Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles

BACKGROUND: The purpose of this study is to evaluate the effectiveness of a standardized mixture of purified enzymes (Liberase), for the isolation of human ovarian follicles. METHODS: This is an experimental prospective study. Ovarian biopsies were obtained from eight young women undergoing laparoscopy for benign gynaecological disease. Follicles were isolated by Liberase or collagenase enzymatic digestion. Follicle quality was assessed by evaluating their general morphology and viability after fluorescent staining, and the ultrastructure by electron microscopy. RESULTS: The number of fully isolated follicles recovered from the Liberase-treated group was lower than from the collagenase group (156 versus 263) despite equal-sized biopsies being taken. A high proportion of follicles (98.6%, 70/71) were viable after Liberase isolation and most follicles were of good morphology with a complete granulosa cell layer (70.4%, 31/44). Ultrastructural studies indicated that Liberase-isolated follicles showed signs of atresia only occasionally and that the oolemma-follicular cell interface was well preserved. CONCLUSIONS: Liberase treatment allows the isolation of highly viable follicles from human ovarian tissue, with an unaltered morphology and ultrastructure. This purified endotoxin-free enzyme preparation is a promising alternative to impure collagenase preparations for the reproducible isolation of intact primordial and primary follicles for culture and grafting purposes.     

The discharge of subthalamic neurons is modulated by inhibiting the nitric oxide synthase in the rat

The effects induced on the discharge of subthalamic spontaneously active neurons by inhibiting the enzyme nitric oxide synthase was studied in two groups of urethane-anesthetized rats. In the first group of animals (n = 10), the activity of subthalamic single units was recorded before and after the systemic administration of 7-nitro-indazole (7-NI, 50 mg/kg i.p.), a selective inhibitor of neuronal nitric oxide synthase. In the second group of rats (n = 15), Nomega-nitro-L-arginine methyl ester (L-NAME), another inhibitor of nitric oxide synthase, was iontophoretically administered while performing single unit extracellular recordings. The activity of most tested spontaneously discharging neurons (8/10) was influenced by 7-NI administration, which always caused a statistically significant decrease in the firing rate of the responsive cells. In contrast, the iontophoretic administration of L-NAME, although influencing many cells (24/32), did not have univocal effects: in fact, 18 cells were inhibited while 6 neurons were excited in a statistically significant manner. We hypothesize that nitric oxide neurotransmission could exert a tonic modulatory influence upon spontaneously discharging subthalamic neurons, with a prevalent excitatory effect.     

Increased spinal cord NGF levels in rats with cobalamin (vitamin B12) deficiency

We have recently demonstrated that the neuropathological morphological alterations caused by cobalamin (Cbl) deficiency in the rat central nervous system are related to the vitamin's inability to modulate the synthesis of some neurotoxic and neurotrophic agents in opposite directions. In the present study, we measured nerve growth factor (NGF) levels in the spinal cord (SC) and cerebrospinal fluid (CSF) of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy (TG) or a Cbl-D diet. In both cases, Cbl deficiency increased SC and CSF NGF levels after the appearance of myelinolytic lesions in the SC white matter (SCWM) (i.e. after the second post-TG month), and these changes were normalised by Cbl treatment in the 4-month-totally-gastrectomised (TGX) rats. Intracerebroventricular (i.c.v.) anti-NGF-antibody treatment prevented the onset of the myelinolytic SCWM lesions in the 2-month-TGX rats (i.e. when SC and CSF NGF levels are still normal) and normalised the ultrastructure of the SCWM in the 4-month-TGX rats, which was however worsened by the i.c.v. administration of NGF. These findings demonstrate that: (i) Cbl deficiency increases SC and CSF NGF levels; and (ii) endogenous NGF seems to play a noxious role in the progression of rat Cbl-D central neuropathy.     

Ductal carcinoma in situ with microinvasion: clinicopathologic study and biopathologic profile

Data regarding the biologic behavior and surgical management, in particular the axillary lymph node excision, of ductal carcinoma in situ with microinvasion (DCIS-MI) are controversial. Therefore, we decided to study the histopathologic characteristics, the biopathologic profile, as well as the follow-up of a group of patients with DCIS-MI. Thirty-one cases of DCIS-MI, 21 of whom were treated with axillary lymph node dissection, were studied. All cases were classified according to the Van Nuys classification, and the extension of DCIS was quantified. The biopathologic profile (ER, PR, MIB 1, p53, c-erbB-2) as well as the follow-up was also investigated. The results did not reveal any statistically significant differences between the two groups, and there was no statistically significant relationship between the extension of DCIS and the number of microinvasion (MI) foci or maximum MI diameter, or between Van Nuys classification of DCIS and again the number of MI foci or maximum MI diameter. DCIS-MI seems associated with good prognosis. None of the patients had relapses or metastases. Our data seem to suggest that the natural history of DCIS-MI resembles DCIS, and we, therefore, suggest that all the surgically removed area should be examined histologically to avoid missing foci of infiltrating breast cancer larger than 1mm.     

Bio-Oss collagen and orthodontic movement for the treatment of infrabony defects in the esthetic zone

The aim of the present study was to evaluate whether it is possible to orthodontically move migrated teeth into infrabony defects augmented with a biomaterial. Three adult patients suffering from chronic periodontitis were treated. Each of the patients presented with an infrabony defect adjacent to a migrated maxillary central incisor. After cause-related therapy was completed, a surgical procedure was performed using the papilla preservation technique. The defects were filled with a collagen bovine bone mineral; after 2 weeks, an orthodontic device was activated using light, continuous forces. Orthodontic treatment time varied from 4 to 9 months; during this period, patients were enrolled in an oral hygiene recall program. At baseline and 6 months after the end of therapy, probing pocket depths (PPD) and clinical attachment levels (CAL) were assessed. In addition, the vertical and horizontal dimensions of the defects were measured on standardized radiographs. Residual mean PPD was 3.33 mm, with a mean reduction of 3.67 mm. Mean CAL gain was 4.67 mm. Radiologic vertical and horizontal bone fills were, on average, 3.17 mm and 2.0 mm, respectively. The present case series shows the effectiveness of a combined periodontic-orthodontic approach for the treatment of infrabony defects. Reduction of PPD to physiologic values, CAL gain, and radiologic defect resolution were obtained. No detrimental effects from the orthodontic movement were observed on the augmentation material.     

Association between hormones and metabolic syndrome in older Italian men

OBJECTIVES: To determine whether low levels of testosterone, sex hormone binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), and dehydroepiandrosterone sulfate (DHEAS) and high levels of cortisol and leptin would be associated with metabolic syndrome (MS). DESIGN: Cross-sectional. SETTING: Population-based sample of older Italian men. PARTICIPANTS: Four hundred fifty-two men aged 65 and older enrolled in the Invecchiare in Chianti (InCHIANTI) study. MEASUREMENTS: Complete data on testosterone, cortisol, DHEAS, SHBG, fasting insulin, IGF-1 and leptin. MS was defined according to Adult Treatment Panel III criteria. RESULTS: MS was present in 73 men (15.8% of the sample). After adjusting for confounders, total testosterone (P < .05) and log (SHBG) (P < .001) were inversely associated, whereas log (leptin) was positively associated with MS (P < .001). Independent of age, log (SHBG) was positively associated with high-density lipoprotein cholesterol (P < .05) and negatively associated with abdominal obesity (P < .001) and triglycerides (P < .001). Log (leptin) was significantly associated with each component of MS. Cortisol, DHEAS, free and bioavailable testosterone, and IGF-1 were not associated with MS. Having three or more hormones in the lower (for hormones lower in MS) or the upper (for hormones higher in MS) quartile was associated with three times the risk of being affected by MS (odds ratio = 2.8, 95% confidence interval = 1.3-6.9) (P = .005), compared with not having this condition. CONCLUSION: Total testosterone and SHBG are negatively and leptin is positively associated with MS in older men. Whether specific patterns of hormonal dysregulation predict the development of MS should be tested in longitudinal studies.     

Risk and predictors of motor-performance decline in a normally functioning population-based sample of elderly subjects: the Italian Longitudinal Study on Aging

OBJECTIVES: To examine risk and predictors of motor-performance (MP) decline targeting subjects performing normally at an initial observation. DESIGN: Cohort study. SETTING: A subsample of the Italian Longitudinal Study on Aging (aged 65-84). PARTICIPANTS: One thousand fifty-two subjects (mean age+/-standard deviation = 71+/-5, 69% men) with normal MP at baseline. MEASUREMENTS: Six tests (standing up from a chair, stepping up, tandem walk, standing on one leg, walking speed, and steps turning 180 degrees ) were used to assess MP at baseline and after 3 years. Baseline characteristics were potential predictors of MP decline. RESULTS: Of the 1,052 subjects performing normally at baseline, 166 (15.8%) had declined in MP at follow-up. Older age, female sex, lower education, symptoms of distal symmetrical neuropathy, cognitive impairment without dementia, parkinsonism, heart failure, anemia, depressive symptoms, worse Mini-Mental State Examination score, and lost activities of daily living and instrumental activities of daily living (IADLs) were significantly associated with MP decline in univariate comparisons. Older age (odds ratio (OR) = 3.84, 95% confidence interval (CI) = 2.14-6.88 comparing age classes > or =80 with 65-69), female sex (OR=1.50, 95% CI = 1.03-2.20), distal symmetric neuropathy (OR = 2.00, 95% CI = 1.03-3.87), depressive symptoms (OR = 1.85, 95% CI = 1.17-2.24), and baseline IADLs (OR = 1.22, 95% CI = 1.08-1.37 for each lost activity) independently predicted MP decline after regression analysis. CONCLUSION: In a population-based cohort of elderly people with normal MP, one-sixth declined in 3 years. Age, sex, distal symmetrical neuropathy, depressive symptoms, and baseline IADLs independently predicted this decline. Distal symmetrical neuropathy is underestimated in the clinical and epidemiological evaluation of motor decline in older people.