Working while unwell: Workplace impairment in people with severe asthma.

BACKGROUND: Severe asthma affects quality of life; however, its impact on workplace productivity is poorly understood. OBJECTIVE: To compare workplace productivity-absenteeism and presenteeism-and impairment in daily activities in severe and non-severe asthma over time and identify characteristics associated with presenteeism in severe asthma. METHODS: The Severe Asthma Web-based Database is an ongoing observational registry from Australia, New Zealand and Singapore. At April 2017, 434 patients with severe asthma and 102 with non-severe asthma were enrolled (18-88 years; 59% female). Participants provided comprehensive clinical and questionnaire data at baseline and were followed-up every 6 months for 24 months. Absenteeism (percentage of time not at work), presenteeism (self-reported impairment at work) and impairment in daily activities outside work due to health problems in the last week were calculated. RESULTS: At baseline, 61.4% of participants with severe asthma and 66.2% with non-severe asthma under 65 years were employed. At younger ages (30-50 years), fewer severe asthma participants were employed (69% vs 100%). Presenteeism and impairment in daily activity were more frequently reported in severe asthma and in participants with poorer asthma control, poorer lung function and more past-year exacerbations (P < .01). Over time, deteriorating asthma control was associated with increasing presenteeism. Although absenteeism was not different between severe and non-severe asthma, worse asthma control was associated with absenteeism (P < .001). In participants with severe asthma, presenteeism was reported more frequently in those with poorer asthma control, poorer asthma-related quality of life and symptoms of depression or anxiety (P < .01). CONCLUSION AND CLINICAL RELEVANCE: Severe asthma was associated with impairment at work and outside the workplace. Improving asthma control and mental health may be important targets for optimizing workplace productivity in severe asthma. Presenteeism and absenteeism may represent key metrics for assessing intervention efficacy in people with severe asthma of working age.

ECFS standards of care on CFTR-related disorders: Identification and care of the disorders.

This is the third paper in the series providing updated information and recommendations for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (CFTR-RD). This paper covers the individual disorders, including the established conditions - congenital absence of the vas deferens (CAVD), diffuse bronchiectasis and chronic or acute recurrent pancreatitis - and also other conditions which might be considered a CFTR-RD, including allergic bronchopulmonary aspergillosis, chronic rhinosinusitis, primary sclerosing cholangitis and aquagenic wrinkling. The CFTR functional and genetic evidence in support of the condition being a CFTR-RD are discussed and guidance for reaching the diagnosis, including alternative conditions to consider and management recommendations, is provided. Gaps in our knowledge, particularly of the emerging conditions, and future areas of research, including the role of CFTR modulators, are highlighted.

Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice.

We report gene transfer to the Edinburgh insertional mutant mouse (cf/cf), delivering CFTR cDNA-liposome complexes into the airways by nebulization. We show full restoration of cAMP related chloride responses in some animals and demonstrate, in the same tissues, human CFTR cDNA expression. Overall, a range of correction was seen with restoration of about 50% of the deficit between wild type mice and untreated cf/cf controls. We report modest correction in the intestinal tract following direct instillation and provide initial encouraging safety data for both the respiratory and intestinal tract following the liposome mediated gene delivery. The non-viral nature and potentially lower immunogenicity of DNA-liposomes suggest that this may offer a therapeutic alternative to adenoviral therapies.

Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

We report the results of a double-blind, placebo-controlled trial in nine cystic fibrosis (CF) subjects receiving cationic liposome complexed with a complementary DNA encoding the CF transmembrane conductance regulator (CFTR), and six CF subjects receiving only liposome to the nasal epithelium. No adverse clinical effects were seen and nasal biopsies showed no histological or immuno-histological changes. A partial restoration of the deficit between CF and non-CF subjects of 20% was seen for the response to low Cl- perfusion following CFTR cDNA administration. This was maximal around day three and had reverted to pretreatment values by day seven. In some cases the response to low Cl- was within the range for non-CF subjects. Plasmid DNA and transgene-derived RNA were detected in the majority of treated subjects. Although these data are encouraging, it is likely that transfection efficiency and the duration of expression will need to be increased for therapeutic benefit.

Detection of occult Scedosporium species in respiratory tract specimens from patients with cystic fibrosis by use of selective media.

Respiratory samples from cystic fibrosis outpatients were cultured on Sabouraud's dextrose agar (SABD) containing antibiotics, Mycosel, and Scedosporium-selective medium (SceSel+). Thirty-two (14.7%) of 218 specimens from 11/69 (15.9%) patients yielded a Scedosporium sp., most frequently Scedosporium aurantiacum (17/218). Scedosporium was recovered on SceSel+, Mycosel, and SABD from 90.6%, 50.0%, and 46.9% of the specimens tested, respectively.

Phenotypic characterisation of patients with intermediate sweat chloride values: towards validation of the European diagnostic algorithm for cystic fibrosis.

BACKGROUND: In patients with symptoms suggestive of cystic fibrosis (CF) and intermediate sweat chloride values (30-60 mmol/l), extensive CFTR gene mutation analysis and nasal potential difference (NPD) measurement are used as additional diagnostic tests and a positive result in either test provides evidence of CFTR dysfunction. To define the phenotype of such patients and confirm the validity of grouping them, patients with intermediate sweat chloride values in whom either additional CF diagnostic test was abnormal were compared with subjects in whom this was not the case and patients with classic CF. METHODS: The phenotypic features of four groups were compared: 59 patients with CFTR dysfunction, 46 with an intermediate sweat chloride concentration but no evidence of CFTR dysfunction (CF unlikely), 103 patients with CF and pancreatic sufficiency (CF-PS) and 62 with CF and pancreatic insufficiency (CF-PI). RESULTS: The CFTR dysfunction group had more lower respiratory tract infections (p = 0.01), more isolation of CF pathogens (p<0.001) and clubbing (p = 0.001) than the CF unlikely group, but less frequent respiratory tract infections with CF pathogens than the CF-PS group (p = 0.05). Patients in the CF-PS group had a milder phenotype than those with PI. Many features showed stepwise changes through the patient groups. CONCLUSION: Patients with intermediate sweat chloride values and two CFTR mutations or an abnormal NPD measurement have a CF-like phenotype compatible with CFTR dysfunction and, as a group, differ phenotypically from patients with intermediate sweat chloride values in whom further CF diagnostic tests are normal as well as from CF-PS and CF-PI patients.

Progression of periodontal disease and interleukin-10 gene polymorphism.

BACKGROUND AND OBJECTIVE: Interleukin-10 is a key immunoregulatory cytokine that may be of significance in the immunopathogenesis of chronic inflammatory diseases such as periodontal disease. Molecular genetic studies have defined a number of haplotypes that may be associated with differing levels of interleukin-10 secretion. The present study investigated the possible association between interleukin-10 gene polymorphism and periodontal disease progression. MATERIAL AND METHODS: Genomic DNA was obtained from 252 adults who were part of a prospective longitudinal study on the progression of periodontal disease in a general adult Australian population. Single nucleotide polymorphisms at positions -592 and -1082 in the interleukin-10 promoter were analysed using an induced heteroduplex methodology and used to determine interleukin-10 promoter haplotypes in individual samples. Periodontitis progression was assessed by measuring probing depths and relative attachment levels at regular intervals over a 5-year period. A generalized linear model was used to analyse the data, with age, gender, smoking status, interleukin-1 genotype and Porphyromonas gingivalis included as possible confounders. RESULTS: There was a significant (p approximately 0.02) main effect of interleukin-10 haplotypes, with individuals having either the ATA/ACC or the ACC/ACC genotype experiencing around 20% fewer probing depths of >or= 4 mm compared to individuals with other genotypes. Age and smoking had significant (p < 0.001) additional effects. CONCLUSION: These data suggest that the interleukin-10 genotype contributes to the progression of periodontal disease.

A single nucleotide polymorphism in the coding region of ABL and its effects on sensitivity to imatinib.

We have identified a gene polymorphism (K247R) within or close to the P-loop of BCR-ABL, which leads to the substitution of arginine for lysine. We investigated the allelic frequency of K247R by screening 157 CML patients and 213 healthy blood donors with conventional sequencing, restriction enzyme digest and single strand conformational polymorphism analysis, and found the arginine allele to be rare. Three out of five CML patients with the arginine allele of K247R failed to achieve a major cytogenetic response to imatinib, suggesting that the arginine allele may have reduced sensitivity. However, despite K247R's position in or near to the P-loop, biochemical and cellular assays of imatinib and dasatinib sensitivity showed no alteration compared to wild type. Clinicians should be aware that possession of the arginine allele of K247R does not reflect a mutation that necessitates a change in the therapeutic strategy, unless there are other signs of inadequate response to drug.

In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Burkitt lymphoma cell lines are prone to recombination in the switch region of the Ig mu heavy chain locus.

Different recombinations have been found at the Ig heavy chain gene loci in a number of sublines of the Burkitt lymphoma (BL) cell line Namalwa, following prolonged in vitro culture. The Namalwa sublines examined are DNA fingerprint-identical and derived from a monoclonal source. Recombinant DNA clones were used to map the Ig heavy chain gene mutations to a region between the VDJ and C mu segment of the locus. This region is associated with Ig heavy chain class switching in normal B cells. Of 24 clones established from one subline, three were found to have additional VDJ-C mu region mutations, indicating a high frequency of mutation at this locus.

Alpha interferon gene deletions in adults, children and infants with acute lymphoblastic leukemia.

DNA from 76 cases of acute lymphoblastic leukemia (ALL) was tested with a cDNA probe encoding the alpha 2B interferon (IFN) gene transcript. Deletions were found in three of ten pre-B, three of 21 T-cell, four of 22 common and one of 23 null ALL cases. Amongst those with null ALL were 20 infants, most with characteristic translocations, none of whom had deletion of alpha IFN genes. The results confirm that alpha IFN gene deletions may occur without visible abnormalities of chromosome 9p and show that they occur across a wide range of ALL phenotypes. The results suggest that alpha IFN gene deletions may be rare events in null ALL of infants but their incidence and cellular consequences remain unknown.

Somatic mitochondrial DNA mutations in adult-onset leukaemia.

Mitochondrial genome instability has recently been demonstrated in a wide variety of human tumours and is implicated in the development of the myelodysplastic syndromes, a heterogeneous group of haematological disorders with an increased risk of malignant transformation. We therefore investigated the incidence of somatic mitochondrial DNA (mtDNA) mutations in patients with adult-onset leukaemia. We sequenced the entire mitochondrial genome from both normal tissue (buccal epithelial cells) and the leukaemia from 24 patients with adult-onset leukaemia. Somatic mtDNA mutation was present in nine individuals ( approximately 40%) and in each case the tumour genome differed from the normal genome sequence by a single sequence change. Using PCR-RFLP analysis and real-time PCR, we have studied in detail the mutation present in one patient with acute lymphatic leukaemia, demonstrating that the mutation is associated specifically with the leukaemia.

Characterisation of non-concordance in the T-cell receptor gamma chain genes at presentation and clinical relapse in acute lymphoblastic leukemia.

We have analysed the structure of the T-cell receptor gamma chain (TCRG) genes in a panel of biopsies taken from 24 patients with acute lymphoblastic leukemia (ALL) (13 cALL, one pre-B ALL, two null ALL and eight T-ALL) at presentation and at clinical relapse. In the majority of cases (18/24) the structure of these genes was concordant, but in a significant minority of cases (6/24) the TCRG genes were in a different conformation at different clinical stages. In three of these patients (one null ALL, two T-ALL) the clonal TCRG rearrangements detected at presentation were absent at relapse possibly as a result of clonal regression. In one other patient (cALL), the TCRG locus at relapse was rearranged to V genes which are located downstream of the V genes found in the presentation rearrangement. This indicates that the relapse leukemic clone is probably the result of clonal evolution. In two patients (one cALL, one T-ALL) there were no clonally dominant rearrangements of the TCRG genes at presentation, but evidence for clonal rearrangements at relapse, possibly as a result of clonal progression. The structure of the IgH genes were determined in four of the six patients with clonal changes in the TCRG genes and were found to be concordant. The changes in TCRG gene structure were not restricted to ALL of any one particular age group, phenotype or duration of first remission. These data indicate that the assignment of clonal specific markers based upon the sequence of TCRG rearrangements at presentation may not always be useful in the detection of minimal residual disease in ALL.

GvHD risk assessment in hematopoietic stem cell transplantation: role of cytokine gene polymorphisms and an in vitro human skin explant model.

This present review concentrates on the recent results investigating the role of certain cytokine gene polymorphisms, including tumor necrosis factor alpha, interferon gamma, interleukin-6 (IL-6), IL-10, and IL-1 receptor antagonist, in allogeneic stem cell transplantation. The review discusses their potential role in predicting outcome and the development of a genetic risk index for graft-versus-host disease in human leukocyte antigen matched sibling transplants. By the comparative use of an in vitro human skin explant model, initial results suggest that certain polymorphisms may be associated with more severe disease.

De novo Ph negative T-cell lymphoblastic leukaemia associated with bcr gene rearrangement.

One case of de novo Ph-negative T-cell acute lymphoblastic leukaemia has been found to have a classical breakpoint cluster region (bcr) rearrangement of the type seen in chronic granulocytic leukaemia. There were no haematological features to suggest a previous chronic phase. This case represents the first report of this rearrangement in Ph negative acute T-lymphoblastic leukaemia at presentation. The implications for various therapeutic options in such patients are discussed.

Oestrogen receptor alpha gene polymorphism associates with occurrence of graft-versus-host disease and reduced survival in HLA-matched sib-allo BMT.

Oestrogen receptors mediate the cellular response to oestrogens and related compounds and promote a wide range of effects on haemopoiesis. Polymorphisms of the oestrogen receptor genes have previously been associated with variation in bone mineral density, likelihood of fractures, risk of developing Alzheimer's disease, endometrial cancer and response to hormone replacement therapy. We examined the polymorphisms in both ERalpha and ERbeta genes in 108 patients receiving a bone marrow transplant from an HLA-matched sibling donor, and compared ER genotype with outcomes of occurrence of graft-versus-host disease (GVHD) and survival using logistic regression analysis. Polymorphism of ERalpha (presence of the PX haplotype (PvuII-XbaI RFLP) of intron 1), but not ERbeta, in the patient genotype associates with occurrence of acute GVHD and with lower overall survival, following correction for known clinical and genotypic risk features. Analysis of ER genotype prior to transplant might therefore inform on a patient's likelihood of developing post-transplant complications. Variation in transplant performance because of ER genotype suggests an underlying role for oestrogens in the pathophysiology of transplant-related complications, and suggests that oestrogen-related therapy may offer a new modality of post-transplant support.

Vitamin D receptor gene polymorphism associates with graft-versus-host disease and survival in HLA-matched sibling allogeneic bone marrow transplantation.

We investigated the role of polymorphism of the vitamin D receptor (VDR) gene in HLA-matched sibling BMT for polymorphisms previously associated with human disease pathology. In intron 8 of the VDR gene, the B and A alleles of the BsmI and ApaI RFLPs were found to associate with reduced aGVHD when present in the patient's genotype. Logistic regression analysis demonstrated that patient VDR genotype, along with previously identified IL-10(-1064) and IFN-gamma genotype to be risk factors for severe acute GVHD. The A allele also associates with increased likelihood of death when present in the donor genotype (AA vs Aa or aa, hazard ratio 2.03, P = 0.0232). In patients who received increased prophylaxis with multi-agent therapy, patients whose graft was from a donor with an AA genotype had a substantially worse survival than patients whose graft was from a donor with a non-AA genotype (hazard ratio 12.93, P < 0.0001). Analysis of VDR genotype in prospective BMT recipients could indicate patients at risk of severe aGVHD. Analysis of VDR genotype in prospective BMT donors may identify individuals who have greater transplant-related mortality, and also allow appropriately restricted use of increased immunosuppressive prophylaxis.

Longstanding proliferation of CD3 negative large granular lymphocytes preceding the development of high grade non-Hodgkin's lymphoma.

A patient with a persistent CD3 negative large granular lymphocyte (LGL) proliferation with immunophenotypic and functional characteristics of natural killer cells is described. The LGL proliferation persisted and six years after diagnosis the patient developed a high grade B cell non-Hodgkin's lymphoma. Molecular studies demonstrated clonal B cell populations in the peripheral blood, distinct from that identified in the lymphoma, both at presentation with non-Hodgkin's lymphoma and at complete remission following combination chemotherapy. It is postulated that T cell dysregulation associated with the CD3 negative LGL proliferation may have led to B cell dysfunction and loss of normal B cell control, with the subsequent development of a clonal B cell lymphoproliferative disorder.

Method for quantifying expression of functionally active topoisomerase II in patients with leukaemia.

AIMS: To produce a method to measure and quantify enzymatically active topoisomerase II in normal and neoplastic human cells. METHODS: A crude cell lysate from density separated mononuclear cells from either peripherial blood or bone marrow was prepared as a source of topoisomerases. Using the lysate, minicircles from the Crithedia kinetoplast DNA complex were decatenated before being separated by agarose gel electrophoresis and visualised using ethidium bromide/ultraviolet fluorescence. RESULTS: Cell number, sample volume and drug inhibition concentration required to produce reliable and reproducible assay conditions were established. Intra- and interassay standards were included which permitted the quantification of active topoisomerase II in matched peripheral blood, bone marrow, presentation, and relapse samples from patients with acute lymphoblastic leukaemia. Active topoisomerase II has been converted to a unit scale which has been used to compare topoisomerase II activities in cells from patients with normal blood and bone marrow samples. CONCLUSIONS: There was no change in topoisomerase II activities between samples taken at presentation and those taken during a recurrence. However, topoisomerase II activity in leukaemic blast populations was increased compared with topoisomerase II activity in normal cells.

c-MYC gene abnormalities in high grade and centroblastic-centrocytic non-Hodgkins lymphoma.

Fifty nine cases of high grade and centroblastic-centrocytic (cc) Non Hodgkins Lymphoma (NHL) were investigated for mutations and gross gene rearrangements in the 5' region of the c-MYC gene. Mutations in this region, and the presence of hypermutated c-MYC genes, have been associated with poor prognostic groups. All cases showed normal c-MYC gene organisation on Southern blot analysis indicating absence of gross gene rearrangements. PCR amplification and restriction digest analysis of the exon 1/intron 1 region revealed point mutations in 7 cases. No evidence for hypermutation was found. Mutations were relatively more common in high grade NHL (6/39) than in cc-NHL (1/20). There was no correlation with disease status at presentation or relapse or the presence of extranodal disease. The cc-NHL case with a c-MYC mutation subsequently transformed to high grade disease. These data suggest that hypermutation of the c-MYC gene is a relatively rare event in cc-NHL and high grade NHL and does not contribute to the aetiology in the majority of cases. Mutation of c-MYC in cc-NHL may predict transformation to high grade disease.