Analysis of changes in cardiac circadian rhythms of RR and QT induced by a 60-day head-down bed rest with and without nutritional countermeasure.

PURPOSE: Prolonged weightlessness exposure generates cardiovascular deconditioning, with potential implications on ECG circadian rhythms. Head-down (- 6°) tilt (HDT) bed rest is a ground-based analogue model for simulating the effects of reduced motor activity and fluids redistribution occurring during spaceflight. Our aim was to evaluate the impact of 60-day HDT on the circadianity of RR and ventricular repolarization (QTend) intervals extracted from 24-h Holter ECG recordings, scheduled 9 days before HDT (BDC-9), the 5th (HDT5), 21st (HDT21) and 58th (HDT58) day of HDT, the 1st (R + 0) and 8th (R + 7) day after HDT. Also, the effectiveness of a nutritional countermeasure (CM) in mitigating the HDT-related changes was tested. METHODS: RR and QTend circadian rhythms were evaluated by Cosinor analysis, resulting in maximum and minimum values, MESOR (a rhythm-adjusted mean), oscillation amplitude (OA, half variation within a night-day cycle), and acrophase (φ, the time at which the fitting sinusoid's amplitude is maximal) values. RESULTS: RR and QTend MESOR increased at HDT5, and the OA was reduced along the HDT period, mainly due to the increase of the minima. At R + 0, QTend OA increased, particularly in the control group. The φ slightly anticipated during HDT and was delayed at R + 0. CONCLUSION: 60-Day HDT affects the characteristics of cardiac circadian rhythm by altering the physiological daily cycle of RR and QTend intervals. Scheduled day-night cycle and feeding time were maintained during the experiment, thus inferring the role of changes in the gravitational stimulus to determine these variations. The applied nutritional countermeasure did not show effectiveness in preventing such changes.

Guidelines for improvement of the procedural aspects of devices and surgical instruments in the operating theatre.

Surgical site infections are a major complication for patients undergoing surgical treatment and a significant cause of mortality and morbidity. Many international guidelines suggest measures for the prevention of surgical site infections (SSI) in perioperative processes and the decontamination of surgical devices and instruments. This document proposes guidelines for improving the perioperative setting in view of the devices and instrumentation required for surgical procedures, aiming to reduce contamination rates and improve clinical performance and management for patients undergoing surgical treatment. This document is intended for doctors, nurses and other practitioners involved in operating theatre procedures, resource management and clinical risk assessment processes, and the procurement, organisation, sterilisation and reprocessing of surgical instruments.

Optimisation of perioperative procedural factors to reduce the risk of surgical site infection in patients undergoing surgery: a systematic review.

Surgical site infections (SSI) are the leading cause of hospital readmission after surgical procedures with significant impact on post-operative morbidity and mortality. Modifiable risk factors for SSI include procedural aspects, which include the possibility of instrument contamination, the duration of the operation, the number of people present and the traffic in the room and the ventilation system of the operating theatre.The aim of this systematic review was to provide literature evidence on the relationship between features of surgical procedure sets and the frequency of SSI in patients undergoing surgical treatment, and to analyse how time frames of perioperative processes and operating theatre traffic vary in relation to the features of the procedure sets use, in order tooptimise infection control in OT. The results of the systematic review brought to light observational studies that can be divided into two categories: evidence of purely clinical significance and evidence of mainly organisational, managerial and financial significance. These two systems are largely interconnected, and reciprocally influence each other. The decision to use disposable devices and instruments has been accompanied by a lower incidence in surgical site infections and surgical revisions for remediation. A concomitant reduction in post-operative functional recovery time has also been observed. Also, the rationalisation of traditional surgical sets has also been observed in conjunction with outcomes of clinical significance.

An optimized histological proceeding to study the female gametophyte development in grapevine.

BACKGROUND: Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte's developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species. RESULTS: Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 µm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined. CONCLUSIONS: The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional.

Dose-response study of recombinant factor VIIa/tissue factor inhibitor recombinant nematode anticoagulant protein c2 in prevention of postoperative venous thromboembolism in patients undergoing total knee replacement.

BACKGROUND: With the best prophylactics now available, venous thromboembolism after total knee replacement remains substantial (25% to 27%). Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent inhibitor of factor VIIa/tissue factor complex that has the potential to reduce this risk. The present study was performed to determine an efficacious and safe dose of rNAPc2 for prevention of venous thromboembolism after elective, unilateral total knee replacement. METHODS AND RESULTS: This open-label, sequential dose-ranging study was conducted in 11 centers in Canada, Europe, and the United States. Five regimens were tested. Injections were administered subcutaneously on the day of surgery (day 1) and days 3, 5, and optionally, day 7. Primary efficacy outcome was a composite of overall deep vein thrombosis based on mandatory unilateral venography (day 7+/-2) and confirmed symptomatic venous thromboembolism recorded

Does the type of hormone replacement therapy influence the risk of deep vein thrombosis? A prospective case-control study.

BACKGROUND: Although hormone replacement therapy (HRT) is associated with an increased risk of deep vein thrombosis (DVT), it is not clear if the risk differs in users of combined estrogen-progestin HRT and estrogen-only HRT. METHODS: We prospectively studied postmenopausal women with suspected DVT in whom HRT use status was ascertained and who subsequently had objective diagnostic testing to confirm or exclude DVT. Cases were patients with idiopathic DVT, in whom there were no DVT risk factors, and controls were patients without DVT, in whom there were also no DVT risk factors. The risk of DVT was determined in users of estrogen-progestin HRT and estrogen-only HRT by comparing the prevalence of current HRT use in cases with idiopathic DVT and controls without DVT (reference group). Multivariable regression analysis was done to adjust for factors that might confound an association between HRT use and the risk of DVT. RESULTS: One thousand one hundred and sixty-eight postmenopausal women with suspected DVT were assessed, from whom 95 cases of idiopathic DVT and 610 controls without DVT and no DVT risk factors were identified. Estrogen-only HRT was associated with an increased risk for DVT that was not statistically significant [odds ratio (OR) = 1.22; 95% confidence interval (CI) 0.57, 2.61]. Estrogen-progestin HRT was associated with a greater than 2-fold increased risk for DVT (OR = 2.70; 95% CI 1.44, 5.07). CONCLUSION: The risk of developing DVT may be higher in users of combined estrogen-progestin HRT than in users of estrogen-only HRT.

Gene transfer to the nigrostriatal system by hybrid herpes simplex virus/adeno-associated virus amplicon vectors.

To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.

Neuroimmunophilin ligand enhances neurite outgrowth and effect of fetal dopamine transplants.

Neuroimmunophilin ligands have been shown to enhance neurite outgrowth in several neuronal systems in culture, including primary dopaminergic neurons from fetal ventral mesencephalon. We investigated the ability of neuroimmunophilin ligands to enhance outgrowth of transplanted fetal dopamine neurons in vivo. Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal dopamine system were transplanted with rat embryonic day 14 ventral mesencephalon into the striatum, then treated orally with a neuroimmunophilin ligand (15mg/kg) or vehicle once per day for 14 days. All transplanted animals regained dopamine function over a 10 week behavioral test period, as indicated by decrease and reversal of amphetamine-induced rotation. In addition, neuroimmunophilin ligand-treated animals showed a more pronounced motor response during the first 10min after amphetamine injection, possibly reflecting increased striatal reinnervation or increased functional capacity. At post-mortem analyses, neuroimmunophilin ligand-treated rats showed a significantly higher density of tyrosine hydroxylase-positive fibers reinnervating the lesioned striatum, both immediately surrounding the transplant (92% of unlesioned density in neuroimmunophilin-treated rats vs 67% of unlesioned levels in vehicle-treated rats) and at some distance from the transplant/host interface. The number of tyrosine hydroxylase-positive cells within the transplants was not different between groups. This study demonstrates that short-term oral administration of a neuroimmunophilin ligand can enhance neurite outgrowth from fetal dopamine neuronal transplants.

Development of striatal patch/matrix organization in organotypic co-cultures of perinatal striatum, cortex and substantia nigra.

Organotypic cultures of fetal or early postnatal striatum were used to assess striatal patch formation and maintenance in the presence or absence of dopaminergic and glutamatergic influences. Vibratome-cut slices of the striatum prepared from embryonic day 19 to postnatal day 4 rat pups were maintained in static culture on clear membrane inserts in Dulbecco's modified Eagle's medium/F12 (1:1) with 20% horse serum. Some were co-cultured with embryonic day 12-16 ventral mesencephalon and/or embryonic day 19 to postnatal day 4 cortex, which produced a dense dopaminergic innervation and a modest cortical innervation. Donors of striatal and cortical tissue were previously injected with bromo-deoxyuridine (BrdU) on embryonic days 13 and 14 in order to label striatal neurons destined to populate the patch compartment of the striatum. Patches of BrdU-immunoreactive cells were maintained in organotypic cultures of late prenatal (embryonic days 20-22) or early postnatal striatum in the absence of nigral dopaminergic or cortical glutamatergic influences. In slices taken from embryonic day 19 fetuses prior to the time of in vivo patch formation, patches were observed to form after 10 days in vitro, in 39% of nigral-striatal co-cultures compared to 6% of striatal slices cultured alone or in the presence of cortex only. Patches of dopaminergic fibers, revealed by tyrosine hydroxylase immunoreactivity, were observed in the majority of nigral-striatal co-cultures. Immunostaining for the AMPA-type glutamate receptor GluR1 revealed a dense patch distribution in nearly all cultures, which developed in embryonic day 19 cultures after at least six days in vitro. These findings indicate that striatal patch/matrix organization is maintained in organotypic culture, and can be induced to form in vitro in striatal slices removed from fetuses prior to the time of in vivo patch formation. Furthermore, dopaminergic innervation from co-cultured pieces of ventral mesencephalon enhances patch formation in organotypic cultures.

Compartmental expression of trkB receptor protein in the developing striatum.

To investigate the role of neurotrophins in the initial formation of striatal patch versus matrix, the spatial and temporal expression of trkB receptors was examined using immunohistochemistry. Polyclonal antibodies, against the C-terminus or the tyrosine kinase domain, revealed trkB-immunoreactive cells and fibers localized to patches beginning on embryonic day 19 in the rat, which co-localized with patchy dopamine fibers, substance P-immunoreactive neurons and glutamate receptors. Patchy striatal trkB expression was maintained after lesioning the nigrostriatal dopamine system. The patchy trkB distribution persisted through postnatal day 14, then became more homogeneous at the same time that nigrostriatal afferents become homogeneous. Later in development, trkB immunoreactivity was most intense in a subpopulation of large striatal cells that were similar in size and frequency to those immunoreactive for choline acetyltransferase. The spatiotemporal expression of trkB receptor in phenotypically distinct striatal patches, as well as evidence that neurotrophins regulate expression of neuronal phenotypic markers during development, may indicate a convergence of neurotrophins and afferent innervation on to future patch cells that may regulate the establishment of striatal compartmentalization.

Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum.

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.

Medial fetal ventral mesencephalon: a preferred source for dopamine neuron grafts.

Currently, fetal tissue transplantation into patients with Parkinson's disease utilizes the entire ventral mesencephalon (VM) as donor tissue. However, the resulting mixture of cell types contains a relatively low proportion of therapeutically relevant dopamine (DA) neurons. We show that differential dissection of a medial region of embryonic day 14 rat VM yields a significantly higher proportion of DA neurons (8-10%) than is found in lateral VM (2%) or whole VM (4-5%). Medial VM also contained a larger number of the specific subpopulation of DA neurons (aldehyde dehydrogenase-positive; AHD) that project to dorsolateral motor region of the striatum. Selective dissection of fetal medial VM selectively enriches DA neurons in cell preparations useful for transplantation in Parkinson's disease.

Glutamate receptor subtypes localize to patches in the developing striatum.

The distribution of glutamate receptors in the developing striatum of the rat was studied using antibodies specific to AMPA and NMDA subtypes. Immunocytochemistry revealed a greater density of GluR1, GluR2/3, NMDAR1, and NMDAR2A/2B receptors in patches that matched the patches of substance P-immunoreactive neurons and dopaminergic terminals. GluR1-immunoreactive patches were the most distinctive and were present already at embryonic day 19.

[Hepatitis C virus and mixed cryoglobulinemia in dialysis]. / Virus dell'epatite C e crioglobulinemia mista in dialisi.

Prevalence of hepatitis C virus (HCV) varies in the dialytic population according to the geographic area (5-85%); the European average is 19%, with marked differences between dialysis units. The means of virus transmission is still not clear and represents one of the major management problems faced by dialysis units. US authors and European study groups have recently demonstrated a high level od HCV infection in patients with essential mixed cryoglobulinemia (EMC), suggesting that the virus plays an etiological role. The authors examined the correlation between mixed cryoglobulinemia and HCV and the possible etiological role of the virus in the pool of patients undergoing dialysis at the Centre. In September 1994 28/63 (44.4%) patients were found to be Ab anti-HCV positive (second and third generation ELISA); circulating cryoglobulins identified as Type II following immunofixation were found in 2/28 patients (7%) using centrifugation and cold incubation techniques. In the absence of the pathologies classically associated with cryoglobulinemia, the patients were diagnoses as suffering EMC. The HCV/EMC correlation in the dialysed population has been analysed in a limited number of studies, with discrepant results (0-47%). This paper highlights the association between HCV and EMC in 7% of chronic dialysed patients and given that the finding of anti-HCV Ab in serum solely documents previous exposure to the virus, we can only indirectly attribute an etiological role to HCV. The search for viral RNA in the serum, cryocrit and surnatant of patients found affected by EMC may provide additional etiopathogenetic information.

[Levels of tumor necrosis factor and interleukin 1 in uremia and in hemodialysis]. / Livelli di tumor necrosis factor ed interleuchina-1 nell'uremia ed in emodialisi.

The present study aimed to assess the serum levels of tumor necrosis factor (TNFalpha) and Interleukin-1 (IL-1) in uremic patients and those patients undergoing extracorporeal replacement therapy in relation to the duration of dialysis and the type of membrane. Serum cytokine values were assayed using an immunoradiometric technique (monoclonal antibodies) in 28 uremic patients and 7 healthy controls. TNFalpha levels were normal in control subjects but increased in uremic patients. Patients undergoing hemodialytic treatment showed increases which were statistically significant in comparison to basal levels, with a trend which was directly correlated to the duration of dialysis. No significant variations were induced by the different types of dialysis membranes used. In virtually all patients IL-1 values could not be assayed using this method.

[Infections from extemporaneous catheters for hemodialysis. Experience in a center]. / Infezione dei cateteri estemporanei per emodialisi. Esperienza di un centro.

BACKGROUND AND AIMS: This study reports a retrospective evaluation of the predominance of infection in 67 dual lumen central venous catheters (CVC), 35 of which were positioned in the femoral vein by the nephrological team and 32 in the subclavian vein by anesthetists. METHODS: The microorganisms responsible for infection, the prevalence of clinically symptomatic infections, the relationship between CVC-correlated infection and the time the catheter remained inserted were evaluated, together with a comparison between the two different insertion sites. RESULTS: Culture tests, performed using Maki's semiquantitative technique, gave positive results in 16/67 (23.8%) cases. The main pathogenic agents found were Staphylococcus epidermidis (37.5%) and Staphylococcus aureus (31.2%). In 3/16 cases (18.78%) infections were clinically symptomatic. The mean permanence of CVC with positive cultures was not statistically different to the mean permanence of CVC with negative cultures (22.44 +/- 13.48 vs 18.38 +/- 17.76). The microorganisms isolated on femoral and subclavian catheters showed a comparable distribution and the prevalence of infection was not statistically different in the two insertion sites. CONCLUSIONS: In conclusion, in the absence of infection, the authors tend to keep working catheters in the site, thus avoiding repeated invasive manoeuvres for replacement and/or repositioning, whereas in the presence of suspected systemic infection they feel it is more prudent to remove the CVC without waiting for the results of the hemoculture, starting first empiric and then specific antibiotic treatment on the basis of the antibiogram.

[Effectiveness of oral supplements of folates, vitamins B12 and B6 in hemodialysed patients. Preliminary results after 5 months of follow-up]. / Efficacia dei supplementi orali di folti, vitamine B12 e B6 in pazienti emodializzati. Risultati preliminari dopo 5 mesi di follow-up.

BACKGROUND: Elevated serum levels of homocysteine have increasingly been associated as a risk factor of cardiovascular disease. Recent reports demonstrated that supplements of folates, vitamin B12 (B12) and vitamin B6 (B6) are effective in correcting serum Hcy levels in hemodialysed patients. AIM: to assess the effectiveness of oral supplements of folates, B12 and B6, in order to reduce serum Hcy levels in our cohort of hemodialysed patients. METHODS: Sixty-one hemodialysed patients have been enrolled in the study (age 68+/-13 years; hemodialysis 62+/-42 months). Oral supplements of calcium folinate (30 mg 3 times a week), B12 (500 mg 3 times a week) and B6 (200 mg 3 times a week) were administered at the end of each hemodialysis session. Serum levels of Hcy, folic acid and B12 were tested at the beginning of the study and at 2 month intervals. RESULTS: After 5 months of follow-up, serum levels of Hcy were normalised in 19% of our patients and in total 70% of them showed a reduction >8% when compared with the basal Hcy levels. No side effects related to folates, B12 or B6 supplementation were observed. CONCLUSIONS: Oral supplements of folates, B12 and B6 are a safe and effective treatment of hyperhomocysteinemia in hemodialysed patients.

Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymorphisms.

The domestication of the Eurasian grape (Vitis vinifera ssp. sativa) from its wild ancestor (Vitis vinifera ssp. sylvestris) has long been claimed to have occurred in Transcaucasia where its greatest genetic diversity is found and where very early archaeological evidence, including grape pips and artefacts of a 'wine culture', have been excavated. Whether from Transcaucasia or the nearby Taurus or Zagros Mountains, it is hypothesized that this wine culture spread southwards and eventually westwards around the Mediterranean basin, together with the transplantation of cultivated grape cuttings. However, the existence of morphological differentiation between cultivars from eastern and western ends of the modern distribution of the Eurasian grape suggests the existence of different genetic contribution from local sylvestris populations or multilocal selection and domestication of sylvestris genotypes. To tackle this issue, we analysed chlorotype variation and distribution in 1201 samples of sylvestris and sativa genotypes from the whole area of the species' distribution and studied their genetic relationships. The results suggest the existence of at least two important origins for the cultivated germplasm, one in the Near East and another in the western Mediterranean region, the latter of which gave rise to many of the current Western European cultivars. Indeed, over 70% of the Iberian Peninsula cultivars display chlorotypes that are only compatible with their having derived from western sylvestris populations.

QTL analysis for fruit yield components in table grapes (Vitis vinifera).

A segregation population of 184 genotypes derived from a pseudo-testcross of table grapes (Vitis vinifera), together with 203 AFLP and 110 SSR markers was used to detect quantitative trait loci (QTLs) for fruit yield components. Diffferent QTLs, a low percentage of phenotypic variance explained by the QTLs detected and QTL instability over years were detected for each fruit yield component. These results confirm the complex genetic architecture of the yield components in grapevine due to the perennial nature of this species, which has to adapt to yearly variations in climate. Phenotypic correlation analyses between fruit yield components were also performed. The negative correlation between berry weight and the number of berries per cluster seems to have an indirect negative effect on cluster weight, as revealed by the path coefficient analysis; however, this negative correlation was not supported at the molecular level because no coincident QTLs were observed between these traits. Nonetheless, the possibility to select seedless genotypes with large berries without affecting cluster weight needs to be substantiated in future experiments because factors such as sample size and heritability might influence QTL identification in table grapes.