1-year probability of survival (.58), compared to those continuing HAART (.24), or never receiving HAART (.00). Higher CD4 cell count were associated with a higher survival probability (.45). At multivariate analysis, a younger age, higher CD4, starting HAART at PML diagnosis, the absence of previous acquired immunodeficiency syndrome (AIDS)-defining events, and the absence of a severe neurologic impairment were all associated with a reduced hazard of death. The use of cidofovir showed a trend towards a reduced risk of death.     

Intermittent etidronate partially prevents bone loss in hirsute hyperandrogenic women treated with GnRH agonist

Treatment with gonadotropin-releasing hormone (GnRH) agonist leads to enhanced bone turnover and accelerated bone loss in premenopausal women with endometriosis, uterine leiomyomatomas and hirsutism. Sodium etidronate is a powerful inhibitor of bone resorption which has been proven efficacious in the prevention and treatment of postmenopausal osteoporosis. The objective of this study was to evaluate the skeletal effects of 6 months of therapy with the depot preparation of the GnRH agonist triptorelin (decapeptil 3.75 mg intramuscularly every 4 weeks) in 24 hirsute patients, aged 24–33 years, with hyperandrogenic chronic anovulation. Ten patients also received cyclical etidronate in an oral dose of 400 mg/day for 2 weeks, followed by an 11-week period of 500 mg/day elemental oral calcium (one cycle). The remaining 14 patients received 500 mg/day of elemental calcium continuously. After 6 months all treatments were discontinued for at least a further 6 months. Bone mineral density (BMD) at lumbar spine and hip (dual-energy X-ray absorptiometry, Sophos LXRA, France) and biochemical markers (serum alkaline phosphatase, osteocalcin, urinary N-telopeptide and hydroxyproline/creatinine ratio) were evaluated at baseline, 6 months and 12 months. In the group given GnRH agonist alone BMD fell significantly at all measured skeletal sites during the first 6 months. In the patients treated with etidronate a significant decrease in BMD was observed at lumbar spine but not in the femoral neck and trochanter, and the changes at lumbar spine and trochanter were significantly smaller than those in the control group. At 6 months bone turnover was also increased in patients treated with GnRH and calcium. Cyclical etidronate prevented the increase in biochemical markers of bone formation and resorption, with the exception of calcium/creatinine excretion, which was significantly increased in both groups. Six months after treatment withdrawal BMD did not recover in either group. Biochemical markers (N-telopeptide, serum alkaline phosphatase) remained increased in those patients previously treated with calcium alone while they remained close to baseline values in the patients treated with cyclical etidronate.Our study indicates that: (1) GnRH agonist therapy causes remarkable bone loss in young individuals with androgen excess who are expected to have increased bone mass; (2) this bone loss can be partially prevented by intermittent cyclical etidronate therapy.     

Hand hygiene and glove use behavior in an Italian hospital.

In an Italian hospital, we observed that hand hygiene was performed in 638 (19.6%) of 3,253 opportunities, whereas gloves were worn in 538 (44.2%) 1,218 of opportunities. We observed an inverse correlation between the intensity of care and the rate of hand hygiene compliance (R2=0.057; P<.001), but no such association was observed for the rate of glove use compliance (R2=0.014; P=.078). Rates of compliance with hand hygiene and glove use recommendations follow different behavioral patterns.     

Idarubicin metabolism and pharmacokinetics after intravenous and oral administration in cancer patients: a crossover study

The pharmacokinetics and metabolism of 4-demethoxydaunorubicin (idarubicin, IDA) were studied in 21 patients with advanced cancer after i.v. (12 mg/m2) and oral (30-35 mg/m2) treatment according to a balanced crossover design. Patients were divided into four groups: subjects who showed normal liver and kidney function (group N), those who presented with normal kidney function and liver metastases (group L), those with kidney dysfunction (creatinine clearance, less than or equal to 60 l/h; group R), and those with both liver and kidney dysfunction (group LR). Five patients showed variations in liver or kidney function after the first treatment and were considered to be nonevaluable for the crossover study but evaluable for the liver/kidney function study; some of them appeared in different groups for the i.v. as opposed to p.o. treatments. After i.v. administration, IDA plasma levels followed a triphasic decay pattern. The main metabolite observed in all patients was the 13C-reduced compound (IDAol), which attained plasma levels 2-12 times higher than those of the parent compound. IDA pharmacokinetics was not dependent on the presence of liver metastases but was related to the integrity of kidney function. Analysis of variance indicated a significant correlation between IDA plasma clearance and creatinine clearance; it was also found that IDA plasma clearance was lower in patients whose creatinine clearance was less than 60 ml/min [group N, 122.8 +/- 44.0 l/h; group L, 104.4 +/- 27.7 l/h (P = 0.58) vs group R, 83.4 +/- 18.3 l/h (P = 0.037)]. The IDAol terminal half-life and mean residence time (MRT) were significantly increased in patients with impaired kidney function [MRT: group N, 63.6 +/- 10.8 h; group L, 69.9 +/- 10.2 h (P = 0.27) vs group R, 83.2 +/- 10.9 h (P = 0.025) and t1/2 gamma: group N, 41.3 +/- 10.1 h; group L, 47.0 +/- 7.4 h (P = 0.31) vs group R, 55.8 +/- 8.2 h (P = 0.025)]. After oral treatment, drug absorption occurred during in the first 2-4 h after IDA administration; a biphasic decay pattern was observed thereafter. The main metabolite observed in all patients was again IDAol. The AUC of IDAol was greater after oral administration than after i.v. treatment in proportion to the AUC of IDA (i.v.: AUC-IDAol/AUC-IDA, 2.4-18.9; p.o.: AUC-IDAol/AUC-IDA, 4.1-21.4). Following oral dosing, a substantial amount of 4-demethoxydaunomycinone (AG1) was found in 11/21 patients.(ABSTRACT TRUNCATED AT 400 WORDS)