DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies.

The development of effective treatment strategies for most forms of acute myeloid leukemia (AML) has languished for the past several decades. There are a number of reasons for this, but key among them is the considerable heterogeneity of this disease and the paucity of molecular markers that can be used to predict clinical outcomes and responsiveness to different therapies. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatment that are based on individual mutational profiles. It is particularly notable that studies by The Cancer Genome Atlas and others have determined that 44% of patients with AML exhibit mutations in genes that regulate methylation of genomic DNA. In particular, frequent mutation has been observed in the genes encoding DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), as well as Tet oncogene family member 2. This review will summarize the incidence of these mutations, their impact on biochemical functions including epigenetic modification of genomic DNA and their potential usefulness as prognostic indicators. Importantly, the presence of DNMT3A, IDH1 or IDH2 mutations may confer sensitivity to novel therapeutic approaches, including the use of demethylating agents. Therefore, the clinical experience with decitabine and azacitidine in the treatment of patients harboring these mutations will be reviewed. Overall, we propose that understanding the role of these mutations in AML biology will lead to more rational therapeutic approaches targeting molecularly defined subtypes of the disease.

Impact of antibiotic treatment for pulmonary exacerbations on bacterial diversity in cystic fibrosis.

BACKGROUND: A diverse array of bacterial species is present in the CF airways, in addition to those recognised as clinically important. Here, we investigated the relative impact of antibiotics, used predominantly to target Pseudomonas aeruginosa during acute exacerbations, on other non-pseudomonal species. METHODS: The relative abundance of viable P. aeruginosa and non-pseudomonal species was determined in sputa from 12 adult CF subjects 21, 14, and 7 days prior to antibiotics, day 3 of treatment, the final day of treatment, and 10-14 days afterwards, by T-RFLP profiling. RESULTS: Overall, relative P. aeruginosa abundance increased during antibiotic therapy compared to other bacterial species; mean abundance pre-antibiotic 51.0±36.0% increasing to 71.3±30.4% during antibiotic (ANOVA: F(1,54)=5.16; P<0.027). Further, the number of non-pseudomonal species detected fell; pre-antibiotic 6.0±3.3 decreasing to 3.7±3.3 during treatment (ANOVA: F(1,66)=5.11; P<0.027). CONCLUSIONS: Antibiotic treatment directed at P. aeruginosa has an additional significant impact on non-pseudomonal, co-colonising species.

Antimicrobial susceptibility and synergy studies of cystic fibrosis sputum by direct sputum sensitivity testing.

Standard disc diffusion antimicrobial susceptibility testing (C+S) on individual Pseudomonas aeruginosa colonial morphotypes cultured from cystic fibrosis (CF) sputum has questionable clinical relevance. Direct sputum sensitivity testing (DSST) is a whole-sputum susceptibility test that removes bias associated with selecting individual colonial morphotypes. We sought to determine whether, in principle, the results from DSST support the possibility of improved clinical relevance compared with C+S. Individual (DSSTi) and combination (DSST) susceptibility to gentamicin, tobramycin, ceftazidime and meropenem were determined on 130 sputum samples referred from CF subjects with antibiotic-resistant chronic Gram-negative endobronchial infection. DSSTi and concurrent C+S were compared for categorical susceptibility, synergistic combinations were evaluated and the combination DSST efficacy index (DEI) calculated. Meropenem and tobramycin were the most active individual antibiotics by DSSTi on 89 P. aeruginosa-predominant samples, with 62 % of samples sensitive to each. C+S and DSSTi showed poor agreement (κ ranging from 0.02 to 0.6), discordance ranging from 20 % (meropenem) to 49 % (tobramycin), with DSSTi demonstrating both increased susceptibility and increased resistance. The combination that most frequently had the highest DEI was tobramycin + meropenem, occurring in 76 % of samples. DSSTi appears to be reproducible, yields different antimicrobial susceptibility results from C+S without simply identifying the most resistant isolates and DSST identifies the most effective in vitro antibiotic combinations, providing preliminary proof of concept of the potentially improved clinical relevance of whole-sputum testing. Future studies will determine whether these potential theoretical advantages translate into clinical benefits.

The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples.

The DNA-based techniques used to detect bacteria in clinical samples are unable to discriminate between live bacteria, dead bacteria, and extracellular DNA. This failure to limit analysis to viable bacterial cells represents a significant problem, leading to false-positive results, as well as a failure to resolve the impact of antimicrobial therapy. The use of propidium monoazide treatment significantly reduces the contribution of dead cells and extracellular DNA to such culture-independent analyses. Here, the increased ability to resolve the impact of antibiotic therapy on Pseudomonas aeruginosa load in cystic fibrosis respiratory samples reveals statistically significant changes that would otherwise go undetected.

Modelling the bacterial communities associated with cystic fibrosis lung infections.

In many human diseases that cystic fibrosis (CF) patients suffer from, for example, lung infections, bacteria have been considered to grow as biofilms. The ability of key CF pathogens such as Pseudomonas aeruginosa to resist antibiotic therapies may be due to the poor drug penetration of these biofilms. The overall aim of this study was to develop biofilm models in vitro that resembled the bacterial species composition of CF sputa. Here, this was a step towards a longer term goal of forming multiple bacterial biofilm models in vitro that would serve, in turn, as better assays of antibiotic susceptibilities than conventionally grown cells. Biofilm models were constructed from 31 CF sputum samples, using a modified microtitre plate assay. Three forms of assessment of these biofilms were made, namely, the mass, microscopic analysis and species composition. Species composition in sputa and biofilms, characterised by terminal restriction fragment length polymorphism (T-RFLP) analysis of ribosomal gene polymerase chain reaction (PCR) products amplified from directly extracted nucleic acids, indicated that the bacterial community in sputa was well reproduced in the biofilm models. Typically, fresh sputa contained 4.6 +/- 2.3 bacterial species, with the species number decreasing to 4.0 +/- 1.6 over 5 days-this was not statistically significant (p = 0.29). This study outlines a novel methodology by which to generate and study bacterial biofilms communities. It is also hoped that the versatility of this in vitro approach, combined with its simplicity and high reproducibility, will make it an effective system to study CF sputum biofilm development and, in the longer term, serve as a means of assessing antibiotic susceptibilities.

Use of 16S rRNA gene profiling by terminal restriction fragment length polymorphism analysis to compare bacterial communities in sputum and mouthwash samples from patients with cystic fibrosis.

The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found.

Catheter-related thrombosis associated with elevated factor VIII levels in cystic fibrosis.

We report a cystic fibrosis (CF) subject with extensive, central venous catheter-associated thrombosis and sustained elevation of factor VIII to levels normally associated with significantly increased risks of deep venous thrombosis. To determine the potential significance of this finding, the prevalence of elevated factor VIII levels in 22 adults with CF was investigated. Mean (S.D.) factor VIII level was 177 (43) U/dl, with 77% of subjects having levels >150 U/dl. The high prevalence of elevated factor VIII levels questions the significance of this finding in CF subjects with catheter-related thrombosis.

Inhaled heparin in cystic fibrosis.

Cystic fibrosis (CF) is characterised by inspissated airway secretions and chronic endobronchial infection associated with exuberant neutrophilic inflammation. Unfractionated heparin may be mucolytic and has demonstrated a number of anti-inflammatory properties; however, further safety data are needed in these subjects who are at risk of airway bleeding. The current study aimed to assess the medium-term safety and tolerability of moderately high-dose inhaled heparin in CF adults and to explore possible in vivo mucolytic and anti-inflammatory outcomes. A randomised, double-blind, placebo-controlled crossover study of twice daily inhalation of 50,000 IU of heparin for 2 weeks was undertaken in CF adults, with a 1-week washout period. Eighteen subjects were randomised and 14 (mean+/-sd age 23+/-7.8 yrs and percentage-predicted forced expiratory volume in one second 52.1+/-15.56%) completed the study protocol. Heparin neither affected blood coagulation parameters nor resulted in any increase in adverse events. Heparin inhalation had no significant effect upon forced expiratory volume in one second, symptoms of sputum clearance or sputum inflammatory markers. The current pilot study demonstrated no evidence of improved sputum clearance with 50,000 IU of inhaled heparin given twice daily to adult cystic fibrosis subjects. However, inhaled heparin was safe and the future evaluation of larger doses over a longer period may be warranted.

characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16s ribosomal DNA terminal restriction fragment length polymorphism profiling.

Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 +/- 7.9 per sample and 13.4 +/- 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

Interleukin 5 release into asthmatic airways 4 and 24 hours after endobronchial allergen challenge: its relationship with eosinophil recruitment.

Interleukin 5 (IL-5), a cytokine with a range of activities on eosinophils, has been implicated in the allergic asthmatic reaction. We have investigated the kinetics of release of this cytokine into asthmatic airways as well as its relationship to eosinophil recruitment following allergen challenge. Twelve asthmatic patients underwent endobronchial allergen challenge and bronchoalveolar lavage (BAL) fluid was obtained either 4 h (n=6) or 24 h (n=6) after challenge. Four hours after challenge, levels of IL-5 were significantly increased in BAL fluid (10-fold concentration obtained from the allergen-challenge site compared with the saline control (median 2.67 pg/ml, range 1.0-7.4 pg/ml vs 1.0 pg/ml <1.0-2.4 pg/ml, P<0.05). At 24 h levels of IL-5 increased further at the allergen site but not at the saline control lavage (31.1 pg/ml, range 3.6-59. 0 pg/ml vs 1.5 pg/ml, range <1.5-4.9 pg/ml, respectively P<0.02). At 4h there was almost a three fold increase in IL-5 level, whereas at 24 h IL-5 levels were 20-fold greater. Differential cell counts showed that eosinophil numbers obtained 4 and 24 h after allergen challenge were 7 and 32 times higher than numbers after saline challenge. The parallel increase of eosinophil numbers and IL-5 concentrations in BAL fluid suggests that this cytokine may contribute to the eosinophil recruitment observed into asthmatic airways after allergen challenge.

Endobronchial allergen challenge.

LEAC offers a very practical means of studying the pathophysiology of asthma. Despite the local nature of the challenge, LEAC often has a significant effect on FEV1 and may cause short-term destabilization of asthma. In common with other bronchoscopic methods used to study human asthma, samples obtained by LEAC show a considerable degree of variability and it is therefore necessary to use groups of 12-15 subjects to minimize the risk of Type II statistical errors. Comparisons between different studies of allergen exposure are made difficult by a variety of technical considerations. Chief among these are subject selection, the technique used for allergen exposure, the timing of sampling, and the analysis techniques. Dose-response studies in nonasthmatic allergic subjects indicate that the degree of BAL eosinophilia is related to the dose of antigen [17] but there is as yet no agreement on how LEAC might be standardized. Notwithstanding these reservations, local endobronchial allergen challenge has already yielded valuable information on the pathophysiology of asthma and will remain a useful complement to other investigational techniques in the future exploration of this disease.

Infected suture anchors in the shoulder.

Symptomatic anterior glenohumeral instability secondary to a Bankart lesion may require surgical reconstruction and repair of labral pathology. In this report, a Bankart repair was performed using metallic suture anchors. An infection developed around the anchors necessitating their removal. To our knowledge, this is the first report of an infection associated with a suture anchor device.

Leukocyte recruitment after local endobronchial allergen challenge in asthma. Relationship to procedure and to airway interleukin-8 release.

We have investigated the profile of cellular recruitment into asthmatic airways after allergen and saline exposure and its relationship to interleukin-8 (IL-8) release. Fiberoptic bronchoscopy was used to instill allergen into the middle lobe while the right upper lobe received a sham saline challenge. Bronchoalveolar lavage (BAL) of both sites was performed either 4 or 24 h later. Neutrophil numbers in BAL fluid obtained 4 and 24 h after challenge were 17 and 48 times higher than prechallenge numbers (p < or = 0.001), but there was no statistically significant difference between the numbers of neutrophils at the two sites. In contrast, eosinophil numbers were increased by 6- and 20-fold, respectively, at 4 and 24 h at allergen-challenged as compared with saline-challenged sites (p < 0.005 and p < 0.02, respectively). Baseline concentrations of IL-8 in BAL fluid were undetectable in most cases. Four hours after allergen or saline exposure, BAL fluid IL-8 concentrations were: median, 200 pg/ml; range, 20 to 750 pg/ml and median, 123 pg/ml; range, < 20 to 800 pg/ml, respectively. These declined to 23 pg/ml (range, < 20 to 126 pg/ml) and 43 pg/ml (range < 20 to 130 pg/ml), respectively, 24 h after exposure. There was a significant correlation between neutrophil numbers and IL-8 concentrations 4 h after saline exposure. These findings indicate that neutrophil infiltration is a nonspecific response to the procedure of bronchoscopy and lavage, in contrast to eosinophil recruitment, which is an allergen-specific phenomenon, and it suggests that IL-8 release may be involved in neutrophil recruitment.

Cellular and mediator responses twenty-four hours after local endobronchial allergen challenge of asthmatic airways.

The effects of acute allergen exposure on bronchoalveolar lavage cells and mediators and mucosal inflammatory cells were evaluated in 10 subjects with atopic asthma who underwent lavage and biopsy 24 hours after segmental endobronchial allergen challenge. Increased numbers of bronchoalveolar lavage eosinophils were retrieved from the allergen-challenged sites compared with the saline-challenged sites (mean 21.4 vs 1.5 x 10(3) cells/ml; p < 0.02). Numbers of neutrophils and proportions of CD4+, CD8+, CD25+, and HLA-DR+ T cells were similar at the saline- and allergen-challenged sites. In contrast to the bronchoalveolar lavage findings, eosinophil numbers were not increased in the bronchial submucosa or epithelium. There was also no significant difference in neutrophils, mast cells, CD3+, CD4+, or CD8+ T cells in the submucosa after allergen challenge, but the number of activated (CD25+) T lymphocytes in the mucosa did increase after allergen challenge. Allergen challenge did not induce any significant change in endothelial expression of P-selectin, E-selectin, intercellular adhesion molecule-1, or vascular cell adhesion molecule-1. CD11a+ and very late antigen-4+ cell numbers were similar in the saline- and allergen-challenged sites. This study suggests that in patients with very mild asthma, local allergen challenge induces persistent bronchoalveolar lavage eosinophilia, but the recruitment process seems to have diminished or ceased by 24 hours.

Safety aspects of local endobronchial allergen challenge in asthmatic patients.

Local endobronchial allergen challenge is being increasingly used to investigate the role of allergic inflammation in asthma. However, little information is available about the safety of this procedure and the changes induced in airway physiology. BAL and biopsy were performed at 10 min and at 4 to 6 h, or 24 h after segmental allergen challenge in 49 patients with atopic asthma. Two hours after challenge, FEV1 was reduced from 97.6 +/- 13.9 (mean +/- SD) to 83.4 +/- 21.7% predicted. FEV1 remained reduced at 4 to 6 h (87.7 +/- 20.4%), but it had nearly returned to baseline by 24 h (93.2 +/- 14.0%). When endobronchial challenge was combined with BAL and biopsy, the initial fall in FEV1 was slightly greater (from 101.8 +/- 14.2 to 78.5 +/- 13.6%). Bronchial responsiveness to methacholine was measured in 10 subjects, and it showed a twofold increase 24 h after local challenge and lavage. Significant changes in FEV1 and methacholine PC20 were still detectable 72 h after challenge. Widespread wheezing occurred in 29% of the subjects, but none of the them had to be admitted to hospital. We conclude that local endobronchial allergen challenge, although producing measurable changes in airway physiology, is in general well tolerated and is an acceptable method to investigate airway pathophysiologic processes in patients with mild to moderate asthma.

Early identification of interleukin-16 (lymphocyte chemoattractant factor) and macrophage inflammatory protein 1 alpha (MIP1 alpha) in bronchoalveolar lavage fluid of antigen-challenged asthmatics.

Accumulation of CD4+ interleukin (IL)-2R+ lymphocytes in the airways of asthmatics is generally attributed to the presence of chemoattractant cytokines. The precise mechanism for the initiation of the earliest CD4+ lymphocyte infiltration and activation is unknown. In this study, we describe for the first time the presence of lymphocyte chemoattractant activity in the bronchoalveolar lavage (BAL) fluid obtained from asthmatics 6 h after antigen challenge. The majority of the chemoattractant activity at this early time point is represented by IL-16 (lymphocyte chemoattractant factor), a CD4+ cell-specific chemoattractant and growth factor. In addition to IL-16, macrophage inflammatory protein 1 alpha (MIP1 alpha) chemotactic bioactivity was detected in significant levels. While IL-16, MIP1 alpha, and IL-8 were all identified by enzyme-linked immunosorbent assay, the great majority of the lymphocyte chemoattractant activity in the BAL fluid after antigen challenge is attributable to IL-16 and MIP1 alpha. There were no detectable levels of IL-16 nor MIP1 alpha in BAL fluid of antigen-challenged normal subjects nor atopic nonasthmatics nor in saline-challenged lobes from the asthmatics. The identification of multiple lymphocyte chemoattractants early after antigen challenge suggests a complex cellular, as well as chemoattractant cytokine, profile in initiating the CD4+ T cell-mediated inflammatory process that is specific for the atopic asthmatic phenotype.

Efficacy of radioprotective agents in preventing small and large bowel radiation injury.

PURPOSE: A variety of adjuvant treatments and cytoprotective agents have been proposed to lessen the toxicity of radiation therapy. The following study was designed to evaluate the benefit of six agents or combinations using anastomotic bursting strength as a measure of transmural radiation injury. METHODS: The 40-Gy study consisted of the following. Seventy-two male Sprague-Dawley rats were divided into eight equal groups: nonradiated control, radiated untreated control, and six radiated treated groups. The radioprotective treatments included ribose-cysteine (Rib-Cys), WR-2721, glutamine, vitamin E, MgCl2/adenosine triphosphate, and RibCys/glutamine in combination. Radiated animals received 40 Gy to the abdomen. Two weeks after radiation, all animals underwent small bowel and colonic resection with primary anastomosis. Animals were sacrificed one week postoperatively, at which time anastomoses were evaluated and bursting strengths determined. The 70-Gy study consisted of the following. The same protocol was repeated for five groups of nine rats divided into nonradiated, radiated untreated, and three radiated treated groups receiving RibCys (8 mmol/kg), RibCys (20 mmol/kg), and WR-2721. All radiated animals received 70-Gy doses. RESULTS: In the 40-Gy group, there were 10 radiation-related deaths and 6 anastomotic leaks among 70 rats studied. None of the differences between groups were significant. Nonradiated control group small bowel and large bowel anastomotic bursting pressures were significantly elevated compared with all radiated groups. Compared with radiated controls, there were significant improvements in small bowel bursting strength in the RibCys, WR-2721, RibCys-glutamine, and vitamin E groups and significant improvement in colonic bursting strength in MgCl2/adenosine triphosphate, WR-2721, and RibCys groups. In the 70-Gy group, all nine nonradiated control rats survived. All eight untreated radiated control rats died, four of eight WR-2721 animals died (P = 0.03), all RibCys (8 mmol/kg) animals died (P = 0.03), and three of nine treated with RibCys (20 mmol/kg) survived (P = 0.08). CONCLUSIONS: WR-2721 and RibCys gave consistent protection against large and small bowel radiation injury. The lower incidence of treatment-related toxicity and potentially equal or greater radioprotective effects may make RibCys more clinically useful than WR-2721.