Phenotypic assessment of Neisseria meningitidis isolates obtained from patients with invasive meningococcal disease in Greece, 1993-2003: implications for serogroup B vaccines based on PorA serosubtype antigens

Serogroup B is the major isolate from patients with invasive meningococcal disease (IMD) in Greece. This study used the whole cell enzyme-linked immuosorbent assay (ELISA) with monoclonal antibodies to screen Neisseria meningitidis isolates obtained from patients with IMD between 1993 and 2003 to determine if serosubtypes included in the hexavalent Por A OMP vaccines being tested in northern Europe were prevalent in Greece. During this period there were significant changes in the proportions of serogroups B and C isolated from patients. Serogroup C was predominant in 1996-1997 but fell sharply with corresponding increases in serogroup B. Of the 591 isolates sent to the National Meningitis Reference Laboratory in Athens during this period, 325 (55%) were serogroup B. Among those tested for serosubtype, porA proteins used for the vaccine being tested in Britain were detected on 85/284 (30%) strains and for the vaccine being tested in the Netherlands 175/284 (62%). P1.14 (58/284, 20%) the predominant serosubtype among the Greek isolates, is not present in either vaccine formulation; 23/284 (8%) strains did not react with any of the monoclonal antibodies. Our results indicate that introduction of the vaccines currently being evaluated in northern Europe would not be warranted in the Greek population.     

Mutation E522K in human DNA topoisomerase IIbeta confers resistance to methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride and methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide but hypersensitivity to etoposide

Human cells express two isoforms of topoisomerase II, alpha and beta, that are both targeted by anticancer drugs. To investigate acridine resistance mediated by topoisomerase IIbeta, we used a forced molecular evolution approach. A library of mutated topoisomerase IIbeta cDNAs was generated by hydroxylamine mutagenesis and was transformed into the yeast JN394 top2-4. Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride (AMCA) selection identified a resistant transformant able to grow in media containing 76 microg/ml AMCA. Topoisomerase IIbeta with a glutamic acid-to-lysine substitution at position 522 was responsible for the approximately 10-fold resistance to AMCA. The transformant was cross-resistant to methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) and mAMCA but hypersensitive to etoposide and ellipticine. In vitro, the betaE522K protein was unable to support acridine-stimulated DNA cleavage, suggesting that resistance to these acridines is caused by reduced drug-stimulated DNA cleavage. However, betaE522K showed DNA cleavage with etoposide, and the cleavable complexes formed with etoposide showed greater stability, thus accounting for the hypersensitivity to etoposide. Drug-independent cleavage of an oligonucleotide by betaE522K was reduced compared with the wild-type enzyme. Decatenation and relaxation activities were reduced to 52 and 61% of the wild-type levels, which may explain the slower growth of yeast strain JN394top2-4 expressing betaE522K at the nonpermissive temperature. This study confirms that topoisomerase IIbeta is a target for AMCA and that resistance to AMCA can be mediated by a point mutation at Glu522 in topoisomerase IIbeta. Residue 522 lies within a Rossmann fold in the B' subfragment of topoisomerase II, a region previously implicated in drug interactions.     

The Bellagio Report: Cardiovascular risks of spaceflight: implications for the future of space travel

BACKGROUND: Long-duration space missions, as well as emerging civilian tourist space travel activities, prompted review and assessment of data available to date focusing on cardiovascular risk and available risk mitigation strategies. The goal was the creation of tools for risk priority assessments taking into account the probability of the occurrence of an adverse cardiovascular event and available and published literature from spaceflight data as well as available risk mitigation strategies. METHODS: An international group of scientists convened in Bellagio, Italy, in 2004 under the auspices of the Aerospace Medical Association to review available literature for cardiac risks identified in the Bioastronautics Critical Path Roadmap (versions 2000, 2004). This effort led to the creation of a priority assessment framework to allow for an objective assessment of the hazard, probability of its occurrence, mission impact, and available risk mitigation measures. RESULTS/CONCLUSIONS: Spaceflight data are presented regarding evidence/ no evidence of cardiac dysrhythmias, cardiovascular disease, and cardiac function as well as orthostatic intolerance, exercise capacity, and peripheral resistance in presyncopal astronauts compared to non-presyncopal astronauts. Assessment of the priority of different countermeasures was achieved with a tabular framework with focus on probability of occurrence, mission impact, compliance, practicality, and effectiveness of countermeasures. Special operational settings and circumstances related to sensitive portions of any mission and the impact of environmental influences on mission effectiveness are addressed. The need for development of diagnostic tools, techniques, and countermeasure devices, food preparation, preservation technologies and medication, as well as an infrastructure to support these operations are stressed. Selected countermeasure options, including artificial gravity and pharmacological countermeasures need to be systematically evaluated and validated in flight, especially after long-duration exposures. Data need to be collected regarding the emerging field of suborbital and orbital civilian space travel, to allow for sound risk assessment.     

Significant differences of lymphocytes isolated from ascites of patients with ovarian cancer compared to blood and tumor lymphocytes. Association of CD3+CD56+ cells with platinum resistance

OBJECTIVES: Tumor infiltrating lymphocytes (TILs) and T regulatory cells (Tregs) have been associated with prognosis in ovarian cancer, but their prognostic significance in ascites has not been studied. We performed a prospective study of T lymphocytes isolated from ascites from patients with ovarian carcinoma and we compared them with the respective populations in blood and tumors. METHODS: Mononuclear cells from ascites (n=71) and blood were isolated by Ficoll, while tumor lymphocytes (n=20) were obtained upon mechanical dissociation. Phenotypic analysis was performed with flow cytometry. Ascites from 10 patients with cirrhosis was used as control. RESULTS: Tregs containing CD4(+)CD25(+) cells, NK-T containing CD3(+)CD56(+) cells and CD69 and HLADR expression of CD4 and CD8 lymphocytes were significantly increased in tumor ascites compared to blood and control ascites. A selective accumulation of these populations in the ascites of cancer patients, was suggested by the significantly higher ascites/blood (A/B) ratios in cancer patients but not controls. Cancer cell content in ascites was correlated with CD4(+)CD25(+), CD4(+)CD69(+), CD4(+)HLADR(+) and CD8(+)CD69(+) cells. There was no correlation of lymphocyte populations between ascites and samples from peritoneal metastases. Higher tumor grade was associated with increased A/B CD4(+)CD25(+) ratio and reduced CD3(+)CD56(+) cells, while platinum resistance was associated with reduced A/B CD3(+)CD56(+) ratio. CONCLUSIONS: There are significant differences of CD3(+)CD56(+) and CD25(+)CD4(+) lymphocytes and increase in lymphocyte activation between blood, ascites and peritoneal metastases from patients with ovarian cancer. The selective accumulation of CD3(+)CD56(+) population in ascites may be a predictive factor for platinum resistance.     

Drug eluting stents versus coronary artery bypass surgery in patients with isolated proximal lesion in left anterior descending artery suffering from chronic stable angina.

OBJECTIVE: To compare the efficacy of drug eluting stents (DES) compared with bypass surgery (CABG) with left internal mammary artery (LIMA) in patients with single vessel disease suffering from chronic stable angina. BACKGROUND: There are a limited number of studies investigating this group of patients. METHODS: We included 257 consecutive patients with isolated lesion in the proximal segment of left anterior descending artery (LAD). All patients suffered from chronic stable angina or from stress-induced ischemia. Of 257 patients, 147 underwent DES implantation and 110 CABG with LIMA. All patients were followed-up clinically for major adverse cardiac events. RESULTS: The baseline demographic and angiographic characteristics were similar between the two groups. In the DES group we used sirolimus-, paclitaxel-, and ABT-578-eluting stents. The mean duration of hospitalization after CABG was 7.86 +/- 3.84 days vs. 1.02 +/- 0.19 days after PCI (P < 0.01). The incidence of MACE was 2.72% in the DES and 2.72% in the surgical group during a mean follow-up period of 18.71 +/- 6.27 months for PCI and 18.70 +/- 7.31 months for CABG (P = 0.99). There was one cardiac related death in the DES group and two in the surgical group (P = 0.58). There were three reinterventions in the DES group versus none in the surgical group (P = 0.26). Recurrence of angina was observed in 4.08% of pts in the DES group versus 6.36% in the CABG group (P = 0.57). CONCLUSIONS: The present study demonstrated that patients suffering from chronic stable angina with isolated lesion in the proximal segment of LAD have excellent long-term outcome in both surgical and DES treatment.     

Quantitative multichannel EEG measure predicting the optimal weaning from ventilator in ICU patients with acute respiratory failure.

OBJECTIVE: The objective of this study was to develop a novel quantitative multichannel EEG (qEEG) based analysis method, called Global Field Damping Time (GFDT), in order to detect potential EEG changes of patients admitted to the ICU with acute respiratory failure, and correlate them to the patients' recovery outcome predicting the optimal time-point to disconnect the patient from mechanical ventilation. METHODS: Twenty-nine adult patients with acute respiratory failure out of 98 admitted to the Intensive Care Unit of Saint Paul General Hospital were enrolled, and among them only 15 completed the study. The patients were classified in 3 groups according to their outcome after 3 months follow-up. The patients were intubated with fraction of inspired oxygen (FiO2) of 100%. Neurological Deficit Scores (NDS) were measured 24 h after intubation to assess patients' neurological condition. Twenty-four hours after patient's intubation, FiO2 was decreased to 40% (weaning session), followed by a 5 min early recovery session, a 5 min recovery 1 session and a 5 min recovery 2 session. EEG recordings were performed during this experimental procedure. Multichannel EEG segments were processed and fitted into a multivariate autoregressive (mAR) model, and single channel EEG segments into a scalar autoregressive (sAR) model. The mAR and the sAR models of arbitrary order p were decomposed into mp and p oscillators and relaxators, respectively. Damping time of each oscillator and each relaxator, and the Global Field Damping Time (GFDT) as a weighted damping time were estimated for both mAR and sAR models. RESULTS: A statistically significant increase of mAR model's GFDT during the weaning session was observed in the subjects of all groups. Comparing the 3 patients' groups, statistically significant differences for mAR model's GFDT were observed for the weaning and early recovery session. Linear regression analysis between NDS and mean mAR model's GFDT showed statistical significance during weaning session, early recovery session, and recovery 1 session. There was no statistical significance for SaO2 in the regression analysis with NDS. The sAR model's GFDT presented worst results in comparison with the mAR modelling GFDT in the identification of hypoxic conditions during weaning session and in the discrimination of patients with acute respiratory failure according to their neurological outcome. CONCLUSIONS: Global Field Damping Time as correlated to the patients' neurological outcome appears to be a simple, compact, and substantial novel indicator of cerebral hypoxia and a potential predictor of the optimal time-point to disconnect the patient from the ventilator. SIGNIFICANCE: Quantitative EEG seems to be an important tool for ICU clinicians assisting them to decide for the patients' optimal time-point to disconnect the patient from the ventilator.     

Incidence of toxoplasmosis in 5532 pregnant women in Crete, Greece: management of 185 cases at risk

OBJECTIVES: To study the incidence of toxoplasmosis in pregnant women in Crete and to test a designed protocol for handling those at risk of delivering congenitally infected infants. STUDY DESIGN: Pregnant women were screened serologically over a period of 5 years. Cases with suspected acute toxoplasmosis were treated, peripheral blood (PB), and amniotic fluid (AF) tested by polymerase chain reaction (PCR) and culture, and fetuses monitored by ultrasonography. The absence of congenital infection in infants was confirmed by serology and clinical evaluation. RESULTS: Of the 5532 pregnant women followed, 70.57% remained seronegative, 29.45% were seropositive, and there was direct evidence of seroconversion in six cases. Acute toxoplasmosis was suspected in 185 cases, maternal parasitemia was detected in five cases and positive amniotic fluid in one case. Congenital infection was excluded in all infants followed, based on the absence of ultrasound findings in utero, lack of clinical symptoms at birth, negative Western blotting (WB) at birth and 3 months later, and descending serology for a year. CONCLUSION: Overall, 29.45% of the pregnant women followed were seropositive, 3.3% with suspected acute toxoplasmosis, and in 0.02% cases there was evidence of maternofetal transmission. The protocol tested allowed differentiation between acute and latent toxoplasmosis, safe management of the cases at risk and assisted in avoidance of unwarranted pregnancy terminations.     

Mutation P732L in human DNA topoisomerase IIbeta abolishes DNA cleavage in the presence of calcium and confers drug resistance

The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase IIbeta. We report here the first selection with mAMSA of resistant human topoisomerase IIbeta. Random mutagenesis of human DNA topoisomerase IIbeta cDNA, followed by selection in yeast for resistance to mAMSA, identified betaP732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. betaP732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that betaP732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.     

Severe fenthion intoxications due to ingestion and inhalation with survival outcome

Two cases of severe fenthion intoxication are presented. The first is a case of a psychiatric patient who attempted suicide with ingestion of the compound, and the second case was of a child exposed to the chemical agent by air spraying. Both patients were treated in the intensive care unit with atropine and pralidoxime and finally survived. Fenthion blood levels on admission were 2.7 and 0.95 microg/mL, respectively. Different concentrations of pralidoxime were added to the first patient's poisoned serum in order to assess in vitro the effect of pralidoxime on cholinesterase reactivation. The clinical and toxicological data of the poisonings are discussed, as well as the potential therapeutic use of pralidoxime in organophosphate intoxication.