Performance of galactomannan, beta-d-glucan, Aspergillus lateral-flow device, conventional culture, and PCR tests with bronchoalveolar lavage fluid for diagnosis of invasive pulmonary aspergillosis.

Galactomannan detection in bronchoalveolar lavage (BAL) fluid samples (GM test) is currently considered the gold standard test for diagnosing invasive pulmonary aspergillosis (IPA). The limitations, however, are the various turnaround times and availability of testing. We compared the performance of GM testing with that of conventional culture, an Aspergillus lateral-flow-device (LFD) test, a beta-d-glucan (BDG) test, and an Aspergillus PCR assay by using BAL fluid samples from immunocompromised patients. A total of 78 BAL fluid samples from 78 patients at risk for IPA (74 samples from Graz and 4 from Mannheim) collected between December 2012 and May 2013 at two university hospitals in Austria and Germany were included. Three patients had proven IPA, 14 probable IPA, and 17 possible IPA, and 44 patients had no IPA. The diagnostic accuracies of the different methods for probable/proven IPA were evaluated. The diagnostic odds ratios were the highest for the GM, PCR, and LFD tests. The sensitivities for the four methods (except culture) were between 70 and 88%. The combination of the GM (cutoff optical density index [ODI], >1.0) and LFD tests increased the sensitivity to 94%, while the combination of the GM test (>1.0) and PCR resulted in 100% sensitivity (specificity for probable/proven IPA, 95 to 98%). The performance of conventional culture was limited by low sensitivity, while that of the BDG test was limited by low specificity. We evaluated established and novel diagnostic methods for IPA and found that the Aspergillus PCR, LFD, and GM tests were the most useful methods for diagnosing the disease by using BAL fluid samples. In particular, the combination of the GM test and PCR or, if PCR is not available, the LFD test, allows for sensitive and specific diagnosis of IPA.

Clinical impact of bone marrow morphology for the diagnosis of essential thrombocythemia: comparison between the BCSH and the WHO criteria.

Essential thrombocythemia (ET) is currently diagnosed either by the British Committee of Standards in Haematology (BCSH) criteria that are predominantly based on exclusion and not necessarily on bone marrow (BM) morphology, or the World Health Organization (WHO) criteria that require BM examination as essential criterion. We studied the morphological and clinical features in patients diagnosed according either to the BCSH (n=238) or the WHO guidelines (n=232). The BCSH-defined ET cohort was re-evaluated by applying the WHO classification. At presentation, patients of the BCSH group showed significantly higher values of serum lactate dehydrogenase and had palpable splenomegaly more frequently. Following the WHO criteria, the re-evaluation of the BCSH-diagnosed ET cohort displayed a heterogeneous population with 141 (59.2%) ET, 77 (32.4%) prefibrotic primary myelofibrosis (prePMF), 16 (6.7%) polycythemia vera and 4 (1.7%) primary myelofibrosis. Contrasting WHO-confirmed ET, the BCSH cohort revealed a significant worsening of fibrosis-free survival and prognosis. As demonstrated by the clinical data and different outcomes between WHO-diagnosed ET and prePMF, these adverse features were generated by the inadvertent inclusion of prePMF to the BCSH group. Taken together, the diagnosis of ET without a scrutinized examination of BM biopsy specimens will generate a heterogeneous cohort of patients impairing an appropriate clinical management.

N-acetylserotonin is a better extra- and intracellular antioxidant than melatonin.

Both melatonin and its precursor N-acetylserotonin have been reported to exert antioxidant properties both in vitro and in vivo. Since little is known about their antioxidant activity in lymphocytes, we investigated their effects on spontaneous and on oxidant-induced reactive oxygen species formation in human peripheral blood lymphocytes in comparison to the antioxidant trolox, a water-soluble analogue of alpha-tocopherol. Both melatonin and N-acetylserotonin exhibited antioxidant properties against t-butylated hydroperoxide- and diamide-induced reactive oxygen species formation in peripheral blood lymphocytes. N-acetylserotonin turned out to be about three times more effective than melatonin. In resting cells, the intracellular reactive oxygen species concentration was only decreased by N-acetylserotonin and trolox, melatonin had no effect. In t-butylated hydroperoxide-mediated cell death, N-acetylserotonin was as effective as trolox in protecting peripheral blood lymphocytes from cell death and required 10-fold lower concentrations than melatonin. Furthermore, in an aqueous cell-free solution, the capacity of N-acetylserotonin to scavenge peroxyl radicals was much higher than that of melatonin. These results clearly indicate N-acetylserotonin to be a much better antioxidant than melatonin.

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Beta-blockade enhances adrenergic immunosuppression in rats via inhibition of melatonin release.

We have recently shown in rats that an in vivo treatment with catecholamines via alpha 2-receptors leads to a pronounced suppression of T- and B-cell mitogen responses of peripheral blood lymphocytes (PBL), provided that a beta-blocker is administered concomitantly. Since melatonin (MEL) reportedly has stress-protective effects on several immune functions, and since the release of MEL from the pineal gland is inhibited by beta-blockade, we tested the effect of MEL substitution on T- and B-cell mitogen responses of PBL in rats treated with two s.c. implanted retard tablets containing noradrenaline (NA) and propranolol. It was found that an oral treatment with MEL (about 40 micrograms/animal) abolished the adrenergic immunosuppression. Furthermore, functional pinealectomy induced by constant light had a similar enhancing effect on the alpha 2-adrenergic immunosuppression as observed with beta-blockers, whereas PBL from animals kept at the regular light/dark interval were resistant to the treatment with the selective alpha 2-agonist clonidine. It is concluded that endogenous MEL effectively protects rat PBL from adrenergic immunosuppression, and that beta-blockers enhance the immunosuppressive property of alpha 2-adrenergic agents via blocking the night-time release of MEL.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat.

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

The Italian SEPSIS study: preliminary results on the incidence and evolution of SIRS, sepsis, severe sepsis and septic shock.

This prospective, multicenter, epidemiological study was carried out in 99 Italian ICUs, distributed throughout the country, from April 1993 to March 1994. In the study, we applied the new ACCP/SCCM classification system for sepsis (SIRS, sepsis, severe sepsis and septic shock) and determined the prevalence, incidence, evolution and outcome of these categories in critically ill patients. The preliminary analysis of 1101 patients showed that on admission SIRS accounted for about half of the diagnoses (52%) with sepsis, severe sepsis and septic shock accounting for 4.5%, 2.1% and 3% of patients, respectively. Patients with severe sepsis or septic shock more frequently had high SAPS scores than patients without sepsis. Mortality rates were similar in patients with SIRS (26.5%) and without SIRS or infection (24%), but rose to 36% in patients with sepsis, to 52% in those with severe sepsis and to 81.8% in those with septic shock. Sepsis, severe sepsis and septic shock were more common in patients with medical diagnoses, and neither severe sepsis nor septic shock was observed in trauma patients. With respect to evolution, the incidence of septic shock was progressively higher in patients admitted with more severe "sepsis-related" diagnoses, while only a trivial difference in rates of incidence was observed between SIRS patients and those admitted without SIRS or any septic disorder (nil). The breakdown of the various ACCP/SCCM "sepsis-related" diagnoses at any time during the study was: SIRS in 58% of the population, sepsis in 16.3%, severe sepsis in 5.5% and septic shock in 6.1%. It seems reasonable to expect from the final evaluation of our study answers to the questions raised by the ACCP/SCCM Consensus Conference about the correlations between "sepsis-related" diagnosis, severity score, organ dysfunction score and outcome.

Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: a controlled double-blind, randomized, multicenter study.

BACKGROUND: ATIII is decreased in sepsis and/or shock and its baseline value correlates with mortality. The efficacy of ATIII therapy on mortality was assessed in a selected group of patients admitted to the intensive care unit (ICU) in a double-blind, randomized, multicenter study. METHODS: 120 patients admitted to the ICU with an ATIII concentration < 70% were randomized to receive ATIII (total dose 24000 units) or placebo treatment for 5 days; 56 patients had septic shock. RESULTS: ATIII concentrations in the treated group remained constant throughout the treatment period (range 97-102%). The Kaplan-Meier analysis showed no difference in overall survival between the two groups: 50 and 46% for ATIII and placebo, respectively. Septic shock and hemodynamic support were unbalanced in the two groups at admission. Therefore the Cox analysis was carried out after adjusting for these two variables. Treatment with ATIII decreases the risk of death with an odds ratio (OR) of 0.56. Of the covariates analyzed, septic shock and the baseline multiple organ failure score were negatively associated with survival and plasma activity level was positively associated with survival with an OR of 0.97 for each 1% increase in the ATIII plasma concentration at baseline. CONCLUSIONS: The results of ATIII treatment in this population of patients suggests that replacement therapy reduces mortality in the subgroup of septic shock patients only.

Lack of calmodulin antagonism of melatonin in T-lymphocyte activation.

Despite various reported effects of the pineal hormone melatonin on the immune system, its mechanism of action on immune cells is still unknown. Since melatonin has been suggested as a physiological antagonist to calmodulin in certain cell types, we investigated effects of melatonin on calmodulin-dependent IL-2 production and proliferation of activated T-lymphocytes. It was found, however, that, in contrast to the calmodulin antagonists trifluoperazine and W7, melatonin neither inhibited the IL-2 production of activated lymphoblastoid Jurkat T-cells nor decreased the mitogen response of peripheral blood mononuclear leukocytes. Preincubation of Jurkat cells with melatonin did not influence trifluoperazine effects on IL-2 production indicating that melatonin does not bind to the same sites of calmodulin as trifluoperazine, as has been postulated. In conclusion, these results did not give any evidence for a calmodulin antagonism of melatonin in T-lymphocyte activation. Thus, melatonin as a calmodulin antagonist appears not to be a universal phenomenon.

[Interaction of the epiphysis and the immune system]. / Die Interaktion von Epiphyse und Immunsystem.

First indications that the pineal gland may be involved in endocrine immunomodulation came from early reports on anti-tumor effects of pineal extracts in animals and humans. In the meantime, evidence has accumulated suggesting that melatonin, the major endocrine product of the pineal gland-as a well preserved molecule during evolution-is indeed involved in the feedback between neuroendocrine and immune functions. At present we are beginning to understand the mechanisms of action by which melatonin affects cellular functions, and from the variety of possible direct and indirect interactions it appears that melatonin may play a complex physiological role in neuroimmunomodulation. In this article we present a critical review of the numerous reports on the influence of melatonin on immune functions and discuss the possible underlying molecular pathways. In addition, a short comment is given on the current public discussion as to the clinical value of melatonin.

Recommendations for anesthesia and perioperative management of patients with neuromuscular disorders.

Patients with neuromuscular disorders are at high risk of intraoperative and postoperative complications. General anesthesia in these patients may exacerbate respiratory and cardiovascular failure due to a marked sensitivity to several anesthetic drugs. Moreover, succinylcholine and halogenated agents can trigger life-threatening reactions, such as malignant hyperthermia, rhabdomyolysis and severe hyperkalemia. Therefore, regional anesthesia should be used whenever possible. If general anesthesia is unavoidable, special precautions must be taken. In particular, for patients at increased risk of respiratory complications (i.e., postoperative atelectasis, acute respiratory failure, nosocomial infections), noninvasive ventilation associated with aggressive airway clearance techniques can successfully treat upper airway obstruction, hypoventilation and airway secretion retention, avoiding prolonged intubation and tracheotomy. Anesthesia and perioperative management of patients with neuromuscular disorders are described in this article. To grade the strength of recommendations and the quality of evidence we adopted the GRADE approach. In case of low-quality evidence, these recommendations represent the collective opinion of the expert panel.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Therapy-related myeloid neoplasms: pathobiology and clinical characteristics.

Therapy-related myeloid neoplasms (t-MNs) are serious long-term consequences of cytotoxic treatments for an antecedent disorder. t-MNs are observed after ionizing radiation as well as conventional chemotherapy including alkylating agents, topoisomerase-II-inhibitors and antimetabolites. In addition, adjuvant use of recombinant human granulocyte-colony stimulating factor may also increase the risk of t-MNs. There is clinical and biological overlap between t-MNs and high-risk de novo myelodysplastic syndromes and acute myeloid leukaemia suggesting similar mechanisms of leukaemogenesis. Human studies and animal models point to a prominent role of genetic susceptibilty in the pathogenesis of t-MNs. Common genetic variants have been identified that modulate t-MN risk, and t-MNs have been observed in some cancer predisposition syndromes. In either case, establishing a leukaemic phenotype requires acquisition of somatic mutations - most likely induced by the cytotoxic treatment. Knowledge of the specific nature of the initiating exposure has allowed the identification of crucial pathogenetic mechanisms and for these to be modelled in vitro and in vivo. Prognosis of patients with t-MNs is dismal and at present, the only curative approach for the majority of these individuals is haematopoietic stem cell transplantation, which is characterized by high transplant-related mortality rates. Novel transplantation strategies using reduced intensity conditioning regimens as well as novel drugs - demethylating agents and targeted therapies - await clinical testing and may improve outcome. Ultimately, individual assessment of genetic risk factors may translate into tailored therapies and establish a strategy for reducing t-MN incidences without jeopardizing therapeutic success rates for the primary disorders.

Reduced-intensity allografting in patients with therapy-related myeloid neoplasms and active primary malignancies.

Therapy-related myeloid neoplasms (t-MNs) are severe long-term consequences of cytotoxic treatments for a primary, often, malignant disorder. So far, the majority of patients eligible for transplantation have undergone myeloablative allo haematopoietic SCT (HSCT) as a potentially curative treatment, but it has been associated with high transplantation-related mortality (TRM) rates. In this retrospective study, we analysed the outcome of patients with t-MNs undergoing HSCT with reduced-intensity conditioning (RIC). Of 55 patients, seen at a single centre over a 10-year period, 17 underwent RIC HSCT with related or unrelated donors. The estimated overall survival was 53% at 1 year and 47% at 3 years, and disease-free survival was 47% at 1 year. At 1 year, the cumulative incidence of relapse and TRM were 24% and 30%, respectively. Of five patients with active primary neoplasms who underwent transplantation, two are alive beyond 1 year and show CR of both t-MNs and the primary malignancy. These data indicate that RIC HSCT is an encouraging approach for patients with t-MNs. The issue of primary malignancies not being in remission at the time of transplantation should be explored in further studies.

Long-term home ventilation of children in Italy: a national survey.

BACKGROUND: Improved technology, as well as professional and parental awareness, enable many ventilator-dependent children to live at home. However, the profile of this growing population, the quality and adequacy of home care, and patients' needs still require thorough assessment. OBJECTIVES: To define the characteristics of Italian children receiving long-term home mechanical ventilation (HMV) in Italy. METHODS: A detailed questionnaire was sent to 302 National Health Service hospitals potentially involved in the care of HVM in children (aged <17 years). Information was collected on patient characteristics, type of ventilation, and home respiratory care. RESULTS: A total of 362 HMV children was identified. The prevalence was 4.2 per 100,000 (95% CI: 3.8-4.6), median age was 8 years (interquartile range 4-14), median age at starting mechanical ventilation was 4 years (1-11), and 56% were male. The most frequent diagnostic categories were neuromuscular disorders (49%), lung and upper respiratory tract diseases (18%), hypoxic (ischemic) encephalopathy (13%), and abnormal ventilation control (12%). Medical professionals with nurses (for 62% of children) and physiotherapists (20%) participated in the patients' discharge from hospital, though parents were the primary care giver, and in 47% of cases, the sole care giver. Invasive ventilation was used in 41% and was significantly related to young age, southern regional residence, longer time spent under mechanical ventilation, neuromuscular disorders, or hypoxic (ischemic) encephalopathy. CONCLUSIONS: Care and technical assistance of long-term HMV children need assessment, planning, and resources. A wide variability in pattern of HMV was found throughout Italy. An Italian national ventilation program, as well as a national registry, could be useful in improving the care of these often critically ill children.

Invasive and non-invasive long-term mechanical ventilation in Italian children.

BACKGROUND: To date, few studies have been published regarding the number of children in Italy who require long-term mechanical ventilation (LTV) and their underlying diagnoses, ventilatory needs and hospital discharge rate. METHODS: A preliminary national postal survey was conducted and identified 535 children from 57 centers. Detailed data were then obtained for 378 children from 30 centers. RESULTS: The estimated prevalence in Italy of this population was 4.3/100000. The majority of children (72.2%) were followed in pediatric units. The primary physicians who cared for these patients were either pediatric intensivists or pediatric pulmonologists. Neurological patients (78.2% of cases) represented the principal disorder category. 57.2% of the patients were non-invasively ventilated, with a nasal mask being the most common interface (85% of cases). The presence of clinical symptoms that were associated with abnormal findings on diagnostic testing was the primary indication for ventilatory support, whereas weaning failure was the primary indication for tracheotomy. Invasive ventilation was significantly related to younger age, longer daily hours on ventilation and cerebral palsy. Ventilatory modes with guaranteed minimal tidal volume were more often used in patients with tracheotomy. Despite their age, illness severity and need for technological care, 98% of the study population were successfully home discharged. CONCLUSION: Managing pediatric home LTV requires tremendous effort on the part of the patient's family and places a significant strain on community financial resources. In particular, neurological patients require more health care than patients in other categories. To further improve the quality of care for these patients, it is essential to establish a dedicated national database.