Biosynthetic scaffolds for partial meniscal loss: A systematic review from animal models to clinical practice.

Acute or degenerative meniscus tears are the most common knee lesions. Meniscectomy provides symptomatic relief and functional recovery only in the short- to mid-term follow-up but significantly increases the risk of osteoarthritis. For this reason, preserving the meniscus is key, although it remains a challenge. Allograft transplants present many disadvantages, so during the last 20 years preclinical and clinical research focused on developing and investigating meniscal scaffolds. The aim of this systematic review was to collect and evaluate all the available evidence on biosynthetic scaffolds for meniscus regeneration both in vivo and in clinical studies. Three databases were searched: 46 in vivo preclinical studies and 30 clinical ones were found. Sixteen natural, 15 synthetic, and 15 hybrid scaffolds were studied in vivo. Among them, only 2 were translated into clinic: the Collagen Meniscus Implant, used in 11 studies, and the polyurethane-based scaffold Actifit®, applied in 19 studies. Although positive outcomes were described in the short- to mid-term, the number of concurrent procedures and the lack of randomized trials are the major limitations of the available clinical literature. Few in vivo studies also combined the use of cells or growth factors, but these augmentation strategies have not been applied in the clinical practice yet. Current solutions offer a significant but incomplete clinical improvement, and the regeneration potential is still unsatisfactory. Building upon the overall positive results of these "old" technologies to address partial meniscal loss, further innovation is urgently needed in this field to provide patients better joint sparing treatment options.

Animal-based measures on fattening heavy pigs at the slaughterhouse and the association with animal welfare at the farm level: a preliminary study.

Monitoring animal welfare (AW) in pig farms requires both proper indicators and a feasible approach. Animal-based measures (ABMs) are well-established AW indicators. Furthermore, AW screening at the slaughterhouses could be useful for identifying problems on farm. The aim of this study was to evaluate ABMs at the slaughterhouse and, when possible, to compare these ABMs with those collected on the farm. The study was carried out in northern Italy in a commercial abattoir and in a sample of farms. Animal-based measures were recorded on pigs from 62 batches of 54 farms, during ante-mortem (n=10 085 pigs) and post-mortem (n=7952 pigs) inspections. Sixteen of 54 farms were selected to compare ABMs collected at the slaughterhouse with ABMs collected on the farm. Overall, 2295 pigs (mean pigs examined per farm 119±45) were inspected at the slaughterhouse (group S) and 420 pigs (mean pigs per farm 26±5) on the farm (group F). Non-animal-based measures were also collected at the 16 farms. Differences between groups S and F, at the animal level, were assessed by a two-tailed paired Wilcoxon-Mann-Whitney test. Differences at the site of observation level (farm and slaughterhouse) were assessed by Fisher's exact test using a hierarchical log-linear modelling for contingency tables. The most frequent ABMs at the slaughterhouse were manure on the body (47.7%), followed by dermatitis (28.0%), white spot (25.4%) and bursitis (24.7%). Recording ABMs at the slaughterhouse and on the farm usually yielded similar results; however, there were some exceptions. In particular, significant differences were found for non-uniformity of size (P<0.05) and dermatitis (P<0.001), which were higher at the slaughterhouse than on the farm. Results of log-linear modelling underlined the effect of the farm of origin on the percentage of pigs with bursitis, manure on the body and ear injuries that were observed at the slaughterhouse. In group S, significant associations between manure on the body and insufficient presence of clean and dry areas in the corresponding farm were found (P<0.05). Although these results should be interpreted with care due to the limited sample of farms, the slaughterhouse could be a feasible site of observation of ABMs, which could then be integrated in monitoring of AW on farm. Considering the number of slaughtered batches per farm, it would be possible to repeat assessments several times throughout the year for each farm, which could help provide an index for the continuous monitoring of AW.

Minimally Manipulated Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis: A Systematic Review of Clinical Evidence.

BACKGROUND: The use of laboratory-expanded mesenchymal stem cells (MSCs) is subject to several restrictions, resulting in "minimal manipulation" methods becoming the current most popular strategy to increase the use of MSCs in an orthopaedic practice. The aim of the present systematic review is to assess the clinical applications of "minimally" manipulated MSCs, either as bone marrow aspirate concentrate (BMAC) or as stromal vascular fraction (SVF), in the treatment of knee osteoarthritis (OA). METHODS: A systematic review of three databases (PubMed, ScienceDirect, and Google Scholar) was performed using the following keywords: "Knee Osteoarthritis" with "(Bone marrow aspirate) OR (bone marrow concentrate)" or with "(adipose-derived mesenchymal stem cells) OR (adipose derived stromal cells) OR (stromal vascular fraction) OR (SVF)" as either keywords or MeSH terms. The reference lists of all retrieved articles were further reviewed for identification of potentially relevant studies. RESULTS: Twenty-three papers were included in the final analysis (10 on BMAC and 13 on SVF). Of these, only 4 were randomized controlled trials (RCTs). Bias risk evaluation, performed using a modified Coleman score, revealed an overall poor quality of the studies. In terms of clinical application, despite the apparent safety of minimally manipulated MSCs and the short-term positive clinical outcomes associated with their use, clinicians reported different preparation and administration methods, ranging from single intra-articular injections to intraosseous applications to administration in combination with other surgical procedures. CONCLUSIONS: The available literature is undermined by both the lack of high-quality studies and the varied clinical settings and different protocols reported in the few RCTs presently published. This prevents any recommendation on the use of either product in a clinical practice. Nevertheless, the use of minimally manipulated MSCs (in the form of BMAC or SVF) has been shown to be safe and have some short-term beneficial effects.

Behavior of Escherichia coli O157:H7 during the manufacture and ripening of Fontina Protected Designation of Origin cheese.

This study was conducted to describe the cheese-making procedure of Fontina Protected Designation of Origin (PDO) cheese and to evaluate the behavior of Escherichia coli O157:H7 during cheese manufacture and ripening. The study was divided into 2 phases: the production of Fontina PDO cheese was monitored at 3 different dairies in the Aosta Valley and an E. coli O157 challenge was conducted at a fourth dairy. The dairies employ different commercial starter cultures for cheese making. The growth of lactic acid bacilli (LAB) and the decrease in pH were slower in the first hours and the LAB concentrations were overall higher in dairy A than in the other 2 dairies. The pH remained substantially unchanged during ripening (range 5.2 to 5.4) in all dairies. Water activity remained constant at around 0.98 until d 21, when it decreased to around 0.97 until d 80 in dairies A and B and 0.95 in dairy E. Whole raw cow milk was used for making Fontina cheese according to the standard procedure. For the experimental production, the milk was inoculated with E. coli O157:H7 at a concentration of approximately 5 log10 cfu/mL and commercial starter cultures were used according to the Fontina PDO regulation. An increase of 2.0 log10 cfu/g in E. coli O157:H7 was observed during the first 9.5 h of cheese making, followed by a decrease at 46 h when pH decreased to 5.4 in all trials. Fresh cheeses were salted and held at 10°C for ripening for 80 d. Water activity was decreased to 0.952 at the end of the ripening stage. The LAB concentrations declined gradually; this trend was more marked for the lactobacilli than either the thermophilic or the mesophilic lactococci. The increase in LAB count and the decrease in pH in the first hours did not seem to affect E. coli O157 growth. Ripening was found to inhibit pathogen survival, however, as seen in the reduction of 3 log10 from the maximum concentration measured during the earlier stages of production.

Identification of a practical and reliable method for the evaluation of litter moisture in turkey production.

1. An experiment was conducted to compare 5 different methods for the evaluation of litter moisture. 2. For litter collection and assessment, 55 farms were selected, one shed from each farm was inspected and 9 points were identified within each shed. 3. For each device, used for the evaluation of litter moisture, mean and standard deviation of wetness measures per collection point were assessed. 4. The reliability and overall consistency between the 5 instruments used to measure wetness were high (α = 0.72). 5. Measurement of three out of the 9 collection points were sufficient to provide a reliable assessment of litter moisture throughout the shed. 6. Based on the direct correlation between litter moisture and footpad lesions, litter moisture measurement can be used as a resource based on-farm animal welfare indicator. 7. Among the 5 methods analysed, visual scoring is the most simple and practical, and therefore the best candidate to be used on-farm for animal welfare assessment.

Hepatitis E Virus: A Cross-Sectional Serological and Virological Study in Pigs and Humans at Zoonotic Risk within a High-Density Pig Farming Area.

An increase in autochthonous hepatitis E virus (HEV) infections has been recorded in Italy suspected to be zoonotically transmitted from pigs; this study was carried out to determinate the seroprevalence and risk factors associated with hepatitis HEV exposition, both in swine and humans working in pig farms, located within a high-density pig farming area in Piedmont region, north-western Italy. The presence of viral RNA in human and swine samples was also evaluated, and phylogenetic analysis was performed on HEV-positive samples. Forty-two swine farms were sampled; 142 workers were enrolled in the study and classified into two groups: (i) 69 workers with occupational contact with swine (including veterinarians and farmers) recruited in the 42 sampled farms; (ii) 73 without occupational contact with swine. Forty-one of 42 (97%) swine farms resulted positive to enzyme-linked immunosorbent assay test for HEV antibodies (Abs). Overall seroprevalence in swine was 50% (441/879), with seropositivity rate higher in sows (333/469, 71%). HEV RNA in stool samples was detected in animals from 13 of 42 tested farms (31%), and a higher positivity resulted in weaners (40/246, 16.3%). Phylogenetic analysis classified all HEV isolates within genotype 3 (subtypes 3f, 3e, 3c). All humans were negative for HEV viral genome in blood. Five of 142 sera were positive for IgG anti-HEV with an overall prevalence of 3.52% with no statistically significant differences in prevalence rates between workers at zoonotic risk and the control group (5.7% versus 1.3%). In contrast, a significant difference (OR 10.1) was observed within the subgroup including subjects exposed for short periods (veterinarians) compared with those who worked for long periods (farmers) suggesting a correlation between the time of exposure and the likelihood of HEV infection. Reporting HEV infection is not mandatory in Italy, but a constant epidemiological surveillance should be ensured to clarify the epidemiology of this disease.

Mutations in NOTCH1 PEST domain orchestrate CCL19-driven homing of chronic lymphocytic leukemia cells by modulating the tumor suppressor gene DUSP22.

Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.

Factors Affecting Seroconversion Rates in Cattle Vaccinated with Two Commercial Inactivated BTV-8 Vaccines Under Field Conditions.

The immunogenicity of two inactivated bluetongue virus serotype 8 (BTV-8) vaccines was evaluated in 880 cattle under field conditions. The effect of selected factors on vaccine performance was also analysed at the herd and animal levels (vaccine, herd size and production, age, sex, time interval between vaccination and blood sampling and veterinary training). The immunogenicity elicited by vaccination with the two vaccines was monitored with the aid of a competitive enzyme-linked immunosorbent assay (c-ELISA) and serum neutralization test (SNT). To investigate whether the selected factors influenced seroconversion at the herd and animal levels, a multilevel logistic regression model developed in a mixed model was applied. Of the 880 cattle vaccinated, 76.0% yielded BTV c-ELISA antibodies, whereas only 25.0% seroconverted based on SNT. Type of vaccine (odds ratio [OR] 4.5; 95% confidence interval [CI], 2.2-9.0 for SNT and OR 3.5; 95% CI, 2.1-5.9 for c-ELISA), veterinary training in vaccine administration (OR 8.1; 95% CI, 4.7-14.1 for SNT and OR 2.4; 95% CI, 1.3-4.2 for c-ELISA), animal age (OR 1.4; 95% CI, 1.1-1.8 for SNT and OR 1.7; 95% CI, 1.4-2.1 for c-ELISA) and days between first vaccine administration and blood collection (OR 1.9; 95% CI, 1.1-3.1 for SNT and OR 2.6; 95% CI, 1.7-3.8 for c-ELISA) were the major factors affecting vaccine performance under field conditions. This is the first study to use multilevel logistic regression in the evaluation of selected risk factors affecting BTV-8 vaccine performance in cattle.

Mosquito surveillance in northwestern Italy to monitor the occurrence of tropical vector-borne diseases.

Mosquito-borne arboviruses (MBV) represent an important health problem, causing diseases and deaths both in human and animals mainly in tropical and subtropical countries. In recent years, they have emerged also in temperate regions where they have caused epidemics. Of mounting concern among public health authorities in Europe are zoonotic mosquito-borne viruses belonging to the Flavivirus genus. The aim of this study was to carry out active surveillance on mosquitoes in two regions of northwestern Italy (Liguria and Piedmont) to gain a better knowledge of the mosquito populations by identifying potential vectors of arboviruses and to investigate arbovirus infection. A network of 61 CO2 CDC traps was placed in the study area; sampling was conducted from May to October 2011. A total of 46,677 mosquitoes was collected, identified to species level, and classified according to their vector competence. Mosquitoes collected from 16 traps, selected according to risk-based factors, were tested by biomolecular analysis to detect flavivirus infection. This study highlights the importance of entomological surveillance in northwestern Italy because most of the mosquitoes collected were found to have high vector competence. Moreover, the risk-based virological surveillance allowed to detect the presence of mosquito flavivirus RNA, phylogenetically closely related to the MMV Spanish isolate, in three pools and USUV RNA in one pool in new areas where it has not been reported previously. The availability of continuous data on mosquito populations provides invaluable information for use in cases of an epidemic emergency. Maintenance of this integrated system for the next years will provide stronger data that can inform the design of a risk-based surveillance for the early detection of the occurrence of outbreaks of tropical MBDs.

ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein.

The ADP-ribosylation factors (ARFs) 1 and 6 are small GTP-binding proteins, highly expressed and activated in several breast cancer cell lines and are associated with enhanced migration and invasiveness. In this study, we report that ARF1 has a critical role in cell proliferation. Depletion of this GTPase or expression of a dominant negative form, which both resulted in diminished ARF1 activity, led to sustained cell-growth arrest. This cellular response was associated with the induction of senescent markers in highly invasive breast cancer cells as well as in control mammary epithelial cells by a mechanism regulating retinoblastoma protein (pRB) function. When examining the role of ARF1, we found that this GTPase was highly activated in normal proliferative conditions, and that a limited amount could be found in the nucleus, associated with the chromatin of MDA-MB-231 cells. However, when cells were arrested in the G(0)/G(1) phase or transfected with a dominant negative form of ARF1, the total level of activated ARF1 was markedly reduced and the GTPase significantly enriched in the chromatin. Using biochemical approaches, we demonstrated that the GDP-bound form of ARF1 directly interacted with pRB, but not other members of this family of proteins. In addition, depletion of ARF1 or expression of ARF1T(31)N resulted in the constitutive association of pRB and E2F1, thereby stabilizing the interaction of E2F1 as well as pRB at endogenous sites of target gene promoters, preventing expression of E2F target genes, such as cyclin D1, Mcm6 and E2F1, important for cell-cycle progression. These novel findings provide direct physiological and molecular evidence for the role of ARF1 in controlling cell proliferation, dependent on its ability to regulate pRB/E2F1 activity and gene expression for enhanced proliferation and breast cancer progression.

Acromegalia: comparación de los niveles séricos de IGF-I por dos inmunoensayos y su correlación con la prueba de tolerancia oral a la glucosa / Acromegaly: Comparison of serum IGF-1 levels measured by two immunoassays and their correlation with the Oral Glucose Tolerance Test

Introducción: La determinación de IGF-I en suero o plasma es una herramienta esencial en el diagnóstico y seguimiento de la acromegalia. Sin embargo, se deben tener presentes algunos inconvenientes en su medición por diferentes inmunoensayos. Objetivos: Evaluar dos inmunoensayos para la determinación de IGF-I y su correlación con el nadir de GH en el TTOG en pacientes acromegalicos. Materiales y métodos: Se analizaron 37 pacientes acromegálicos, 20 mujeres y 17 hombres. IGF-I fue determinada por Immulite 1000, (IMM) y por IRMA (DSL). Se realizó el TTOG y se determinó glucosa y GH en todos los tiempos (basal, 30, 60, 90 y 120min). Se consideró respuesta normal un nadir de GH <1ng/ml. Nueve pacientes se encontraban bajo tratamiento y 28 sin tratamiento. Análisis estadístico: se utilizaron el test de Wilcoxon, de Bland y Altman y curvas ROC. Se consideró significativa una p<0,05. Resultados: Las concentraciones basales de glucosa fueron 97,86±10,91 mg/dl, de GH 2,8 (1,59-14,4) ng/ml, de IGF-I por IMM 602±318 ng/ml y por DSL 1006±596 ng/ml. IGF-I por IMM y DSL mostró una diferencia significativa con p <0,01 y un bias de - 403,2 ng/ml con valores menores por IMM. IGF-I elevada por IMM y DSL, se encontró en el 84% y en el 97% respectivamente. IGF-I elevada con nadir de GH >1ng/ml se encontró en el 70%, con nadir de GH normal en el 13,5%. IGF-I normal con nadir >1ng/ml en el 2,7% y con nadir de GH normal en el 13,5%. El área bajo las curvas ROC no mostró diferencias significativas. Conclusiones: Los niveles de IGF-I determinados por IMM y DSL fueron significativamente diferentes mostrando un bias negativo para IMM. La mayoría de los valores del nadir de GH fueron consistentes con los niveles de IGF-I observándose una discrepancia en el 30% de los pacientes, estuvieran o no bajo tratamiento.

Introduction: IGF-I determination in serum or plasma is an essential tool in the diagnosis and follow-up of acromegaly. Hepatic production of IGF-I is regulated by GH and circulates bound to several IGF-I binding proteins which extends its half life. IGF-I is not released in a pulsatile pattern and has no significant variability in 24 h. Objective: To evaluate two different methodologies in IGF-I levels determination and their correlation with GH nadir in OGTT in acromegalic patients. Material and methods: We analyzed 37 acromegalic patients, 20 women and 17 men, mean age was 45±12 years. IGF-I levels were assayed by Immulite 1000, DPC (IMM) and DSL-5600 ACTIVE® IGF-I Coated-Tube IRMA (DSL) and OGTTs (at baseline and at 30, 60, 90 and 120 minutes) were performed by measuring plasma glucose and GH assay by immunochemiluminometric assay (Access); we considered a nadir <1ng/ml as normal response. Nine patients were under medical treatment (cabergoline: 4, octeotride: 4, and cabergoline plus octeotrite: 1) and 28 without treatment. Statistical analysis: Wilcoxon and, Bland and Altman tests and ROC curves. Differences were considered significant at p< 0.05. Results: Basal glucose levels were 97.86±10.91 mg/dl and mean GH was 2.8 (1.59-14.4) ng/ml. Mean IGF-I levels performed by IMM were 602±318 ng/ml and 1006±596 ng/ml by DSL. There was a statistically significant difference between both methodologies (p<0.01). Bland and Altman test showed a bias of - 403.2 ng/ml with lower values by IMM. We observed elevated IGF-I levels in 84% by IMM and in 97% by DSL, and only one patient had normal levels with both methodologies. Elevated IGF-I levels and GH nadir >1ng/ml were observed in 70% of the patients, increased IGF-I with normal GH nadir in 13.5%, normal IGF-I with GH nadir >1ng/ml in 2.7% and normal IGF-I with normal GH nadir in 13.5%. Patients under treatment: 3 showed normal GH nadir with elevated IGF-I levels, in 2 of them by both methodologies, and in the other one it was normal by IMM and elevated by DSL; the other 6 showed GH nadir > 1ng/ml, 5 of them presented elevated IGF-I by both methodologies and the other one showed discrepancy in IGF-I levels. The under ROC curve area and confidence interval (CI) of 95% for IGF-I IMM and DSL were 0.96 (0.90-1.00) and 0.91 (0.82-1.00) respectively. Differences between the ROC curves areas were not significant Conclusions: IGF-I levels determined by IMM and DSL were statistically significantly different. IGF-I levels showed a negative bias by IMM. Most of the results of GH nadir were consistent with IGF-I levels but we observed discrepancy in 30% of the patients, regardless of whether they were under treatment or not.

Lipid dynamics in exocytosis.

Regulated exocytosis of neurotransmitter- and hormone-containing vesicles underpins neuronal and hormonal communication and relies on a well-orchestrated series of molecular interactions. This in part involves the upstream formation of a complex of SNAREs and associated proteins leading to the eventual fusion of the vesicle membrane with the plasma membrane, a process that enables content release. Although the role of lipids in exocytosis is intuitive, it has long been overlooked at least compared to the extensive work on SNAREs. Here, we will present the latest advances in this rapidly developing field revealing that lipids actually play an active role in exocytosis by focusing on cholesterol, 3'-phosphorylated phosphoinositides and phosphatidic acid.

The Coffin-Lowry syndrome-associated protein RSK2 and neurosecretion.

Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked mental retardation, characterized in male patients by psychomotor and growth retardation and various skeletal anomalies. CLS is caused by mutations in the RPS6KA3 gene, which encodes RSK2, a growth factor-regulated protein kinase. Cognitive deficiencies in CLS patients are prominent, but markedly variable in severity, even between siblings. However, the vast majority of patients are severely affected, with mental retardation ranging from moderate to profound. We used a RSK2-KO mouse model that shows no obvious brain abnormalities at the anatomical and histological levels to study the function of RSK2 in neurosecretion. Behavioral studies revealed normal motor coordination, but a profound retardation in spatial learning and a deficit in long-term spatial memory, providing evidence that RSK2 plays similar roles in mental functioning both in mice and human. We found that associative LTP at cortical inputs to the lateral amygdala was blocked in Rsk2 KO mice. Using an RNA interference rescue strategy in PC12 cells, we were able to demonstrate that RSK2 regulates catecholamine release through the phosphorylation of PLD. These results provide the first molecular evidence that RSK2 could regulate neurotransmitter release by activating PLD production of lipids required for exocytosis.

The Rho guanine nucleotide exchange factors Intersectin 1L and ß-Pix control calcium-regulated exocytosis in neuroendocrine PC12 cells.

GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and ß-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.

Estudio retrospectivo sobre las posibles complicaciones maternofetales de la exposición a cabergolina / Retrospective Study of any potencial Adverse Effects of Cabergoline on Pregnancy and Embryofetal Development

Objetivos: Estimar la frecuencia de complicaciones maternofetales en mujeres que se embarazaron durante el tratamiento con cabergolina (CAB). Estimar la frecuencia de patología detectada posnacimiento en los niños producto de dichos embarazos. Material y métodos: Estudio retrospectivo y multicéntrico de 86 embarazos en 78 mujeres con hiperprolactinemia idiopática (7) o tumoral (44 micro y 27 macro), en tratamiento con CAB en el momento de la concepción. Edad: 20 a 45 años; PRL inicial: 30 a 1429 ng/ml; duración del tratamiento previo al embarazo 1 a 120 meses; dosis: 0.125 a 4 mg/semana. El rango de exposición embriofetal a la CAB fue de 3 a 27 semanas, el 96.39% de las pacientes la recibió durante el primer trimestre y el 3.61% hasta el segundo. Resultados: No hubo complicaciones mayores durante el embarazo. Se registraron 7 abortos espontáneos (8.1%) y 75 partos, de los cuales 49 fueron vaginales y 26 cesáreas. Se registraron 69 recién nacidos, 63 fueron a término y 6 pretérmino (8.8%), ninguno bajo peso para la edad gestacional. En 3 (5.2%) recién nacidos se observó: 1 malformación mayor (Síndrome de Down) y 2 menores (hernia umbilical e inguinal). Se obtuvo seguimiento de 42 recién nacidos; se diagnosticó epilepsia refractaria en uno y un trastorno generalizado del desarrollo en otro. No se halló una mayor frecuencia de complicaciones en los embarazos ni en los recién nacidos expuestos a CAB que en la población normal. Sería necesario mayor número de pacientes para concluir sobre la seguridad de CAB durante el embarazo.

Objectives: To assess the rate of any potential adverse effects on pregnancy and embryo-fetal development in women who became pregnant under treatment with cabergoline (CAB). To follow up medical data of children who were born from mothers exposed to Cab in early weeks of gestation. Material and methods: Observational, retrospective and multicenter study on 86 pregnancies in 78 women with idiopathic or tumoral hyperprolactinemia. All patients were under Cab at conception. The average age was 29 (range: 20-45). Pituitary images at diagnosis showed 44 microadenomas, 27 macroadenomas and 7 were normal. Serum PRL at baseline was between 30 and 1429 ng/ml. Duration of therapy before pregnancy ranged from 1 to 120 months. Maternal and fetal exposure to cabergoline and doses ranged from 0.125 to 4 mg/week. The mean serum PRL level under which patients achieved pregnancy was 17 ng/ml. Fetal exposure ranged from 3 to 27 weeks; 96.39% of patients received CAB during the first trimester of pregnancy and 3.61% until the second one. Results: No significant complications during pregnancy were found. Seven women (8.1%) had spontaneous abortions. Term deliveries were recorded in 63/69, preterm in six (8.8%), none of them with low weight for gestational age. Neonatal abnormalities were observed in 3 (5.2%): 1 major (Down syndrome) and 2 minor malformations (umbilical and inguinal hernia). Two out of 42, developed abnormalities during the follow- up, one of them was a refractory epilepsy during the second month of life, the other presented a Pervasive Developmental Disorder diagnosed in the third year of life. Conclusion: No significantly higher frequency of complications was found in pregnancies and/or offspring exposed to CAB than in normal population. Larger series of patients are needed to asses the safety.

Frequent sampling allows detection of short and rapid surges of exhaled ethane during cardiac surgery.

During cardiopulmonary bypass (CPB), hypoperfusion and reperfusion may cause oxidative stress and lipid peroxidation that generates ethane. The aim of this pilot study was to assess the feasibility of frequent sampling of exhaled ethane during cardiac surgery. After approval of the Research Ethics Committee, 10 patients undergoing combined aortic valve and coronary artery bypass surgery were enrolled. Breath samples were drawn in the perioperative period and analyzed by a rapid, sensitive and validated gas-chromatographic method. Increased exhaled ethane was regularly seen following sternotomy, after the start of CPB and after aortic clamp removal, whereas no change was seen after termination of bypass. In one patient, the maximum increase in exhaled ethane was 30-fold. Peak durations lasted only 2-4 min. This study demonstrates that frequent sampling of breath ethane is feasible in a clinical setting, allowing detection of rapid ethane surges of short duration.

The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders.

We have applied a new method of genetic analysis, called 'minisequencing', to preimplantation genetic diagnosis (PGD) of monogenic disorders from single cells. This method involves computer-assisted mutation analysis, which allows exact base identity determination and computer-assisted visualization of the specific mutation(s), and thus facilitates data interpretation and management. Sequencing of the entire PCR product is unnecessary, yet the same qualitative characteristics of sequence analysis are maintained. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. To evaluate the reliability of this method for subsequent application to PGD, we analysed PCR products from 887 blastomeres including 55 PGD cases of different genetic diseases, such as cystic fibrosis, beta-thalassaemia, sickle cell anaemia, haemophilia A, retinoblastoma, and spinal muscular atrophy. Minisequencing was found to be a useful technique in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation. The method was also efficient, providing interpretable results in 96.5% (856/887) of the blastomeres tested. Fifteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 10 healthy babies have already been born. Its applicability to PGD could be helpful, particularly in cases in which the mutation(s) involved are difficult to assess by restriction analysis or other commonly used methods.

A role for phospholipase D1 in neurotransmitter release.

Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca(2+)-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.

Clinical evaluation of St Jude Medical Hemodynamic Plus versus standard aortic valve prostheses: The Italian multicenter, prospective, randomized study.

OBJECTIVE: Hemodynamic and clinical performances of 21-mm and 23-mm St Jude Medical Hemodynamic Plus aortic valves (St Jude Medical, Inc, St Paul, Minn) were compared with those of 21-mm and 23-mm St Jude Medical standard cuff aortic valves in the first such multicenter, prospective, randomized study. Hemodynamic Plus valves are mechanical, bileaflet prostheses suitable for the small aortic anulus. METHODS: Patients with 21-mm and 23-mm anulus diameters were randomized to receive either a Hemodynamic Plus or a standard cuff valve. Postoperatively and at 6 months after the operation, patients underwent 2-dimensional Doppler echocardiography. Ejection fraction, cardiac output, peak gradient, mean gradient, effective orifice area, effective area index, and performance index were calculated. Postoperative and 6-month echocardiographic measurements and their variations across observation times were analyzed statistically. RESULTS: Of the 140 patients enrolled, 5 died at operation and 1 died of aortic dissection during the follow-up period. Eight patients were lost to follow-up. A total of 125 patients completed the study. In 1 patient a sewing cuff escaped intraoperatively. At 6 months the 21-mm and 23-mm Hemodynamic Plus valves showed significantly lower peak gradients and mean gradients than those of the 21-mm and 23-mm standard cuff valves. The 21-mm Hemodynamic Plus valves had gradients similar to those of the 23-mm Hemodynamic Plus valves. The effective orifice area did not differ significantly between the Hemodynamic Plus and standard cuff valves at either measurement. No valve mismatch was found in the 4 groups of patients. A more enhanced decrease of peak gradients and mean gradients and a more enhanced increase of effective orifice areas, effective area indices, and performance indices were found across observation times for patients with Hemodynamic Plus valves compared with those with standard cuff valves. CONCLUSIONS: Clinical hemodynamic performances of 21-mm and 23-mm St Jude Medical Hemodynamic Plus valves correspond closely with those of standard cuff valves, and gradients are substantially better than those of standard cuff valves of the same diameter. Therefore, use of this valve may minimize the need for aortic anulus enlargement. Early follow-up results with the Hemodynamic Plus valves were excellent, although more time is required to confirm this outcome.

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis.

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.