Adaptation and psychometric evaluation of the Swedish version of the Assessment of Inter professional Team Collaboration Scale (AITCS-S) for use in occupational health services.

Interprofessional team collaboration (ITC) in the Swedish Occupational Health Service is an important part of the service given to the customer. The Occupational Health Service (OHS) could be more competitive if they were able to show how successful is their ITC. The Assessment of Interprofessional Team Collaboration Scale (AITCS) is an instrument that measures ITC in teams working with the client as part of the team. The aim of this study was to adapt the Swedish version of the instrument for use in OHS and to evaluate the psychometric properties of the adapted version and the adapted short version. The study included 472 participants from different OHSs, all members of the trade association of occupational health care in Sweden. Face and content validity of the instrument were assessed, and floor and ceiling effects were measured. Internal consistency was measured with Cronbach's alpha and an exploratory factor analysis was conducted on the 42-item adapted instrument and the short, 24-item version. The exploratory factor analysis gave a three-factor solution with an eigenvalue >1 and explaining a total variance of 57.1% and 62.3% for the short version. The study concludes that AITCS-S-(OHS) as well as the short version, is a reliable and valid questionnaire. Further development of the AITCS-S-(OHS) needs to be undertaken and assessed by confirmatory factor analysis.

Unwanted mainstream nitritation-denitritation causing massive N2O emissions in a continuous activated sludge process.

Nitrous oxide emissions can contribute significantly to the carbon footprint of municipal wastewater treatment plants even though emissions from conventional nitrogen removal processes are assumed to be moderate. An increased risk for high emissions can occur in connection with process disturbances and nitrite (NO2-) accumulation. This work describes the findings at a large municipal wastewater treatment plant where the levels of NO2- in the activated sludge process effluent were spontaneously and strongly increased on several activated sludge lines which was suspected to be due to shortcut nitrogen removal that stabilized for several months. The high NO2- levels were linked to a dramatic increase in nitrous oxide (N2O) emissions. As much as over 20% of the daily influent nitrogen load was emitted as N2O. These observations indicate that highly increased NO2- levels can occur in conventional activated sludge processes and result in high nitrous oxide emissions. They also raise questions concerning the risk of increased greenhouse gas (GHG) emissions of the nitritation-denitritation processes - although the uncontrolled nature of the event described here must be taken into consideration - and underline the importance of continuous monitoring and control of N2O emissions.

Ibrutinib induces rapid down-regulation of inflammatory markers and altered transcription of chronic lymphocytic leukaemia-related genes in blood and lymph nodes.

In chronic lymphocytic leukaemia (CLL) patients, treatment with the Bruton tyrosine kinase inhibitor ibrutinib induces a rapid shift of tumour cells from lymph nodes (LN) to peripheral blood (PB). Here, we characterized in depth the dynamics of ibrutinib-induced inflammatory, transcriptional and cellular changes in different compartments immediately after treatment initiation in seven relapsed/refractory CLL patients. Serial PB and LN samples were taken before start and during the first 29 days of treatment. Changes in plasma inflammation-related biomarkers, CLL cell RNA expression, B-cell activation and migration markers expression, and PB mononuclear cell populations were assessed. A significant reduction of 10 plasma inflammation markers, the majority of which were chemokines and not CLL-derived, was observed within hours, and was paralleled by very early increase of CD19+ circulating cells. At the RNA level, significant and continuous changes in transcription factors and signalling molecules linked to B-cell receptor signalling and CLL biology was observed in both PB and LN CLL cells already after 2 days of treatment. In conclusion, ibrutinib seems to instantly shut off an ongoing inflammatory response and interfere with diverse sensitive pathways in the LN.

Substitution scanning identifies a novel, catalytically active ibrutinib-resistant BTK cysteine 481 to threonine (C481T) variant.

Irreversible Bruton tyrosine kinase (BTK) inhibitors, ibrutinib and acalabrutinib have demonstrated remarkable clinical responses in multiple B-cell malignancies. Acquired resistance has been identified in a sub-population of patients in which mutations affecting BTK predominantly substitute cysteine 481 in the kinase domain for catalytically active serine, thereby ablating covalent binding of inhibitors. Activating substitutions in the BTK substrate phospholipase Cγ2 (PLCγ2) instead confers resistance independent of BTK. Herein, we generated all six possible amino acid substitutions due to single nucleotide alterations for the cysteine 481 codon, in addition to threonine, requiring two nucleotide substitutions, and performed functional analysis. Replacement by arginine, phenylalanine, tryptophan or tyrosine completely inactivated the catalytic activity, whereas substitution with glycine caused severe impairment. BTK with threonine replacement was catalytically active, similar to substitution with serine. We identify three potential ibrutinib resistance scenarios for cysteine 481 replacement: (1) Serine, being catalytically active and therefore predominating among patients. (2) Threonine, also being catalytically active, but predicted to be scarce, because two nucleotide changes are needed. (3) As BTK variants replaced with other residues are catalytically inactive, they presumably need compensatory mutations, therefore being very scarce. Glycine and tryptophan variants were not yet reported but likely also provide resistance.

Cells release subpopulations of exosomes with distinct molecular and biological properties.

Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.

mRNA and microRNA transcriptomics analyses in a murine model of dystrophin loss and therapeutic restoration.

Duchenne muscular dystrophy (DMD) is a pediatric, X-linked, progressive muscle-wasting disorder caused by loss of function mutations affecting the gene encoding the dystrophin protein. While the primary genetic insult in DMD is well described, many details of the molecular and cellular pathologies that follow dystrophin loss are incompletely understood. To investigate gene expression in dystrophic muscle we have applied mRNA and microRNA (miRNA) microarray technology to the mdx mouse model of DMD. This study was designed to generate a complete description of gene expression changes associated with dystrophic pathology and the response to an experimental therapy which restores dystrophin protein function. These datasets have enabled (1) the determination of gene expression changes associated with dystrophic pathology, (2) identification of differentially expressed genes that are restored towards wild-type levels after therapeutic dystrophin rescue, (3) investigation of the correlation between mRNA and protein expression (determined by parallel mass spectrometry proteomics analysis), and (4) prediction of pathology associated miRNA-target interactions. Here we describe in detail how the data were generated including the basic analysis as contained in the manuscript published in Human Molecular Genetics with PMID 26385637. The data have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE64420.

Multi-level omics analysis in a murine model of dystrophin loss and therapeutic restoration.

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder, a model disease for genomic studies and a priority candidate for regenerative medicine and gene therapy. Although the genetic cause of DMD is well known, the molecular pathogenesis of disease and the response to therapy are incompletely understood. Here, we describe analyses of protein, mRNA and microRNA expression in the tibialis anterior of the mdx mouse model of DMD. Notably, 3272 proteins were quantifiable and 525 identified as differentially expressed in mdx muscle (P < 0.01). Therapeutic restoration of dystrophin by exon skipping induced widespread shifts in protein and mRNA expression towards wild-type expression levels, whereas the miRNome was largely unaffected. Comparison analyses between datasets showed that protein and mRNA ratios were only weakly correlated (r = 0.405), and identified a multitude of differentially affected cellular pathways, upstream regulators and predicted miRNA-target interactions. This study provides fundamental new insights into gene expression and regulation in dystrophic muscle.

Splice-correction strategies for treatment of X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Agammaglobulinemia: causative mutations and their implications for novel therapies.

Agammaglobulinemias are primary (inherited) immunodeficiencies characterized by the lack of functional B-cells and antibodies, and are caused by mutations in genes encoding components of the pre-B-cell or B-cell receptor, or their signaling pathways. The known genetic defects do not account for all agammaglobulinemic patients, suggesting that novel mutations underlying the disease remain to be found. While efficient, the current life-maintaining therapy with immunoglobulins and antibiotics is non-curative, prompting research into alternative treatment strategies that aim at rescuing the expression of the affected protein, thus giving rise to functional B-cells. These include gene therapy, which could be used to correct the defective gene or replace it with a functional copy. For a number of genetic defects, another alternative is to modulate the splicing of the affected transcripts. While these technologies are not yet ready for clinical trials in agammaglobulinemia, advances in genomic targeting are likely to make this option viable in the near future.

Expression analysis in multiple muscle groups and serum reveals complexity in the microRNA transcriptome of the mdx mouse with implications for therapy.

MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions including Duchenne muscular dystrophy (DMD). We have compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx mouse relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as a number of changes common between tissues, including previously identified dystromirs. In the case of miR-31 and miR-34c, upregulation of primary-miRNA transcripts, precursor hairpins and all mature miRNAs derived from the same transcript or miRNA cluster, strongly suggests transcriptional regulation of these miRNAs. The most striking differences in differential miRNA expression were between muscle tissue and serum. Specifically, miR-1, miR-133a, and miR-206 were highly abundant in mdx serum but downregulated or modestly upregulated in muscle, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalized in response to peptide-phosphorodiamidate morpholino oligonucleotide (PMO) mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable in the development of novel therapeutics and for monitoring their efficacy.

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells.

BACKGROUND: The Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays. RESULTS: The largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found. CONCLUSION: Taken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells.

Transcriptional pathways controlling the development of CD44(hi) memory phenotype (MP) T cells with "innate-like" functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44(hi), but not in CD44(lo), CD4(+) T cells. Transgenic expression of PLZF during T cell development and in CD4(+) and CD8(+) T cells induced a T cell intrinsic program leading to an increase in peripheral CD44(hi) MP CD4(+) and CD8(+) T cells and a corresponding decrease of naïve CD44(lo) T cells. The MP CD4(+) and CD8(+) T cells produced IFNgamma upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4(+) transgenic T cells showed reduced IL-2 and IFNgamma production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44(hi)CD62L(+) subset. Our data indicate that PLZF is a novel regulator of the development of CD44(hi) MP T cells with a characteristic partial innate-like phenotype.

Expression profiling of chicken DT40 lymphoma cells indicates clonal selection of knockout and gene reconstituted cells.

The DT40 cell-line has been extensively used to create deletion mutants, one of which lacks Bruton's tyrosine kinase (Btk). Btk is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. It was previously shown that there are differences in gene expression between wild-type and Btk-deficient animals. Global gene expression profiling of the avian B-lymphoma DT40 cell-line was used as a model to differentiate among Btk knockout (KO) and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell-line, sub-clones are selected, which have multiple changes in their gene expression pattern (p<0.01). Although other parameters could also influence the expression profile, this potentially has important implications when interpreting microarray data from gene-deleted cell-lines.

Using undergraduate nursing students as mediators in a knowledge transfer programme for care for patients with advanced cancer.

Nursing today faces numerous challenges. Societal changes lead to reorganization of health care, changing workloads with sicker patients in hospital and home care, and limited economic resources. The increasing and changing nature of knowledge needed for expert care provision challenges nurses to continually update their competencies. These are issues demanding proactive and dynamic changes in the way nurses conceive their mandates and practice. The aim of the action-research project presented here was to foster improved quality of care for patients with advanced cancer through collaborative endeavours integrating cancer nursing clinical practice, research and education in a knowledge exchange programme. The programme was based on input about caregiving needs from multi-professional staff caring for patients with advanced cancer in a variety of healthcare settings. Undergraduate baccalaureate nursing students were then engaged in literature studies to help address these needs. Results of the studies were communicated back to the involved clinicians in a variety of ways. In this paper, we discuss what we have experienced as opportunities and obstacles in conducting the project, based on our reflections and external evaluations. This is linked to a broader discussion of ways of integrating cancer nursing research, education and practice.

Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-kappaB.

Bruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[kappa]B (NF-kappaB). Activation of NF-kappaB is a hallmark of B cells, and NF-kappaB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-kappaB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-kappaB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-kappaB-binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-kappaB signaling), as well as NF-kappaB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-kappaB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-kappaB.

Defective Toll-like receptor 9-mediated cytokine production in B cells from Bruton's tyrosine kinase-deficient mice.

Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-27, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.

Gene expression signatures in primary immunodeficiencies: the experience from human disease and mouse models.

Extensive research on molecular genetics in recent decades has provided a wealth of information regarding the underlying mechanisms of primary immunodeficiency diseases. The microarray technology has made its entry into the molecular biology research area and hereby enabled signature expression profiling of whole species genomes. Perhaps no other methodological approach has transformed molecular biology more in recent years than the use of microarrays. Microarray technology has led the way from studies of the individual biological functions of a few related genes, proteins or, at best, pathways towards more global investigations of cellular activity. The development of this technology immediately yielded new and interesting information, and has produced more data than can be currently dealt with. It has also helped to realize that even a 'horizontally exhaustive' molecular analysis is insufficient. Applications of this tool in primary immunodeficiency studies have generated new information, which has led to a better understanding of the underlying basic biology of the diseases. Also, the technology has been used as an exploratory tool to disease genes in immunodeficiency diseases of unknown cause as in the case of the CD3Delta-chain and the MAPBPIP deficiency. For X-linked agammaglobulinemia, the technique has provided better understanding of the genes influenced by Btk. There is considerable hope that the microarray technology will lead to a better understanding of disease processes and the molecular phenotypes obtained from microarray experiments may represent a new tool for diagnosis of the disease.

Distinct gene expression signature in Btk-defective T1 B-cells.

Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important for B-lymphocyte maturation. Mutations in Btk give rise to the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in man and X-linked immunodeficiency (Xid) in mice. Recent studies have subdivided the mouse immature, or transitional, B-cells into two distinct subsets according to their respective surface markers. Transitional type 1 (T1) and transitional type 2 (T2) cells are also located in distinct anatomic locations. Based on a limited number of markers it has previously been reported that the earliest phenotypic sign of Btk deficiency is manifested at the T2 stage in mice. Here, we report on distinct genome-wide transcriptomic signature differences found in T1 B-lymphocytes from Btk-defective compared to normal mice and demonstrate that Btk deficiency is visible already at this stage.

Differential expression and molecular characterisation of Lmo7, Myo1e, Sash1, and Mcoln2 genes in Btk-defective B-cells.

PURPOSE: Bruton's tyrosine kinase is crucial for B-lymphocyte development. By the use of gene expression profiling, we have identified four expressed sequence tags among 38 potential Btk target genes, which have now been characterised. METHODS: Bioinformatics tools including data mining of additional unpublished gene expression profiles, sequence verification of PCR products and qualitative RT-PCR were used. Stimulations targeting the B-cell receptor and the protein kinase C were used to activate whole B-cell splenocytes. RESULTS: Target genes were characterised as Lim domain only 7 (Lmo7); Myosin1e (Myo1e); SAM and SH3 domain containing 1 (Sash1); and Mucolipin2 (Mcoln2). Expression was found in cell lines of different origin and developmental stages as well as in whole B-cell splenocytes and Transitional type 1 (T1) splenic B-cells from wild type and Btk-defective mice, respectively. By the use of semi-quantitative RT-PCR we found Sash1 not to be expressed in the investigated haematopoietic cell lines, while transcripts were found in whole splenic B-cells from both wild type and Btk-defective mice, whereas Lmo7, Myo1e, and Mcoln2 were expressed in both B-cell lines and primary B-lymphocytes. Except for Lmo7, the transcript level was similarly affected by stimulation in control and Btk-defective cells.