Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation.

Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.

Codon 129 polymorphism of prion protein gene in sporadic Alzheimer's disease.

Codon 129 polymorphism of the prion protein gene represents a major genetic risk factor for Creutzfeldt-Jakob disease (CJD). Both CJD and Alzheimer's disease (AD) are brain amyloidoses and it would be possible that codon 129 polymorphism plays a role in the susceptibility to AD. In order to investigate this polymorphism in AD the distribution of polymorphic codon 129 of the PRNP gene in 194 probable AD and 124 controls selected in Italy and 109 neuropathologically verified AD and 58 matched controls recruited in the USA was studied. No significant association was found for the PRNP polymorphism in AD compared to controls either in Probable or in Definite AD series even after stratification for APOE polymorphism. This study does not support a role of PRNP polymorphism as a susceptibility factor for AD.

IGF-II promoter methylation and ovarian cancer prognosis.

PURPOSE: The insulin-like growth factor-II (IGF-II) gene has four promoters that produce distinct transcripts which vary by tissue type and developmental stage. Dysregulation of normal promoter usage has been shown to occur in cancer; DNA methylation regulates promoter use. Thus, we sought to examine if DNA methylation varies among IGF-II promoters in ovarian cancer and if methylation patterns are related to clinical features of the disease. STUDY DESIGN: Tumor tissue, clinical data, and follow-up information were collected from 215 patients diagnosed with primary epithelial ovarian cancer. DNA extracted from tumor tissues was analyzed for IGF-II promoter methylation with seven methylation specific PCR (MSP) assays: three for promoter 2 (P2) and two assays each for promoters 3 and 4 (P3 and P4). RESULTS: Methylation was found to vary among the seven assays: 19.3% in P2A, 45.6% in P2B, 50.9% in P2C, 48.4% in P3A, 13.1% in P3B, 5.1% in P4A, and 6.1% in P4B. Methylation in any of the three P2 assays was associated with high tumor grade (P = 0.043), suboptimal debulking (P = 0.036), and disease progression [hazards ratio (HR) = 1.73, 95% confidence interval (CI) 1.09-2.74]. When comparing promoter methylation patterns, differential methylation of P2 and P3 was found to be associated with disease prognosis; patients with P3 but not P2 methylation were less likely to have disease progression (HR = 0.39, 95% CI 0.17-0.91) compared to patients with P2 but not P3 methylation. CONCLUSIONS: This study shows that methylation varies among three IGF-II promoters in ovarian cancer and that this variation seems to have biologic implications as it relates to clinical features and prognosis of the disease.

Can cognitive and behavioural disorders differentiate frontal variant-frontotemporal dementia from Alzheimer's disease at early stages?

Frontal variant-Frontotemporal dementia (fvFTD) and Alzheimer's disease (AD) patients matched for severity of dementia at the Clinical Dementia Rating (CDR) received neuropsychological testing in order to explore if the dysexecutive disorder might characterise fvFTD at early stage, when AD is dominated by the episodic memory defect. We also determined if the behavioural syndrome was more severe in fvFTD than AD, and if specific patterns of behavioural symptoms could differentiate the two types of dementia, using the Neuropsychiatry Inventory (NPI). AD patients performed worse than fvFTD not only in memory but also in executive tasks. Apathy and eating disorders proved to be more severe or frequent in fvFTD even if the two groups did not differ in the total NPI score. CDR score significantly correlated with the NPI score in fvFTD and with the MMSE in AD. Our data confirm that the memory disorders may differentiate the two types of dementia; however, the dysexecutive syndrome is as severe, and even more severe in AD. The severity of the behavioural syndrome is comparable in the two groups but the nature of the behavioural disorders may vary to some extent. We conclude that AD dementia at early stage is a behavioural-cognitive syndrome, while in fvFTD the behavioural disorders appear when the cognitive deficit is still relatively mild.

Chlamydia pneumoniae in PBMC: reproducibility of the OMPA nested touchdown PCR.

The aim of our study was to evaluate whether the replicate PCR testing may provide more accurate estimates of C. pneumoniae DNA prevalence in PBMC of patients undergoing carotid endarterectomy. Clinical sensitivity and reproducibility of ompA nested touchdown PCR was also performed. Clinical sensitivity and reproducibility was examined by testing C. pneumoniae-negative PBMC spiked with serial dilutions of semipurified C. pneumoniae elementary bodies (from 8 to 0.002 IFU/ml). Detection of C. pneumoniae DNA was performed by ompA nested touchdown PCR. Each clinical and spiked PBMC DNA specimen was analyzed in replicates of 1, 3, 5 and 10. PCR results of serial dilutions of C. pneumoniae DNA performed in replicates of 10 were analysed by probit analysis. C. pneumoniae DNA was detected in 14 of the 30 (46.7 %) PBMC clinical specimens examined when 10 replicates were tested. When we analyzed 1, 3 and 5 replicates, 4 (13.3 %), 7(23.3 %), 12(40 %) of the 30 specimens were positive, respectively. The limit of detection of ompA nested PCR touchdown was 0.008 IFU/ml when 10 replicates were tested. The ompA nested PCR had reproducibility scores of 10 for 10 from 8 to 4 IFU/ml concentration, but scores decreased for smaller numbers of IFU/ml. Our results showed that repeat testing of the same specimen increased clinical sensitivity as well as reproducibility of the ompA nested touchdown PCR. In conclusion the replicate PCR testing improves the performance of ompA nested touchdown PCR and provides a more accurate estimates of the prevalence of C. pneumoniae in PBMC of patients with atherosclerotic cardiovascular disease.

Predictors of survival in sporadic Creutzfeldt-Jakob disease and other human transmissible spongiform encephalopathies.

A collaborative study of human transmissible spongiform encephalopathies has been carried out from 1993 to 2000 and includes data from 10 national registries, the majority in Western Europe. In this study, we present analyses of predictors of survival in sporadic (n = 2304), iatrogenic (n = 106) and variant Creutzfeldt-Jakob disease (n = 86) and in cases associated with mutations of the prion protein gene (n = 278), including Gerstmann-Sträussler-Scheinker syndrome (n = 24) and fatal familial insomnia (n = 41). Overall survival for each disease type was assessed by the Kaplan-Meier method and the multivariate analyses by the Cox proportional hazards model. In sporadic disease, longer survival was correlated with younger age at onset of illness, female gender, codon 129 heterozygosity, presence of CSF 14-3-3 protein and type 2a prion protein type. The ability to predict survival based on patient covariates is important for diagnosis and counselling, and the characterization of the survival distributions, in the absence of therapy, will be an important starting point for the assessment of potential therapeutic agents in the future.

Survival after AIDS among Italian haemophiliacs with HIV infection. The Italian Group on Congenital Coagulopathies.

OBJECTIVES: To estimate survival trends for persons with haemophilia and HIV/AIDS. DESIGN AND METHODS: Survival analysis conducted among the cohort of HIV-positive haemophiliacs with AIDS at the Italian Haemophilia Registry. Kaplan-Meier method was used to estimate survival times, stratifying for demographic and clinical covariates. Cox proportional hazards model was applied in order to identify factors independently associated with survival. RESULTS: Median survival from the first AIDS diagnosis to death was estimated to be 17.0 months for 176 individuals with AIDS. Median survival after AIDS diagnosis increased from 12.0 months in December 1983-December 1988 to 17.0 months in January 1989-May 1990 and to 25.0 months in June 1990-December 1991. Median survival times were significantly (P < 0.001) lower for individuals diagnosed with non-infective AIDS indicator diseases (lymphoma, AIDS-associated neurological disease, Kaposi's sarcoma, wasting syndrome: 4.0 months), in comparison with haemophiliacs diagnosed with Pneumocystis carinii pneumonia (PCP; 18.0 months) or other infections (35.0 months). Antiretroviral treatment after AIDS diagnosis was associated with a longer survival than that estimated for individuals with no treatment after AIDS; the same was true for PCP prophylaxis. Younger age at HIV seroconversion and at AIDS diagnosis were associated with a longer survival. Multivariate analysis showed that factors independently associated with survival were type of AIDS indicator disease and antiretroviral administration after AIDS diagnosis. CONCLUSIONS: This study indicates an increasing survival from AIDS diagnosis to death over time, also as a result of the introduction of antiretroviral therapy. Survival trends are similar to those reported among homosexual men and intravenous drug users with AIDS, suggesting a similar access to the health-care system for individuals with AIDS. Survival studies may improve our understanding of the natural history of HIV infection and may indicate the impact of preventive measures.

Increased brain synthesis of prostaglandin E2 and F2-isoprostane in human and experimental transmissible spongiform encephalopathies.

The levels of 2 arachidonic acid metabolites formed either by enzymatic activity of cyclooxygenase, i.e. prostaglandin E2 (PGE2), or by free radical-catalyzed peroxidation, i.e. F2-isoprostane 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), were measured in the CSF of subjects with sporadic and familial Creutzfeldt-Jakob disease (CJD) and in brain homogenates of scrapie-infected mice. The CSF levels of both metabolites were increased in sporadic CJD (n = 52) and familial CJD (n = 10) patients when compared with a group of patients with noninflammatory disorders. Similarly, PGE2 and 8-epi-PGF2alpha levels were higher in brain homogenates obtained from C57BL/6J mice infected with the ME7 scrapie strain than in brain homogenates from control animals. As PGE2 is 1 of the most abundant prostaglandins released during inflammation and 8-epi-PGF2alpha is a quantitative marker of lipid peroxidation, our results provide in vivo biochemical evidence for the occurrence of inflammation and oxidative stress in human and experimental transmissible spongiform encephalopathies (TSEs), a concept so far based mainly on histopathological and in vitro evidence. Interestingly, in sporadic CJD patients, high CSF levels of PGE2, but not 8-epi-PGF2alpha, correlated with short survival time, suggesting that the inflammatory response correlates with the clinical duration of disease.

Increased CSF levels of prostaglandin E(2) in variant Creutzfeldt-Jakob disease.

The concentration of the cyclooxygenase product prostaglandin E(2) was sixfold higher in CSF samples from 18 cases of variant Creutzfeldt-Jakob disease (CJD) than in a group of eight subjects with other noninflammatory neurologic diseases, and comparable to those found in a group of six patients affected by diseases with a known inflammatory component. This finding suggests that cyclooxygenase activity may have a role in variant CJD pathogenesis, as previously reported in sporadic CJD.

Selective neuroinhibitory effects of taurine in slices of rat main olfactory bulb.

Taurine is abundant in the main olfactory bulb, exceeding glutamate and GABA in concentration. In whole-cell patch-clamp recordings in rat olfactory bulb slices, taurine inhibited principal neurons, mitral and tufted cells. In these cells, taurine decreased the input resistance and caused a shift of the membrane potential toward the chloride equilibrium potential. The taurine actions were sustained under the blockade of transmitter release and were reversible and dose-dependent. At a concentration of 5 mM, typically used in this study, taurine showed 90% of its maximal effect. GABA(A) antagonists, bicuculline and picrotoxin, blocked the taurine actions, whereas the glycine receptor antagonist strychnine and GABA(B) antagonists, CGP 55845A and CGP 35348, were ineffective. These findings are consistent with taurine directly activating GABA(A) receptors and inducing chloride conductance. Taurine had no effect on periglomerular and granule interneurons. The subunit composition of GABA(A) receptors in these cells, differing from those in mitral and tufted cells, may account for taurine insensitivity of the interneurons. Taurine suppressed olfactory nerve-evoked monosynaptic responses of mitral and tufted cells while chloride conductance was blocked. This action was mimicked by the GABA(B) agonist baclofen and abolished by CGP 55845A; CGP 35348, which primarily blocks postsynaptic GABA(B) receptors, was ineffective. The taurine effect most likely was due to GABA(B) receptor-mediated inhibition of presynaptic glutamate release. Neither taurine nor baclofen affected responses of periglomerular cells. The lack of a baclofen effect implies that functional GABA(B) receptors are absent from olfactory nerve terminals that contact periglomerular cells. These results indicate that taurine decreases the excitability of mitral and tufted cells and their responses to olfactory nerve stimulation without influencing periglomerular and granule cells. Selective effects of taurine in the olfactory bulb may represent a physiologic mechanism that is involved in the inhibitory shaping of the activation pattern of principal neurons.

Sodium current in periglomerular cells of rat olfactory bulb in vitro.

The capacity of periglomerular cells (PGc) to give fast, Na-dependent action potentials is a crucial and debated issue for the comprehension of how sensory information is processed in the olfactory bulb (OB). Using patchclamp whole cell recording in thin slices of rat OB (P8-P20) we showed that fast sodium conductance is present in all the PGc studied, that this current is sufficiently large to generate action potentials and that action potentials can be evoked in these cells by direct stimulation of the olfactory nerve. A comprehensive kinetic characterization of INa is also presented.

Direct inhibitory effect of taurine on relay neurones of the rat olfactory bulb in vitro.

Whole-cell recordings in rat olfactory bulb slices showed that bath application of 5 mM taurine produces a potent and reversible inhibition of identified mitral and tufted cells. Under current-clamp conditions, a shift of the membrane potential toward the chloride equilibrium potential and a 75% reduction in the membrane resistance were observed. These effects were strongly blocked by bicuculline (10 microM), but not by GABA(B) antagonist and strychnine, and completely maintained under the blockage of synaptic transmission. The results suggest that inhibition of bulbar relay neurons produced by taurine is primarily due to direct activation of somatic GABA(A) receptors and initiation of chloride conductance. This study demonstrates for the first time the actions of taurine in the olfactory system.

Potassium currents in periglomerular cells of frog olfactory bulb in vitro.

Voltage-activated currents have been recorded from periglomerular cells in thin slices of frog olfactory bulb. Cells were examined with whole-cell patch clamp methods. The voltage-dependent potassium currents were studied after pharmacological block of inward currents. Depolarising steps from -130 mV gave an early transient, A-type, outward current and a delayed rectifier K+ current (IKV). The two currents could be isolated on the basis of the differences in their kinetic properties. The A-current developed following a third-order kinetics when the membrane was depolarised to potentials more positive than -40 mV after preconditioning to potentials more negative than -60 mV. Once activated (tau a 2.5 ms at 0 mV), IA inactivated following a single exponential (tau ha about 60 ms). IKV activated with a second-order kinetics above -30 mV with a time constant of 4 ms at 0 mV. IA and IKV were sensitive, respectively, to 4-aminopyridine (4-AP) and tetraethylammonium (TEA).

Response of CD-1 mice to the chemical defence of a common arthropod (Ommatoiulus sabulosus).

In order to set up a novel and ethologically relevant methodology that could be applied to the study of olfactory capabilities in transgenic mice, we analysed the behavioural responses of sexually mature male and female CD-1 mice individually exposed to a striped millipede, Ommatoiulus sabulosus (L.), a very common myriapod species that secretes a repulsive and persistent odour in the presence of a predator. As control, we exposed mice to a larva of the lepidopteran Greater wax moth, Galleria mellonella (L.), which closely resembles the millipede in shape and dimensions but which does not secrete a repulsive odour in defence. We recorded and analysed a wide spectrum of behavioural responses including both those of avoidance and nonavoidance such as attempts to eat the arthropod. Behavioural responses were measured for 10 min upon first exposure to the millipede or wax moth. The procedure was repeated for 3 consecutive days. Upon exposure to a millipede, mice of both sexes showed a dramatic increase in the avoidance behaviour of digging. Moreover, millipedes were repulsive to mice and though they were sniffed frequently and sometimes caught, they were never eaten. In comparison, mice exposed to a wax moth almost always ate it. Sex differences emerged only for locomotion with female appearing to be more active. These results suggest that mice are able to discriminate between ethologically relevant odours and that the behavioural responses they display in this more natural context differ from those observed in response to odours of predators.

Lung cancer in young patients.

Despite the increasing frequency of lung cancer, the percentage occurring in young patients is very low (1.3-5.5% of all lung cancers). In 1992, of the 78,124 cases observed in Italy, 2.8% involved patients under 40 years of age. We reviewed a series of 800 patients with histologically proven lung cancer, candidates to a long-term follow-up. Of these, 23 (2.9%) were under 40 years of age, with a low male/female ratio (1.87:1). Fifty-two percent were smokers and 82.6% presented symptoms as the time of diagnosis. The most frequent histologic types were adenocarcinoma and large-cell type, which carried a better outcome (10-year survival of 28.5%) than epidermoid and small-cell types (p = 0.013). These tumors detected in 13% and 17.4% of cases, were unresectable (except for one epidermoid carcinoma), with a survival expectancy of 0% at two years. Considering all patients, resection was possible in nine cases, being curative in seven, with an overall 10-year survival rate of 44.4% (p = 0.002 vs non-resected patients). Stage I-II had the best prognosis with a 10-year survival rate of 80% (p = 0.022 vs resected stage III-IV). Patients undergoing primary chemotherapy and/or radiotherapy had the worst prognosis with no survivors at 30 months. In young patients clinical and pathological parameters had almost the same distribution except for sex and histologic type and offered almost the same survival probability as in patients over 40 years of age. When prognostic findings were tested by univariate analysis, only resectability was found to have an independent favourable impact on survival (hazard risk: 7.47; 95% confidence interval: 1.50-37.14).

Local and/or distant recurrences in T1-2/N0-1 non-small cell lung cancer.

Data from a series of 181 patients subjected to long-term follow-up after surgical resection for non-small cell stage I and II lung cancer were analyzed to evaluate the statistical incidence and the prognostic factors of recurrence. The recurrence rate/year was particularly high in the first 2 years after surgery: the 2-year recurrence rate was 35.1% in stage I tumors and 51.8% in stage II, whereas the 5- and 7-year recurrence rates were 46.1 and 55.9% and 65.8 and 70.7%, respectively, for the same groups. Recurrences were observed more frequently in non-epidermoid carcinomas with multiple nodules (100% at 5 years) and in carcinomas classified as stage II (70.7% at 5-7 years), particularly when defined as adenocarcinoma (100% at 3 years). In the overall recurrence rate we observed no significant difference dependent on the type of resection even though limited segmental or wedge resection appeared to be related to a higher risk rate (true recurrence rate ratio: 0.6). Over two-thirds of the first observed recurrences were located at a distant site, with a slightly higher incidence of non-epidermoid carcinoma (72.5%). Isolated local recurrence mostly occurred in epidermoid carcinoma (47.6%). The most frequent sites of recurrence were the brain, bone and mediastinum. On multivariate analysis, independently significant adverse prognostic factors regarding the recurrence incidence were: tumor size greater than 3 cm, bronchial or hilar lymph node involvement, tumor histologically defined as adenocarcinoma, and the presence of satellite nodules.

Neurobehavioral effects of prenatal lamivudine (3TC) exposure in preweaning mice.

The present study provides a characterization of the behavioral changes induced in preweaning mice by prenatal exposure to lamivudine (3TC), an antiviral drug recently entered in the clinical practice to treat HIV patients. Pregnant CD1 mice were given per os bidaily either 3TC at different doses (125, 250, or 500 mg/kg) or vehicle solution (saline 0.9%) from pregnancy day 10 to delivery. Data on reproductive performance, such as gestation length, litter size, and offspring viability, were collected. Offspring were then examined for a series of different somatic and behavioral end points, including sensorimotor development, ontogenetic pattern of ultrasonic vocalization, passive avoidance learning, and locomotor activity. In the absence of gross changes in somatic and sensorimotor development, a slight change in ultrasound emission was found on postnatal day (PND) 3, with 125 and 500 mg/kg 3TC-treated offspring emitting a lower number of ultrasounds. Learning and retention performances of a passive-avoidance task on PND 20-21 were unaffected by 3TC treatment, while decreased habituation in an automated locomotor activity test was evident in male offspring exposed to 250 and 500 mg/kg 3TC.

Behavioural effects of endocrine disrupting chemicals on laboratory rodents: statistical methodologies and an application concerning developmental PCB exposure.

Appropriate behavioural tests and adequate statistical tools may help to establish the ED properties of a given compound by pointing out the alterations of selected behavioural endpoints. Frequently, laboratory collected data consist of frequencies and/or durations of specific items, and the analysis of variance (ANOVA) technique is performed to assess whether the investigated factors affect these behavioural endpoints. Moreover, when numerous aspects of behaviour are investigated simultaneously, Principal Component Analysis (PCA), a multivariate technique, may be very useful to reduce the overwhelming number of correlated original variables to a few orthogonal artificial variables (factors). Continuous Time Markov Chain (CTMC) models may be applied to analyse the time structure of a behavioural pattern when data consist of sequences of events and the time points at which they occur. Moreover, the Cox Proportional Hazard Model, a methodology originally developed for the analysis of failure time data, may help to evidence the effects of a given treatment on behavioural sequences when the assumptions of CTMC models are not fully satisfied. Analyses on data from mice of the outbred CD-1 strain (controls in a study of toxicity and exposed to PCB during development) are presented as examples to show how adequate statistical analyses and appropriate behavioural tests may reveal relevant effect of treatments otherwise not easily detected.

Lung cancer surgery in elderly patients.

AIMS AND BACKGROUND: Bronchogenic carcinoma is the major cancer-related cause of death in patients aged 70 years and over, and its incidence is rising. The aim of our study was to compare the incidence and the prognostic effect of the parameters characterizing resected patients with non-small-cell lung cancer (NSCLC) when stratified by age. Of 283 NSCLC patients candidates to a long-term follow-up program and who underwent pulmonary resection in our Unit, 34 (12%) were older than 70 years. METHODS: All patients had been preoperatively selected to exclude those with severe or multiple organ system disease and staged in accordance with the UICC classification. RESULTS: When univariate and multivariate analyses were performed within the elderly group, exclusively epidermoid carcinoma and multiple tumor nodules emerged as independent poor prognostic factors (hazard risk, 5.77 and 7.33, respectively). In comparing the older and younger groups, a higher incidence of previous primary neoplastic disease (P = 0.001), epidermoid carcinoma (P < 0.05) and multiple tumor nodules (P < 0.001) was observed in the elderly. Postoperative death was similar (3% vs 4.8%) in the two age groups, as was survival expectancy when stratified by stage. However, univariate analysis showed that epidermoid carcinoma (P = 0.001) and pneumonectomy (P = 0.00001) had a worse outcome in the older early stage subset than in the younger group. When multivariate analysis was performed in all early stage patients, only lymph node involvement and multiple tumor nodules were independently related to survival (hazard risk, 1.82 and 3.76, respectively) and had a poor prognosis. In more advanced disease, elderly and younger patients had a similar outcome. CONCLUSIONS: Our results confirm that a patient's advanced age is not a risk factor in deciding on pulmonary resection, at least for stage I and II NSCLC, and suggest that in all patients, irrespective of age, stage and histologic cell type, the presence of multiple tumor nodules is the only true prognostic factor with a very low survival rate.