Skin donations after suicide by acute poisoning.

The demand for skin tissue allografts to treat burns and other types of injuries increases each year to the extent that categories of donors formerly deemed "unsuitable", such as victims of suicide by polytrauma or poisoning, are now considered. Patients who died by ingestion of/exposure to toxic substances can be accepted as tissue donors after assessment of graft safety to rule out any risks of transferring toxic substances to the recipient. A cadaveric skin donation was obtained from a 57-year-old woman who died from intoxication after ingesting colchicine tablets (0.2 mg/kg). To determine the safety of cadaveric skin allografts, high-performance liquid chromatography-mass spectrometry (LC-MS/MS) was used to identify and quantify colchicine in procured skin. Results revealed that colchicine concentrations were lower than the instrument limit of detection (LOD) of 0.5 ng/mg both in epidermis and dermis. Cell viability assessed through the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) test was within standard limits. Thanks to accurate tests performed, which are routinely applied also in clinical diagnostics and forensic toxicology, it was possible to ascertain the safety and suitability of skin tissue for donation.

Cerebral microemboli detected by transcranial doppler in patients treated with extracorporeal membrane oxygenation.

INTRODUCTION: Cerebrovascular complications rate in patients treated with extracorporeal membrane oxygenation (ECMO) is about 7%. Ischemic stroke may be caused by solid or gaseous microemboli due to thrombosis within the circuit or cannula. Transcranial Doppler (TCD) is the only method able to detect microembolic signals (MES) in real time. The objective of this study was to detect possible MES by TCD in patients treated with veno-venous (VV) and veno-arterial (VA) ECMO and to test for a relation between the number of MES and the 6-month clinical outcome of these patients. METHODS: This is a monocentric observational prospective study in patients consecutively admitted and treated with ECMO at our regional ECMO referral center in 18 months. TCD detection of MES was performed in patients upon initiation of treatment and then repeated during treatment. RESULTS: Two hundred and forty-eight TCD monitoring were performed in 42 VV and 11 VA ECMO patients. MES were detected in 26.2% of VV ECMO patients and in 81.8% of VA ECMO patients (P < 0.001). In both subgroups of patients, no correlation was found between MES detection and extracorporeal flow velocities or aPTT values. In VA ECMO patients, an inverse correlation between left ventricular ejection fraction and MES grading was found (P = 0.037). In both groups, no clinical neurological impairments correlated to MES detection were found at 6 months follow-up. CONCLUSIONS: MES were found in both ECMO configurations; independently from their pathophysiology, MES do not seem to influence clinical outcome. Multicenter studies are still required with more extensive cases to confirm these results.

REFINE: a randomized trial comparing cyclosporine A and tacrolimus on fibrosis after liver transplantation for hepatitis C.

REFINE was a 12-month, prospective, open-label study in 356 patients receiving de novo liver transplantation for hepatitis C virus (HCV) cirrhosis, randomized to cyclosporine A (CsA) or tacrolimus with (i) no steroids, IL-2 receptor antibody induction and mycophenolic acid, or (ii) slow steroid tapering. The primary analysis population based on availability of liver biopsies comprised 165 patients (88 CsA, 77 tacrolimus). There was no difference in the primary endpoint, fibrosis stage ≥2 at 12 months, which occurred in 63/88 CsA-treated patients (71.6%) and 52/77 tacrolimus-treated patients (67.5%) (odds ratio [OR] 1.11; 95% CI 0.56, 2.21; p = 0.759). Similarly, no significant between-group difference occurred at month 24 (OR 1.15; 95% CI 0.47, 2.80; p = 0.767). Among steroid-free patients, fibrosis score ≥2 was significantly less frequent with CsA versus tacrolimus at month 12 (7/37 [18.9%] vs. 16/38 [42.1%]; p = 0.029). HCV viral load was similar in both the tacrolimus- and CsA-treated cohorts. Mean blood glucose was significantly higher with tacrolimus from day 15 onward. Biopsy-proven acute rejection, graft loss and death were similar. These results showed no differences in posttransplant HCV-induced liver fibrosis between patients treated with CsA or tacrolimus in steroid-containing regimens, whereas CsA in steroid-free protocols was associated with reduced severity of fibrosis progression at 1 year posttransplant.

Genetic structure in the Mediterranean seagrass Posidonia oceanica: disentangling past vicariance events from contemporary patterns of gene flow.

The Mediterranean Sea is a two-basin system, with the boundary zone restricted to the Strait of Sicily and the narrow Strait of Messina. Two main population groups are recognized in the Mediterranean endemic seagrass Posidonia oceanica, corresponding to the Western and the Eastern basins. To address the nature of the East-West cleavage in P. oceanica, the main aims of this study were: (i) to define the genetic structure within the potential contact zone (i.e. the Strait of Sicily) and clarify the extent of gene flow between the two population groups, and (ii) to investigate the role of present water circulation patterns vs. past evolutionary events on the observed genetic pattern. To achieve these goals, we utilized SSR markers and we simulated, with respect to current regime, the possible present-day dispersal pattern of Posidonia floating fruits using 28-day numerical Lagrangian trajectories. The results obtained confirm the presence of the two main population groups, without any indices of reproductive isolation, with the break zone located at the level of the Southern tip of Calabria. The populations in the Strait of Sicily showed higher affinity with Western than with Eastern populations. This pattern of genetic structure probably reflects historical avenues of recolonization from relict glacial areas and past vicariance events, but seems to persist as a result of the low connectivity among populations via marine currents, as suggested by our dispersal simulation analysis.

Characterization of Follicular Atresia Responsive to BPA in Zebrafish by Morphometric Analysis of Follicular Stage Progression.

Bisphenol A is an industrial chemical compound, pervasively polluting the environment and diet, classified as an endocrine disruptor because of its interference effects on the endocrine system. In zebrafish, BPA exposure induces follicular atresia. To acquire knowledge on this atretic effect, using a qualitative and quantitative histomorphological approach, we studied zebrafish ovarian follicular stage development in response to low BPA concentrations. Results show that BPA interferes with follicular progression by affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) increases follicular recruitment by acting on primary stage follicles, (ii) forces the follicular transition from stage III to stage IV producing enlarged stage IV follicles, and (iii) induces atresia by producing atretic follicles that are peculiarly enlarged (i.e., big atretic follicles). We suggest that BPA induces atresia by the primary effect on recruitment of stage I follicles. This forces follicular progression and produces stage IV follicles that are peculiarly enlarged that undertake the atretic development.

The tumor suppressor p16Ink4a regulates T lymphocyte survival.

In contrast to other cell cycle inhibitors, the tumor suppressor p16Ink4a is not detectable or expressed at very low levels in embryonic and adult mouse tissues, and therefore it has often been considered as a specialized checkpoint protein that does not participate in the control of normal cell cycle progression. However, Ink4a-/- mice possess increased thymus size and cellularity, thus suggesting the involvement of p16(Ink4a) in the control of thymocyte proliferation. In this study, we found increased numbers of CD8 and CD4 T lymphocytes in thymus and spleen from Ink4a-/- mice. Unexpectedly, this was not related to an increase in T-cell division rates, which were similar in lymphoid organs of Ink4a-/- and wild-type mice. In contrast, T-cell apoptosis rates were significantly decreased in thymus and spleen from Ink4a-/- mice. Moreover, whereas p16Ink4a-deficient and wild-type T cells were equally sensitive to Fas or TCR-mediated apoptosis, the former were clearly more resistant to apoptosis induced by oxidative stress or gamma irradiation. Our results indicate that p16Ink4a function is associated with T-cell apoptosis, and subsequently contributes to the control of T-cell population size in lymphoid organs.

New benzo(b)thiophenesulphonamide 1,1-dioxide derivatives induce a reactive oxygen species-mediated process of apoptosis in tumour cells.

In this work, we describe the process of cell death induced by a series of new benzo(b)thiophenesulphonamide 1,1-dioxide derivatives (BTS) that have been selected as candidate antineoplastic drugs. Human leukaemic CCRF-CEM cells incubated with BTS undergo a typical apoptotic process that includes cell shrinkage, phosphatidylserine translocation to the cell surface, mitochondrial dysfunction, caspase activation, chromatin condensation and internucleosomal DNA degradation. Mitochondrial alterations included dissipation of the mitochondrial membrane potential, oxidation of the phospholipid cardiolipin, release of cytochrome c and uncoupling of the mitochondrial respiratory chain, leading to a decrease of the intracellular ATP pool. Activation of caspase-8, -9 and -3 takes place during BTS-induced apoptosis. Either the addition of the specific caspase-8 inhibitor Z-IETD-fmk, or the overexpression of the antiapoptotic protein Bcl-2 significantly prevented BTS-induced apoptosis, suggesting the involvement of both caspase-8-regulated and mitochondria-dependent signalling pathways in this process. BTS induce a significant increase in the production and accumulation of intracellular reactive oxygen species (ROS) that can be observed within minutes after drug addition. Moreover, cytochrome c release, caspase-3 activation and cell death can be completely abrogated by a previous incubation with the antioxidant N-acetyl-cysteine. These results suggest that ROS are essential mediators in BTS-induced apoptosis.

Alkyl- and arylthiodediazoniations of dry arenediazonium o-benzenedisulfonimides. Efficient and safe modifications of the stadler and ziegler reactions to prepare alkyl aryl and diaryl sulfides

The reaction between dry arenediazonium o-benzenedisulfonimides 1 and sodium thiolates in anhydrous methanol represents an efficient and safe procedure, of general validity, for the preparation of unfunctionalized or variously functionalized alkyl aryl and diaryl sulfides. As a rule, the reaction temperature was maintained at 0-5 degrees C for the alkylthiodediazoniations and at room temperature (20-25 degrees C) for the arylthiodediazoniations. The sulfide yields are generally high; of the 63 considered examples, 43 gave yields greater than 80% and 13 were between 70% and 80%. Lower yields were obtained only when sterically hindered diazonium salts or thiols were used. A good amount of the o-benzenedisulfonimide (8) was always recovered from the reactions and could be reused to prepare salts 1. The copious experimental data collected in homogeneous conditions have offered several starting points for the study of the mechanism of these reactions.

The role of erythrocytapheresis in secondary erythrocytosis therapy.

In chronic respiratory insufficiency secondary erythrocytosis (SPC), causing pulmonary hypertension and dx ventricular insufficiency, is often noticed. An alternative therapy to phlebotomy for SPC is isovolemic large volume erythrocytapheresis performed with cell separator (CSE) in order to quickly remove a large volume of red blood cells (RBC) while saving plasma proteins and clotting factors. In order to evaluate the efficiency and safety of CSE in SPC we reported a retrospective analysis of our experience with 61 SPC patients: from April 1996 to May 1998 we performed 208 CSE using Haemonetics MCS3P (TAE protocol). Before every apheresis procedure we verified Hb (in median 18.8 g/dl), Ht (in median 58.4%), viscometry, coagulation test, EGA, PFR and ECG. 11 patients were treated with 1 CSE, 12 with 3, 29 with 4 and 9 with 5. The mean volume of RBC removed was 576 ml (range 426-800); Hb post CSE averaged 14.4 g/dl and Ht post CSE averaged 42.7%; hematic viscosity post CSE was significantly reduced while tissue oxygen tension increased: the improvement of symptomatology and hematochemical parameters was maintained on the average for 6.5 months. All the procedures were well tolerated and light side effects (paresthesias citrate-depending in 27 apheresis) were easily controlled. CSE, compared to phlebotomy, has the advantage of selectively removing RBC without loss of clotting factors, platelets and plasma proteins. Although CSE has relatively high costs we noticed a decrease of hospital recurrence (about 50-65%) in SPC patients treated with apheresis.

The role of extracorporeal membrane oxygenation in donation after circulatory death.

Donor scarcity and the increased need for organ transplantation has prompted the development of an alternative source of donors to the more conventional brain dead donor. While in a Beating-Heart donor, abdominal and intrathoracic organs are perfused, in a non-beating heart donor (NHBD, or DCD), perfusion should be maintained, after confirmation of death, by means of ECMO and inflation of intra-aortic balloon accordingly to the localization of the organs that should be transplanted. In this setting, ECMO allows selective perfusion of the organs which should be transplanted ("compartmental ECMO"). The present review is aimed at summarizing the rationale for ECMO use in organ donation in DCD and the available evidence on this topic, as well as available evidence (in clinical studies) on normothermic organ preservation using ECMO in adults. Despite the fact that available studies suffer from methodological limitations (small cohorts, retrospective analysis, not always comparative), they all reach the same conclusion: the concept of extracorporeal support with oxygenation in DCD seems very promising since it has been reported to increase the available organ supply by approximately 20% to 25%2 by increasing the number of donors by approximately 33%. Centres with ECMO facilities should implement local programmes for donation after cardiac death (both in the emergency department and intensive care) using ECMO taking into account that this technique has been proven to increase donor pool.

Dr. Zompo: an online data repository for Zostera marina and Posidonia oceanica ESTs.

As ecosystem engineers, seagrasses are angiosperms of paramount ecological importance in shallow shoreline habitats around the globe. Furthermore, the ancestors of independent seagrass lineages have secondarily returned into the sea in separate, independent evolutionary events. Thus, understanding the molecular adaptation of this clade not only makes significant contributions to the field of ecology, but also to principles of parallel evolution as well. With the use of Dr. Zompo, the first interactive seagrass sequence database presented here, new insights into the molecular adaptation of marine environments can be inferred. The database is based on a total of 14 597 ESTs obtained from two seagrass species, Zostera marina and Posidonia oceanica, which have been processed, assembled and comprehensively annotated. Dr. Zompo provides experimentalists with a broad foundation to build experiments and consider challenges associated with the investigation of this class of non-domesticated monocotyledon systems. Our database, based on the Ruby on Rails framework, is rich in features including the retrieval of experimentally determined heat-responsive transcripts, mining for molecular markers (SSRs and SNPs), and weighted key word searches that allow access to annotation gathered on several levels including Pfam domains, GeneOntology and KEGG pathways. Well established plant genome sites such as The Arabidopsis Information Resource (TAIR) and the Rice Genome Annotation Project are interfaced by Dr. Zompo. With this project, we have initialized a valuable resource for plant biologists in general and the seagrass community in particular. The database is expected to grow together with more data to come in the near future, particularly with the recent initiation of the Zostera genome sequencing project.The Dr. Zompo database is available at http://drzompo.uni-muenster.de/

Correlation between the release of IL-2 and granule-associated DNase activity in human lymphomononuclear cells stimulated with immunomodulating peptides and proteins. Role of different antigen-presenting cells.

The authors have already described that a series of short peptides, modelled after sequences related to human extracellular matrix (ECM) proteins and sharing some common structural features, activate Th1 clones through a process involving peptide presentation in HLA-DR proteins. Those peptides induce also LAK- and NK-dependent cytotoxicity as well as activation of monocytes/macrophages present in human peripheral blood mononuclear cell (PBMC) populations. The release of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) by Th cells present in PBMC depleted of macrophages, or B cells is reported, after incubation in the presence of those peptides, fibronectin or Staphylococcus aureus protein A. The authors found that all the molecules tested needed at least the presence of a type of antigen-presenting cell (APC) to exert their stimulatory effect. Some peptides seem to be preferentially presented to Th cells by B cells, while others seem to depend on monocyte/macrophages for this presentation. The dependence on one or another APC seems to be due to differences in the sequences of these peptides. The immunomodulatory agents studied also gave rise to a clear increase in a DNase activity associated with secretion granules of PBMC. That there is a correlation between the release of IL-2 and this DNase activity when using a complete PBMC population, B cell-depleted PBMC or macrophage-depleted PBMC stimulated with the peptides tested has been found.

New cytotoxic benzo(b)thiophenilsulfonamide 1,1-dioxide derivatives inhibit a NADH oxidase located in plasma membranes of tumour cells.

A series of benzo(b)thiophenesulfonamide 1,1-dioxide derivatives (BTS) have been designed and synthesized as candidate antineoplastic drugs. Several of these compounds have shown in vitro cytotoxic activity on leukaemic CCRF-CEM cells. The cytotoxic BTS, but not the inactive ones, were able to inhibit a tumour cell-specific NADH oxidase activity present in the membrane of CCRF-CEM cells.

Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential.

In most human somatic cells telomeres progressively shorten with each cell division eventually leading to chromosomal instability and cell senescence. The loss of telomere repeats with cell divisions may also limit the replicative life span of antigen-specific T lymphocytes. Recent studies have shown that the replicative life span of various primary human cells can be prolonged by induced expression of the telomerase reverse transcriptase (hTERT) gene. To test whether introduction of hTERT can extend the life span of primary human T lymphocytes, naive CD8(+) T lymphocytes were transfected with retroviral vectors containing the hTERT gene. Transduced T-cell clones expressed high levels of telomerase and either maintained or elongated their telomere lengths upon culture for extended periods of time. Two of the transduced subclones retained a normal cloning efficiency for more than 170 population doublings (PDs). In contrast, T-cell clones transfected with control vectors exhibited progressive telomere length shortening and stopped proliferation at around 108 PDs. Telomerase-positive T clones had a normal 46,XY karyotype, maintained their cytotoxic properties, and showed very little staining for the apoptotic marker annexin-V. These results indicate that ectopic hTERT gene expression is capable of extending the replicative life span of primary human CD8(+) cytotoxic T lymphocytes. (Blood. 2001;98:597-603)

Synthesis and cytotoxic activity of lipophilic sulphonamide derivatives of the benzo[b]thiophene 1,1-dioxide.

In the search of new compounds with antineoplastic activity, we have analysed the effect of several structural modifications on the nucleus 6-benzo[b]thiophenesulphonamide 1,1-dioxide on its cytotoxic activity on tumour cells. Lipophilic substituents on the sulphonamide group significantly increased the cytotoxic activity measured using a panel of human tumour cell lines. Only slight variations on cytotoxicity were obtained when the sulphonamide group occupied the position 5 of the system. The most active compound was the N-4-methoxyphenyl derivative 15, which showed GI(50) values of 1-9 nM against HT-29, CCRF-CEM, K-562 and MEL-AC cells and of 200 nM against HTB-54 cells. Free access to the 3-position of the heterocyclic system seems to be required to obtain cytotoxic derivatives. Derivative 15 was also active at the same level of commercial Doxorubicine against cultured normal human lung fibroblasts.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Synthesis and cytotoxic activity of N-(2-pyridylsulfenyl)urea derivatives. A new class of potential antineoplastic agents.

Starting from a 3D-model for the antineoplastic activity of diarylsulfonylureas several new features were proposed and tested. Both types of assayed compounds, the N-(2-pyridylsulfonyl)urea and N-(2-pyridylsulfenyl)urea derivatives, inhibited by 50% the growth of the CCRF-CEM cell line at a dosage near to 1 microM. The N -(2-pyrimidinyl) derivative of the sulfenylurea 6c showed a better profile against HT-29, K-562 and HTB-54 tumor cell lines than the corresponding sulfonylurea 6b. Structural modifications on aryl systems affected differently to the cytotoxic activity shown by the compounds against each cell line.

Familial aggregation of blood pressure in a paediatric population.

Familial aggregation of blood pressure in childhood and the difference between males and females were studied in a random sample of a nursery and school-age population in Milan. Age, sex, height, weight, skinfold thicknesses and heart rate were obtained. Blood pressure was measured in accordance with the recommendations of the Task Force of Blood Pressure Control in Children. After adjusting for age, significant correlation coefficients were found between mother and son (r = 0.17 and r = 0.11), mother and daughter (r = 0.11 and r = 0.15), and father and son (r = 0.16 and r = 0.17) for systolic and diastolic pressure, respectively; the father-daughter correlations were not statistically different (r = 0.08 and r = 0.03). Multiple regression analysis underlined the difference between males and females; height was an important determinant in the males and body weight in the females. In conclusion, anthropometric measurements should be considered in evaluations of blood pressure status. Our results suggest important differences between males and females. The BP pattern of the parents also seems more important for sons, and there seems to be a lower correlation between fathers and daughters.