Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 2: Optimisation of cell line assays.

During the production of clostridial vaccines large numbers of mice are used for various in-process control tests. Replacement in vitro assays had been developed for the testing of the toxins and toxoids of several clostridial species, but none of these assays had been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), a project on clostridial vaccines for veterinary use was started as part of the EDQM-co-ordinated Biological Standardisation Programme (BSP). Within the framework of this project (coded BSP130) a collaborative study was organised to evaluate Vero cell-based alternative methods to the current mouse tests used to measure: i) the toxicity of Clostridium septicum toxin, ii) the absence of toxicity of C. septicum toxoid and iii) the antigenicity of C. septicum toxoid. The principal aims of the study were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the in vitro and current in vivo tests. The study results demonstrated good concordance, but the information gathered through the study (later on called Part 1) and the participants' workshop prompted the extension of the project in order to further optimise the in vitro protocols and improve their repeatability and reproducibility, which were comparable to but not better than those of the in vivo assays in Part 1. The 3 in vitro assays to be optimised in the extension of the BSP130 project were : i) the in vitro toxin neutralisation equivalence plus (TNE+), as a replacement for the in vivo minimum lethal dose (MLD) test for quantification of the toxicity of toxin; ii) the in vitro MLD, as a replacement for the in vivo MLD test for detection of residual toxicity associated with toxoid; iii) the in vitro total combining power (TCP), as a replacement for the in vivo TCP test for quantification of the antigenicity of toxoid. At this point, the Analytical Method Transfer Laboratory of Ceva-Phylaxia (Hungary), supported by the project management team, developed suitable SOPs for the 3 in vitro assays. These optimised methods were further assessed in BSP130 through a second international collaborative study (Part 2) aimed at defining repeatability and reproducibility in different laboratories and determining the levels of improvement compared with the original in vivo tests and the initial in vitro assays used in Part 1 of the project. Fourteen laboratories, comprising 4 public sector and 10 manufacturers' medicines control laboratories, from 11 countries participated in the collaborative Part 2 study, each testing 6 different C. septicum toxins and 6 C. septicum toxoids. Improved repeatability and reproducibility were observed for the optimised assays. The results of this study confirm the suitability of these assays for in-process control of C. septicum vaccines, with better repeatability and reproducibility than their in vivo equivalents. It is expected that, with appropriate minor changes and the use of relevant reagents, these optimised in vitro assays could be used not only for the assessment of C. septicum toxins and toxoids but for all cytotoxin-based clostridial antigens. The development and implementation of such in vitro assays would offer a great opportunity to significantly reduce animal usage, shorten the duration of QC test procedures and increase the precision of toxicity and antigenicity assays in clostridial veterinary vaccine in-process control. This would also provide more accurate and reproducible dosing of antigens in the final vaccine products, help to promote compendial acceptance and to proffer a basis for improved international harmonisation across this area of product testing.

Patterns of violence and coercion with mental health among female and male trafficking survivors: a latent class analysis with mixture models.

AIMS: Human trafficking is a crime and a human rights violation that involves various and simultaneous traumatic events (sexual and physical violence, coercion). Yet, it is unknown how the patterning of violence and coercion affects the mental health of female and male trafficking survivors. METHODS: We conducted a cross-sectional study using a sample of 1015 female and male survivors of trafficking who received post-trafficking assistance services in Cambodia, Thailand or Vietnam. We assessed symptoms of anxiety and depression with the Hopkins Symptoms Checklist and symptoms of post-traumatic stress disorder (PTSD) with the Harvard Trauma Questionnaire. Violence was measured with questions from the World Health Organization International Study on Women's Health. Latent class analysis (LCA) was used to identify distinct patterns of violence and coercion in females and males. Novel multi-step mixture modelling techniques were employed to assess the association of the emergent classes with anxiety, depression and PTSD in females and males. RESULTS: LCA identified two distinct classes of violence and coercion experiences in females (class I: severe sexual and physical violence and coercion (20%); class II: sexual violence and coercion (80%)) and males (class I: severe physical violence and coercion (41%); class II: personal coercion (59%)). Females in class I had a two-fold increase in the odds of anxiety (OR = 2.10; 95% CI: 1.57-2.81) and PTSD (OR = 2.07; 95% CI: 1.03-4.17) compared with females in class II, but differences in the prevalence of anxiety, depression and PTSD were not significant when comparing males in class I to class II. CONCLUSIONS: Specific patterns of violence and coercion provide a more in-depth understanding of the role of gender in the experience of violence and coercion and its association with mental health in survivors of trafficking. This information could be useful to target comprehensive mental health services for female and male trafficking survivors.

Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe.

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on grapevines, either as agents of pre- or post-harvest infections, including Phomopsis cane and leaf spot, cane bleaching, swelling arm and trunk cankers. In this study we explore the occurrence, diversity and pathogenicity of Diaporthe spp. associated with Vitis vinifera in major grape production areas of Europe and Israel, focusing on nurseries and vineyards. Surveys were conducted in Croatia, Czech Republic, France, Hungary, Israel, Italy, Spain and the UK. A total of 175 Diaporthe strains were isolated from asymptomatic and symptomatic shoots, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates were determined. Preliminary pathogenicity tests were performed on green grapevine shoots with representative isolates. The most commonly isolated species were D. eres and D. ampelina. Four new Diaporthe species described here as D. bohemiae, D. celeris, D. hispaniae and D. hungariae were found associated with affected vines. Pathogenicity tests revealed D. baccae, D. celeris, D. hispaniae and D. hungariae as pathogens of grapevines. No symptoms were caused by D. bohemiae. This study represents the first report of D. ambigua and D. baccae on grapevines in Europe. The present study improves our understanding of the species associated with several disease symptoms on V. vinifera plants, and provides useful information for effective disease management.

A hyperparasite affects the population dynamics of a wild plant pathogen.

Assessing the impact of natural enemies of plant and animal pathogens on their host's population dynamics is needed to determine the role of hyperparasites in affecting disease dynamics, and their potential for use in efficient control strategies of pathogens. Here, we focus on the long-term study describing metapopulation dynamics of an obligate pathogen, the powdery mildew (Podosphaera plantaginis) naturally infecting its wild host plant (Plantago lanceolata) in the fragmented landscape of the Åland archipelago (southwest Finland). Regionally, the pathogen persists through a balance of extinctions and colonizations, yet factors affecting extinction rates remain poorly understood. Mycoparasites of the genus Ampelomyces appear as good candidates for testing the role of a hyperparasite, i.e. a parasite of other parasites, in the regulation of their fungal hosts' population dynamics. For this purpose, we first designed a quantitative PCR assay for detection of Ampelomyces spp. in field-collected samples. This newly developed molecular test was then applied to a large-scale sampling within the Åland archipelago, revealing that Ampelomyces is a widespread hyperparasite in this system, with high variability in prevalence among populations. We found that the hyperparasite was more common on leaves where multiple powdery mildew strains coexist, a pattern that may be attributed to differential exposure. Moreover, the prevalence of Ampelomyces at the plant level negatively affected the overwinter survival of its fungal host. We conclude that this hyperparasite may likely impact on its host population dynamics and argue for increased focus on the role of hyperparasites in disease dynamics.

H2S preconditioning of human adipose tissue-derived stem cells increases their efficacy in an in vitro model of cell therapy for simulated ischemia.

AIMS: A major limitation of cell-based therapies for ischemia-reperfusion injury is the excessive loss of administered cells. We investigated whether H2S can improve the survival and efficacy of therapeutic cells in an in vitro model of cell-based therapy for simulated ischemia. MAIN METHODS: H9c2 rat cardiomyoblasts were exposed to oxygen-glucose deprivation and NaHS (3-30 µM) pretreated human adipose tissue derived stem cells (hASCs) were added after reoxygenization. Viability of both cell lines was assessed with flow cytometry after 24h. The effects of H2S on antioxidant defense, proliferation, AKT and ERK1/2 phosphorylation and mitochondrial activity were analyzed in hASCs. Proliferation was evaluated using propargylglycine, an inhibitor of endogenous H2S synthesis. KEY FINDINGS: NaHS pretreatment decreased the ratio of necrotic therapeutic cells by 41.8% in case of 3 µM NaHS and by 34.3% with 30 µM NaHS. The ratio of necrotic postischemic cardiomyocytes decreased by 35%, but only with the use of 3 µM NaHS. Antioxidant defense mechanisms and ERK-phosphorylation were enhanced after 3 µM NaHS treatment while AKT-phosphorylation was suppressed. NaHS dose-dependently increased the proliferation of hASCs while pretreatment with propargylglycine decreased it. SIGNIFICANCE: NaHS pretreatment can increase the survival of therapeutically used human adipose tissue-derived stem cells via increased antioxidant defense and improves the postischemic cardiac derived cells' survival as well. Proliferation of human adipose tissue-derived stem cells is enhanced by H2S. The underlying mechanisms involve enhanced ERK-phosphorylation and decreased AKT-phosphorylation. Pretreatment with NaHS may represent a simple pharmacological step that may enhance the efficacy of cell-based therapies.

Intraosseous structural graft technique: a new surgical concept in the treatment of superolateral defects in case of dysplastic acetabulum, during hip replacement surgery biomechanical and cadaver experimentations.

PURPOSE: The replacement of the dysplastic hip, despite the numerous available surgical techniques, is not entirely solved. One of the reasons for this is the relative lack of bone in the acetabulum. In this study, we present a new concept (intraosseous structural graft technique), with the aim to improve the primary stability and biological environment of the graft. METHODS: During the procedure in the cranial quadrant of the acetabulum, a proximally hinged cortico-spongious plate is fashioned that is opened laterally and a wedge-shaped graft made from the femoral head is impacted. The precisely fitted graft has good primary rotational and cranial stability which is further improved by a bicortical screw. RESULTS: The hypothesis is supported by examination on 3D models. Cadaver experimentations were performed on 19 hips of 10 cadavers, and stability testing was done on a further 20 hips of 10 cadavers with a material testing apparatus. Also the analysis of the biological and mechanical properties of the graft and the implant were examined. CONCLUSION: Based on our results, it can be concluded that our technique provides good primary stability with a more favorable biological condition for bony incorporation compared to other bulk bone graft techniques.

Surgical strategies in severe acute pancreatitis (SAP): indications, complications and surgical approaches.

BACKGROUND: Severe acute pancreatitis (SAP) is still related to high mortality rates. This study evaluated the various surgical strategies for treatment of suspected infected necroziting pancreatitis (INP). METHODS: This retrospective study included 212 patients with SAP and INP, who had surgical treatment during the period between January 2000 - December 2012 at the Ist Surgical Clinic. Surgical approaches included laparostomy with continous postoperative retropancreatic lavage, open abdomen strategy, laparotomy with primary abdominal closure accompanied or not by laparostomy (marsupialization), retroperiostomy with retroperitoneal lavage. RESULTS: The overall mortality rate was 34.0 percent, with 24 percent in laparotomy with continous retropancreatic lavage,11 percent for retroperitoneostomy and retroperitoneal continous lavage, 71 percent for the open abdomen strategy,and 43 percent for laparotomy with closed abdomen(p 0.001). Acute operations, alcoholic origin, Apache II scores of ‰¥10 organ dysfunction on admission were independent factors that predisposed patients to complications.Colonic necrosis with high mortality rates (53 percent), however seemed to be of prognostic relevance. CONCLUSIONS: The conservative approach in severe acutepancreatitis is a promising therapeutic concept. Delaying surgery up to the third week after onset of disease significantly improves the patients€™ survival. Complications are common in severe necrotizing pancreatitis leading to organ failure and need for acute operations. Colonic necros is is an independent prognostic factor for survival.

Transthoracic versus Transhiatal esophagectomy: a permanent dilemma. our 15-year experience.

OBJECTIVE: Controversy still exists regarding the optimal surgical management of esophageal cancer. This study was performed to determine and compare early and late morbidity,mortality and overall survival after transthoracic (TTE) and transhiatal esophagectomies (THE). METHODS: Between 1997-2011, 100 patients underwent TTE or THE for squamous esophageal carcinoma (90 patients)and adenocarcinoma (10 patients). Assessed parameters included patient demographics, operative data, pathology results, postoperative morbidity and mortality and 1-3 year survival. RESULTS: Thoracic approach was preferred in cases of more advanced tumors, located in the upper and mid-third of the esophagus, in patients with a better cardiopulmonary status. Perioperative blood loss was significantly higher after transthoracic resections (p=0.0004) and these surgeries took significantly longer than transhiatal esophagectomies(p=0.02). We identified complications in 70.7% patients who under went TTE and in 59.3% patients with transhiatal approach. Respiratory complications were statistically significant in the TTE- group (p-0.0003). The 30-day mortality rates were 12.2% for patients in TTE group and 10.1% in THE patients group, respectively. The mortality ratefor the entire period of the study has been calculated at 84.4%.We have identified a survival rate after 1 year of 62.2%, after 2 years of 39.3% and after 3 years - 15.1%. CONCLUSIONS: According to the results of this study, both procedures appear to be acceptable depending on surgeon preference and appropriate patient selection.

Pressure dependence of magnetic properties in Fe-Mn-B amorphous alloys: evidence for inhomogeneous ferromagnetism.

The pressure dependence of the saturation magnetization and Curie temperature was studied in melt-spun Fe60Mn20B20, Fe56Mn24B20 and Fe75B25 amorphous alloys up to 0.9 GPa, corresponding to volume changes up to 0.45%. In addition, in situ high-pressure (up to 40 GPa) x-ray diffraction was performed to determine the compressibility of the latter two alloys. Both the Curie temperature TC (at atmospheric pressure TC = 201 ± 3 and 159 ± 3 K) and the low-temperature saturation magnetization M5 K,5 T decrease remarkably with increasing pressure: dTC/dp =- 31 ± 0.5 and -32 ± 5 K GPa(-1) and dlnM5 K,5 T/dp =- 0.15 ± 0.02 and -0.13 ± 0.03 GPa(-1) for xMn = 20 and 24 at.%, respectively. Compared to dlnM5 K,5 T/dp =- 0.016 ± 0.003 GPa(-1) measured for Fe75B25, the pressure dependence of M5 K,5 T is one order of magnitude larger in the ternary alloys. The bulk moduli for the Fe56Mn24B20 and Fe75B25 glasses were measured to be 152 GPa and 173 GPa, respectively. These data are also compared with the pressure dependence of the hyperfine field and theoretical calculations of the saturation moment for Fe-B alloys reported in the literature. The results were interpreted within an inhomogeneous itinerant-electron model of ferromagnetism.

Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis.

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated a global activity in medical and interdisciplinary sciences. Most of the diverse variety of emerging techniques are based on research-grade instruments, sophisticated reagent-based assays or rely on expertise. Here, we suggest an alternative optical methodology with an easy-to-use and cost-effective instrumentation based on unique properties of malaria pigment reported previously and determined quantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble microcrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropy distinctly from any other components of blood. As a consequence, they can simultaneously act as magnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gain importance not only in malaria diagnosis and therapies, where hemozoin is considered as drug target or immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopic scale.

A COMPARATIVE ANALYSIS OF FIRST DAY NEONATAL MORTALITY BETWEEN ADOLESCENTS AND ADULT FEMALES GIVING BIRTH AT LIGULA HOSPITAL IN MTWARA, SOUTH EASTERN TANZANIA 2008-2009.

OBJECTIVE: Compare first day neonatal mortality between adolescents and adults delivering at the main referral hospital in Mtwara, Tanzania DESIGN: Cross-sectional chart review SETTING: The study was conducted at the main referral hospital in Mtwara, Tanzania. Rates of adolescent pregnancy at the hospital were 15.5% in 2009 and 14.3% in 2010 SUBJECTS: A total of 450 adolescent and adult females delivering at Ligula Hospital between 2008 and 2009 were included in the study. OUTCOME MEASURES: First day neonatal mortality between adolescents and adults was the primary outcome. Secondary outcomes included neonatal birth weight, parity, gravidity, prematurity, HIV and neonates delivered. RESULTS: First day neonatal mortality was 5.56%. Birth weight was the only risk factor significantly associated with neonatal mortality CONCLUSION: Younger women have predisposal to neonatal mortality due to underlying causal mechanisms. In order to validate the results of this study, further research on risk and causes of first day neonatal mortality at facilities is warranted.

Magnetic multilayers as a way to increase the magnetic field responsiveness of magnetocaloric materials.

The magnetocaloric response of Ni-Cu based multilayers has been studied with the aim of optimizing their magnetic field dependence. In contrast to the behavior of single phase materials, whose peak magnetic entropy change follows a power law with exponents close to 0.75, multilayering leads to exponents of -1 for an extended temperature span close to the transition temperature. This demonstrates that nanostructuring can be a good strategy to enhance the magnetic field responsiveness of magnetocaloric materials.

Reverse-mode NCX current in mouse airway smooth muscle: Na(+) and voltage dependence, contributions to Ca(2+) influx and contraction, and altered expression in a model of allergen-induced hyperresponsiveness.

AIM: We examined the electrophysiological properties of reverse-mode Na(+) /Ca(2+) exchange (NCX) in mouse airway smooth muscle (ASM), assessing its contributions to regulation of [Ca(2+) ], and its expression in acute and chronic airway hyperresponsiveness (AHR). METHODS: Membrane currents were studied in single murine ASM cells under voltage clamp at -60 mV using ramp depolarizing commands to +80 mV. Confocal fluorimetric and RT-PCR techniques were used to monitor changes in cytosolic [Ca(2+) ] and NCX expression, respectively. RESULTS: With standard KCl-containing electrode, 30 µm KB-R7943 (an inhibitor of reverse-mode NCX activity) exhibited variable effects on membrane current, indicating modulation of more than one conductance. KB-R7943 activated outwardly rectifying current that was inhibited by 100 µm iberiotoxin (blocker of large-conductance Ca(2+) -dependent K(+) channels), indicating a direct enhancing effect of KB-R7943 on those K(+) channels. After obviating K(+) currents, we found that a current sensitive to 4-4'-diisothiocyanostilbene-2,2'-disulfonic acid (blocker of Ca(2+) -dependent Cl- channels) was markedly increased by elevating [Na(+) ] in the electrode solution to 13, 15.5 and 18 mm and suppressed by KB-R7943, indicating Ca(2+) influx via reverse-mode NCX activity. With conditions preventing Ca(2+) influx through voltage-dependent Ca(2+) channels but promoting that through NCX, we found that introduction of Ca(2+) led to marked but transient KB-R7943-sensitive elevation of [Ca(2+) ]. Additionally, KB-R7943 suppressed cholinergically evoked Ca(2+) waves. Finally, NCX1 expression was not significantly changed in allergen-induced AHR acute model but increased approx. 2.5-fold in a chronic model. CONCLUSION: Reverse-mode NCX activity leads to a physiologically relevant increase in [Ca(2+) ] even under control conditions, and this may be exaggerated in allergen-induced AHR and asthma.

Prospective assessment of C4d deposits on circulating cells and renal tissues in lupus nephritis: a pilot study.

Complement activation plays a key role in the pathogenesis of lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE). We prospectively evaluated 15 LN subjects and two control groups: 13 non-SLE renal subjects (control A) and 239 SLE subjects without LN (control B). All had C4d levels on circulating erythrocytes (E-C4d), reticulocytes (R-C4d) and platelets (P-C4d) measured by flow cytometry, while C4d deposition in renal tissue was semiquantitatively assessed in LN subjects and control A using immunoperoxidase staining. Compared with control A, LN biopsies had higher glomerular-C4d scores (p = 0.003), which were associated with more frequent granular glomerular immunofluorescence staining and electron dense deposits (p < 0.001). Compared with control A and B groups, LN subjects had higher E-C4d (p = 0.002 and p = 0.005) and R-C4d levels (p = 0.002 and p = 0.008), respectively. LN subjects were more likely to have P-C4d compared with control A (p = 0.016). In LN, only E-C4d correlated with National Institutes of Health (NIH) activity index (r = 0.55, p = 0.04). In conclusion, LN biopsies showed frequent glomerular-C4d staining associated with immune complex deposits. LN subjects had higher E-C4d and R-C4d levels compared with both control groups. E-C4d levels also correlated with NIH activity index. These findings suggest a potential role of C4d on circulating cells as a biomarker for lupus nephritis.

Early clinical remission and normalisation of CRP are the strongest predictors of efficacy, mucosal healing and dose escalation during the first year of adalimumab therapy in Crohn's disease.

BACKGROUND: Adalimumab is a fully human monoclonal antibody targeting tumour necrosis factor with proven efficacy in the treatment of Crohn's disease (CD). AIM: To investigate the predictors of medium-term clinical efficacy and mucosal healing during adalimumab therapy, in patients with CD, in specialised centres approved for biological therapy in Hungary. METHODS: Data capture of the 201 CD patients was standardised and prospective (male/female: 112/89, median age: 33.0 years, duration: 8 years). Previous infliximab therapy had been administered in 48% of patients, concomitant steroids in 41%, azathioprine in 69% and combined therapy in 27% of patients. RESULTS: Overall clinical response and remission rates at 24 weeks were 78% and 52%, respectively; at 52 weeks were 69% and 44%, respectively. Endoscopic improvement and healing were achieved in 43% and 24% of patients. In a logistic regression model, clinical efficacy and CRP at week 12, need for combined immunosuppression at induction, shorter disease duration and smoking were identified as independent predictors for 12-month clinical outcome, whereas CRP at week 12, clinical remission at week 24, inflammatory parameters and nonsmoking were associated to endoscopic improvement/healing. Intensification to weekly dosing was needed in 16% of patients. Parallel azathioprine therapy and clinical remission at week 12 were inversely associated with dose escalation. CONCLUSIONS: Clinical efficacy and normalised CRP at week 12 (early deep clinical remission) are associated with medium-term clinical efficacy and mucosal healing during adalimumab therapy, whereas need for combined immunosuppression at induction and smoking status are predictors for non-response. Parallel azathioprine therapy may decrease the probability for dose escalation.

Muscle regeneration is undisturbed by repeated polytraumatic injury.

INTRODUCTION: Clinical observations suggest that repeated injury within a week after a traumatic event impairs the regeneration of tissues. Our aim was to investigate the effect of repeated trauma on the proliferation of satellite cells in skeletal muscle tissue. MATERIALS AND METHODS: Cold lesion injury was performed in the soleus muscle and in the motor cortex of anesthetized male Wistar rats 0, 1, or 2 times with 7 day intervals between the interventions. Following the last operation, 5-bromo-2'-deoxyuridine was injected i.p. for 6 or 12 days to label dividing cells. Gut epithelium was used as positive control. Immunohistochemistry was performed 1 and 5 weeks after the last injury and the sections were analyzed with confocal microscopy. RESULTS: In the case of repeated trauma, the percentage of proliferating cells remained the same compared to single hit animals after 1 week (28.0 ± 2.5% and 29.6 ± 3.0%) as well as after 5 weeks (13.9 ± 1.8% and 14.5 ± 2.2%). CONCLUSION: The second hit phenomenon is probably due to systemic factors rather than to a diminished regenerating potential of injured soft tissues.

Pathological evidence in support of total mesorectal excision in the management of rectal cancer.

BACKGROUND: Pelvic recurrence following conventional rectal resection for cancer is common. Preoperative iradiation has been shown in prospective randomized studies to halve this risk. AIM: This multiinstitutional study aimed to assess the necesity of total mesorectal excision in rectal cancer. PATIENTS AND METHOD: Pathological resections from 50 consecutive patients with adenocarcinoma of the rectum within 12 cm of the anal verge who underwent currative resection incorporating total mesorectal excision were examined. The resection specimen was examined by one of two pathologists. Some 50 total mesorectal excision specimens were examined following rectal excision for cancer. Some 38 had total mesorectal excision as a component of a low anterior resection and 12 with abdomino-perineal resection. "Cure" was defined as absence of metastatic disease and the excision of entire macroscopic tumor tissue with negative proximal and distal borders. TME was performed as described by Heald et al. The mesorectum was evaluated for lymph nodes and tumor deposists in three areas: deep to the tumor, in the proximal mesorectum and in the distal mesorectum. RESULTS: Six patients had Dukes A lesions. Of 21 patients with Dukes B tumors, five had discrete foci of adenocarcinoma in the mesorectum, with no evidence of lymph node metastasis. Dukes C lesions were more heterogeneous, but 12 out of 23 patients had distinct mesorectal deposists in addition to mesorectal node involvement. Circumferential margin involvement was rare, but mesorectal tumor deposits were present in 17 of 44 patients with pT3 tumors, and 23 of 44 had mesorectal nodal involvement. No patient with a pT2 tumor had mesorectal involvement. Failure to excise the mesorectum completely has the potential to leave gross or microscopic residual disease that may in theory predispose to local failure. CONCLUSION: Total mesorectal excision is necessary to avoid incomplete pathological evaluation of the mesorectum and understaging of rectal cancer.

Atrial remodeling and novel pharmacological strategies for antiarrhythmic therapy in atrial fibrillation.

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. It can occur at any age, however, it becomes extremely common in the elderly, with a prevalence approaching more than 20% in patients older than 85 years. AF is associated with a wide range of cardiac and extra-cardiac complications and thereby contributes significantly to morbidity and mortality. Present therapeutic approaches to AF have major limitations, which have inspired substantial efforts to improve our understanding of the mechanisms underlying AF, with the premise that improved knowledge will lead to innovative and improved therapeutic approaches. Our understanding of AF pathophysiology has advanced significantly over the past 10 to 15 years through an increased awareness of the role of "atrial remodeling". Any persistent change in atrial structure or function constitutes atrial remodeling. Both rapid ectopic firing and reentry can maintain AF. Atrial remodeling has the potential to increase the likelihood of ectopic or reentrant activity through a multitude of potential mechanisms. The present paper reviews the main novel results on atrial tachycardia-induced electrical, structural and contractile remodeling focusing on the underlying pathophysiological and molecular basis of their occurrence. Special attention is paid to novel strategies and targets with therapeutic significance for atrial fibrillation.

Role of Ca²+-sensitive K+ currents in controlling ventricular repolarization: possible implications for future antiarrhytmic drug therapy.

Normal heart function and repolarization of the cardiac action potential (AP) is to a high extent subjective to synchronized activity of sarcolemmal K(+) channels, expressed in both ventricular and atrial myocardium, largely contributing to regulation of the resting potential, the pacemaker activity, and the shape and duration of the AP. Clinical observations and experimental studies in cardiomyocytes and multicellular preparations provided firm evidence for the sensitivity of some major outward K+ currents and the corresponding ion channels to shifts in intracellular Ca(2+) concentration ([Ca(2+)](i)). Direct regulation via interaction between [Ca(2+ )](i) and the channel protein or indirect modulation via Ca(2+ ) signaling pathways of these currents have strong implications to mechanical and electrical performance of the heart, and its physiological adaptation to altered load. It may also lead to severe cardiac dysfunction, if [Ca(2+ )](i) handling is disturbed in a variety of pathological conditions. In this review we attempt to summarize the present state of the topic on two ubiquitous repolarizing K(+) currents (I(to1) and I(K1)) with documented Ca(2+)-sensitivity and critical significance in cellular antiarrhythmic defense, to highlight fields where clue data are missing, and discuss the apparently unsolved "mystery" of the cardiac small conductance Ca(2+ )-activated K(+ ) (SK) channels. We have collected the available information on the known novel, although usually still not enough selective inhibitors and activators of these currents justifying the need for more selective ones. Finally, we emphasize a few related therapeutical perspectives to be considered for future experimental research and particularly in pharmaceutical development.