GPs' prescription patterns, experience, and attitudes towards medicinal cannabis-a nationwide survey at the early stage of the Danish test scheme.

BACKGROUND: On 1 January 2018 a four-year test scheme concerning use of medicinal cannabis (MC) was enacted. It has recently been extended for four more years by the Danish Parliament permitting all Danish physicians to prescribe MC to their patients. Previous studies have shown that general practitioners (GPs) have varying prescription experience, little knowledge, and mixed attitudes about MC. However, the present evidence is still limited, and no studies exist about Danish GPs' prescription experience, knowledge, and attitudes towards MC. Therefore, our aim was to examine Danish GPs' prescription experience, knowledge, and attitudes towards MC. METHODS: A national online survey-based study addressing Danish GPs was performed from September 2018 to July 2019. We performed separate multivariable logistic regression analyses including GPs' prescription experience, knowledge, and attitudes towards MC as outcome variables. RESULTS: A total of 427 (38.4%) of 1112 GPs completed the questionnaire. Of these, 37 (8.7%) had experience in prescribing MC. The majority had little or no knowledge about MC (80.6%) as well as a negative view on prescription of MC (71.4%) to patients. Factors associated with prescribing MC to patients were: Single-handed practices (OR = 1.6, 95% CI 1.1;1.8) and perception of having quite some knowledge about MC (OR = 4.8, 95% CI 2.2;10.4). Factors associated with having quite some knowledge about MC were: having a positive attitude towards prescribing MC (OR = 5.2, 95% CI 1.9;14.0), being male (OR = 1.7, 95% CI 1.4;1.8), and being at least 60 years of age (OR = 2.8, 95% CI 1.3;6.0). Factors associated with having a positive attitude towards prescribing MC were: having quite some knowledge about MC (OR = 5.2, 95% CI 2.2;12.5) and GPs being male (OR = 1.7, 95% CI 1.1;1.9). CONCLUSION: In this first study on prescription experience, knowledge, and attitudes about MC among Danish GPs, conducted one year after the Danish test scheme was enacted, we find a very low proportion of prescribers, little knowledge, and an overall negative attitude towards MC. Among the prescribing GPs, four in ten have little to no knowledge and a negative attitude towards MC. We stress that prescribing patterns, knowledge, and attitudes may change throughout the remaining time of the test scheme.

Challenges in the cross-sectoral collaboration on vulnerable pregnant women: a qualitative study among Danish general practitioners.

BACKGROUND: Vulnerable pregnant women, defined as women threatened by social, psychological, or physical risk factors, need special support during pregnancy to prevent complications in pregnancy, birth, and childhood. Proper cross-sectoral collaboration in antenatal care is paramount to delivering sufficient supportive care to these women. General practitioners (GPs) often face barriers when assessing vulnerable pregnant women and may; as a result, under-identify and underreport child abuse. Little is known about how the cross-sectoral collaboration in antenatal care affects the GP's opportunities of managing vulnerable pregnant women. This study explores GPs' perceived barriers and facilitators in the antenatal care collaboration on vulnerable pregnant women and in the reporting of these women to social services. METHODS: A qualitative study with semi-structured focus group interviews among twenty GPs from the Region of Southern Denmark. A mixed inductive and deductive analytic strategy was applied, structured according to the Theoretical Domains Framework (TDF). RESULTS: Three themes emerged: I) collaborative experience, II) motivation, and III) organizational working conditions. Barriers were lacking experience, i.e. knowledge, skills, and attention to antenatal care collaboration and reporting, inadequate organizational working contexts, i.e. insufficient pathways for communication between health care and social care systems, and laws restricting feedback on the consequences of reporting. This decreased the GPs motivation, i.e. poor confidence in navigating the system, fear of breaking the patient alliance when collaborating in antenatal care and reporting with the social services. GPs motivation to collaborate and report was increased by knowing the working contexts of their collaborative partners in the antenatal care and social services system and by a strong doctor-patient relationship enabling them to describe the vulnerability to collaborators. CONCLUSIONS: GPs experience system-related barriers to collaborating and reporting on vulnerable pregnant women within the health care sector and in the interplay with the social services sector. Organizational development of cross-sectoral antenatal care collaboration should imply user involvement of all collaborative partners. Results suggest that health authorities should consider establishing accessible communication pathways between the GPs and the social services to improve options for proper cross-sectoral communication and feedback to GPs, thereby improving care trajectories of vulnerable pregnant women.

General practitioners' perceived indicators of vulnerability in pregnancy- A qualitative interview study.

OBJECTIVE: To explore general practitioners' (GPs') perceived indicators of vulnerability among pregnant women in primary care. DESIGN: A qualitative study with semi-structured in-depth focus group interviews. SETTING: General practices located in a mixture of urban, semi-urban and rural practices throughout the Region of Southern Denmark SUBJECTS: Twenty GPs. MAIN OUTCOME MEASURES: Through qualitative analysis with systematic text condensation of the interview data, the following themes emerged: (1) obvious indicators of vulnerability-i.e. somatic or psychological illnesses, or complex social problems and 2) intangible indicators of vulnerability - i.e. identification depended on the GPs' gut-feeling. From the GPs' perspective, the concept of vulnerability in pregnancy were perceived as the net result of risk factors and available individual and social resources, with a psychosocial etiology as the dominant framework. CONCLUSIONS: The GPs demonstrated a broad variety of perceived indicators of vulnerability in pregnancy; most importantly, the GPs were aware of a group of pregnant women with intangible vulnerability mainly representing low resilience. Despite not fitting into the GPs' perceived concept of vulnerability, the GPs had a strong gut feeling that these women might be vulnerable. Misjudging the resources of pregnant women due to their physical appearance could delay the GPs' identification of vulnerability. Future studies should explore the challenges GPs experiences when assessing vulnerability among pregnant women.

The E3 ubiquitin ligase SMURF1 regulates cell-fate specification and outflow tract septation during mammalian heart development.

Smad ubiquitin regulatory factor 1 (SMURF1) is a HECT-type E3 ubiquitin ligase that plays a critical role in vertebrate development by regulating planar cell polarity (PCP) signaling and convergent extension (CE). Here we show that SMURF1 is involved in mammalian heart development. We find that SMURF1 is highly expressed in outflow tract cushion mesenchyme and Smurf1-/- mouse embryos show delayed outflow tract septation. SMURF1 is expressed in smooth muscle cells of the coronary arteries and great vessels. Thickness of the aortic smooth muscle cell layer is reduced in Smurf1-/- mouse embryos. We show that SMURF1 is a negative regulator of cardiomyogenesis and a positive regulator of smooth muscle cell and cardiac fibroblast differentiation, indicating that SMURF1 is important for cell-type specification during heart development. Finally, we provide evidence that SMURF1 localizes at the primary cilium where it may regulate bone morphogenetic protein (BMP) signaling, which controls the initial phase of cardiomyocyte differentiation. In summary, our results demonstrate that SMURF1 is a critical regulator of outflow tract septation and cell-type specification during heart development, and that these effects may in part be mediated via control of cilium-associated BMP signaling.

Continuous professional development for GPs: experience from Denmark.

BACKGROUND AND OBJECTIVES: Continuous professional development (CPD) for Danish general practitioners (GPs) is voluntary and based on funded accredited activities. There is an ongoing discussion on how to improve this current system by introducing mandatory elements. To inform this debate, we set out to identify GPs' current use of CPD and to explore the motives behind their choices. METHODS: A mixed-methods study with a combined qualitative and quantitative approach was used. In 2012, two focus group interviews were conducted, followed up the same year by an online questionnaire sent to 1079 randomly chosen Danish GPs. RESULTS: Focus groups: CPD activities are chosen based on personal needs analysis, and in order to be professionally updated, to meet engaged colleagues and to prevent burnout. GPs also attend CPD to assess their own pre-existing level of competence. CPD activities need to be experienced as being both meaningful and relevant in order to have an impact. Questionnaire: The response rate was 686/1079 (63%). GPs spend on average 10.5 days per year on accredited, voluntary CPD activities. Workplace-related CPD activities and practice-based small group learning played a significant role. The main motivation for choice of CPD activities included academic interest, experience of patient-related problems in their own surgeries and medical topics where the GPs felt insufficiently confident. CONCLUSIONS: Danish GPs are frequent users of voluntary accredited CPD. Their CPD choices are motivated by topics strengthening their professional capacity and preventing burnout. There would seem to be no need for a mandatory system.

Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study.

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia: the case of SF3B1 and subset #2.

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

The immunological functions of the vitamin D endocrine system.

The discoveries that activated macrophages produce 1alpha25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), and that immune system cells express the vitamin D receptor (VDR), suggested that the vitamin D endocrine system influences immune system function. In this review, we compare and contrast how 1alpha,25-(OH)2D3 synthesis and degradation is regulated in kidney cells and activated macrophages, summarize data on hormone receptor function and expression in lymphocytes and myeloid lineage cells, and discuss how locally-produced 1alpha,25-(OH)2D3 may activate a negative feed-back loop at sites of inflammation. Studies of immunity in humans and animals lacking VDR function, or lacking vitamin D, are viewed to gain insight into the immunological functions of the vitamin D endocrine system. The strong associations between poor vitamin D nutrition, particular VDR alleles, and susceptibility to chronic mycobacterial infections, together with evidence that 1alpha,25-(OH)2D3 served as a vaccine adjuvant enhancing antibody-mediated immunity, suggest a model wherein high levels of 1alpha,25-(OH)2D3-liganded VDR transcriptional activity may promote the CD4+ T helper 2 (Th2) cell-mediated and mucosal antibody responses to cutaneous antigens in vivo. We also review a diverse and rapidly growing body of epidemiological, climatological, genetic, nutritional and biological evidence indicating that the vitamin D endocrine system functions in the establishment and/or maintenance of immunological self tolerance. Studies done in animal models of multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), inflammatory bowel disease (IBD), and transplantation support a model wherein the 1alpha,25-(OH)2D3 may augment the function of suppressor T cells that maintain self tolerance to organ-specific self antigens. The recent progress in infectious disease, autoimmunity and transplantation has stimulated a gratifying renaissance of interest in the vitamin D endocrine system and its role in immunological health.

Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures.

Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7, P <.04). The maximal rate of DNA synthesis was reached at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a significant increase of DNA synthesis was detected already at doses of 1 ng/mL (P =.03). HuCD40LT never inhibited the basal level of DNA synthesis. These findings established 400 ng/mL of huCD40LT for 4 days as standard conditions in the system. Under these conditions, huCD40LT significantly increased the proportion of cells in the S/G(2)/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged (n = 7). HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2, and CD95/Fas and up-regulated the expression of HLA-DR (n = 6). With the use of bromodeoxyuridine incorporation in triple-color flow cytometric analysis, it was found that huCD40LT induced cell-cycle progression in light chain-restricted cells only, of which a median of 14% (range, 0.5% to 29%; n = 4) returned to G(0/1) phase DNA content after bromodeoxyuridine incorporation, demonstrating completion of at least one cell cycle in the presence of huCD40LT. Thus, primary clonal MCL cells are activated and can proliferate in the presence of huCD40LT as a single agent.

Analysis of the function of a putative 2,3-diphosphoglyceric acid-dependent phosphoglycerate mutase from Bacillus subtilis.

A Bacillus subtilis gene termed yhfR encodes the only B. subtilis protein with significant sequence similarity to 2, 3-diphosphoglycerate-dependent phosphoglycerate mutases (dPGM). This gene is expressed at a low level during growth and sporulation, but deletion of yhfR had no effect on growth, sporulation, or spore germination and outgrowth. YhfR was expressed in and partially purified from Escherichia coli but had little if any PGM activity and gave no detectable PGM activity in B. subtilis. These data indicate that B. subtilis does not require YhfR and most likely does not require a dPGM.

Characterization of ywhE, which encodes a putative high-molecular-weight class A penicillin-binding protein in Bacillus subtilis.

The Bacillus subtilis genome sequencing project [Kunst et al., Nature 390 (1997) 249-256] identified ywhE as a gene that potentially encodes a high-molecular-weight class A penicillin-binding protein. Analysis of the expression of a translational ywhE-lacZ fusion showed that ywhE expression is sporulation-specific, and is controlled predominantly by the forespore-specific sigma factor sigma(F), and to a lesser extent by sigma(G). Primer extension analysis identified two transcription start sites located 26 and 27 nucleotides upstream of the ywhE translational initiation codon. Sequences located in the -10 and -35 regions relative to the transcription start sites showed good homology to the consensus sequences for promoter elements of sigma(F)-dependent genes. An insertional mutation in ywhE had no significant effect on growth, morphology, and sporulation, and ywhE spores had normal heat-resistance, cortex structure, and germination and outgrowth properties. However, overexpression of ywhE in Escherichia coli resulted in cell lysis.

Penicillin-binding protein-related factor A is required for proper chromosome segregation in Bacillus subtilis.

Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI = 10.1) with no significant sequence homology to proteins with known functions. Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of approximately 0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37 degrees C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells. In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli.

Septal localization of penicillin-binding protein 1 in Bacillus subtilis.

Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis.

The pbp gene (renamed dacC), identified by the Bacillus subtilis genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional dacC-lacZ fusion revealed that dacC expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor sigmaH; and (iii) appears to be initiated from a promoter located immediately upstream of yoxA, a gene of unknown function located upstream of dacC on the B. subtilis chromosome. A B. subtilis dacC insertional mutant grew and sporulated identically to wild-type cells, and dacC and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics. Expression of dacC in Escherichia coli showed that this gene encodes an approximately 59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.

Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species.

Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of the zymogen, (ii) the rate of zymogen autoactivation, or (iii) the unreactivity of the zymogen's single SH group. Removal of 13 amino-terminal residues resulted in a partially active enzyme whose SH group was as reactive as the fully active enzyme. These findings suggest that at least a part of the propeptide blocks access to the enzyme's active site. However, the free propeptide did not inhibit the enzyme.

Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1.

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro. Our results imply that DNA condensation is not the only function of Hc2.

The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.

Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro.

The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single-stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant concentrations. These results were found to coincide with the formation of condensed Hc1-DNA and Hc1-RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1 in vivo are discussed.