JAK2T875N is a novel activating mutation that results in myeloproliferative disease with features of megakaryoblastic leukemia in a murine bone marrow transplantation model

Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia associated with a poor prognosis. However, there are relatively few insights into the genetic etiology of AMKL. We developed a screening assay for mutations that cause AMKL, based on the hypothesis that constitutive activation of STAT5 would be a biochemical indicator of mutation in an upstream effector tyrosine kinase. We screened human AMKL cell lines for constitutive STAT5 activation, and then used an approach combining mass spectrometry identification of tyrosine phosphorylated proteins and growth inhibition in the presence of selective small molecule tyrosine kinase inhibitors that would inform DNA sequence analysis of candidate tyrosine kinases. Using this strategy, we identified a new JAK2T875N mutation in the AMKL cell line CHRF-288-11. JAK2T875N is a constitutively activated tyrosine kinase that activates downstream effectors including STAT5 in hematopoietic cells in vitro. In a murine transplant model, JAK2T875N induced a myeloproliferative disease characterized by features of AMKL, including megakaryocytic hyperplasia in the spleen; impaired megakaryocyte polyploidization; and increased reticulin fibrosis of the bone marrow and spleen. These findings provide new insights into pathways and therapeutic targets that contribute to the pathogenesis of AMKL.     

Amino acid variations in hepatitis C virus p7 and sensitivity to antiviral combination therapy with amantadine in chronic hepatitis C

BACKGROUND: Formation of transmembrane ion channels by hepatitis C virus (HCV) p7 and abrogation of channel function by amantadine was demonstrated in vitro. The relevance of HCV p7 amino acid (aa) variations for response to antiviral therapy with amantadine is unknown. METHODS: HCV p7 was sequenced in 86 individuals who were infected with HCV genotype 1. Thirty-six of 86 patients received amantadine within an interferon-alpha (IFN-alpha)-based antiviral therapy. Helical wheel modelling for HCV p7 was performed. RESULTS: No significant correlation of overall aa variations within HCV p7 was observed with response to IFN-alpha-based therapy with amantadine in HCV genotype 1alpha/b infected patients. When analysis was restricted to non-conservative aa variations, a higher number of aa substitutions within complete HCV p7 and transmembrane helix 2 was associated with non-response in HCV-1b-infected patients receiving therapy with amantadine (P=0.015 and P=0.037, respectively), without amantadine (P=0.106 and P=0.118, respectively), and in the total cohort of HCV-1b-infected patients (P=0.00007 and P=0.011, respectively). Furthermore, substitution L20F was observed more often in non-responders than responders with HCV-1b infection and therapy with amantadine (P=0.099). By in silico modelling, aa 20 was located toward the p7 channel lumen. Substitution L20F may impair amantadine action by altering the shape of the ion channel pore. CONCLUSION: Substitution L20F within HCV p7 may be associated with non-response to combination therapy specifically with amantadine in HCV-1b-infected patients. Non-responders with HCV-1b infection showed higher numbers of non-conservative aa variations within HCV p7 than responders, irrespective of the application of amantadine.     

Erosive and inflammatory joint changes in hereditary hemochromatosis arthropathy detected by low-field magnetic resonance imaging

The aim of the article is to describe and characterize the hemochromatosis arthropathy of the hand by low-field MRI in symptomatic and asymptomatic patients. Forty-nine patients with hereditary hemochromatosis (37 with and twelve without arthropathy) were examined clinically and by low-field MRI of the hands. The examination showed heterogeneous degenerative and inflammatory joint changes such as erosions (in 84 % of all symptomatic patients), synovitis (77 %), bone marrow edema (38 %), subchondral cysts (30 %), tenosynovitis (30 %), joint space narrowing (73 %) and osteophytes (59 %) including hook-shaped osteophytes at MCP joints (32 %). Mild joint changes were also seen in a lower percentage of asymptomatic patients. This is the first larger study addressing the joint changes of the hand in hereditary hemochromatosis using low-field MRI. Our study emphasizes the inflammatory and destructive character of the arthropathy besides the well-known degenerative joint changes described in conventional X-ray. The impact of joint changes in asymptomatic patients deserves further investigation.     

Internal quality control of histological diagnosis in pathology. A nine-year experience

Internal quality control (IQC) is a necessary component of total quality management. We report our experience with an internal audit scheme focusing on the histological diagnosis. We outline other strategies of IQC and analyze the causes of errors and ways to prevent them. Some practical guidelines to initiate this type of procedure are presented. Our audit was designed to check the accuracy of diagnosis, the clarity and completeness of the report, the quality of the documents leading to the diagnosis, and the turn-around time. It consisted of a retrospective analysis of 4185 randomly selected cases (representing 2% of all cases), over nine years. The control took place once a week and was done by two pathologists working as a team. The mean time spent by each pathologist was 45 minutes per week. Errors were scored using a 3-level grading scheme depending on their potential harm or impact on patient care. The overall rate of errors was 1.1%, and 0.1% of errors were potentially harmful to the patients. A single case (0.02%), in which a cancer was missed, had a real impact on patient care. Retrospective analysis of randomly selected cases mirrors the overall activity of a surgical pathology department. Nevertheless, each lab has to develop its own strategy of IQC, based on its size, its functioning, and its objectives. Although it may be difficult to initiate quality assurance when medical time is already limited, it is a helpful procedure in a more and more demanding medical and societal context and a pragmatic step towards "culture of quality".     

Disturbed granulocyte macrophage-colony stimulating factor priming of phosphatidylinositol 3,4,5-trisphosphate accumulation and Rac activation in fMLP-stimulated neutrophils from patients with myelodysplasia

The production of reactive oxygen species (ROS) by human neutrophils is imperative for their bactericidal activity. Proinflammatory agents such as granulocyte macrophage-colony stimulating factor (GM-CSF) can prime ROS production in response to chemoattractants such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In neutrophils from patients suffering from Myelodysplastic syndromes (MDS), a clonal, hematological disorder characterized by recurrent bacterial infections, this GM-CSF priming is severely impaired. In this study, we set out to further delineate the defects in neutrophils from MDS patients. We examined the effect of GM-CSF priming on fMLP-triggered activation of Rac, a small GTPase implicated in neutrophil ROS production. In contrast to healthy neutrophils, activation of Rac in response to fMLP was not enhanced by GM-CSF pretreatment in MDS neutrophils. Furthermore, activation of Rac was attenuated by pretreatment of neutrophils with the phosphatidylinositol 3-kinase (PI-3K) inhibitor LY294002. Unlike healthy neutrophils, fMLP-induced accumulation of the PI-3K lipid product PI(3,4,5)trisphosphate was not increased by GM-CSF pretreatment in MDS neutrophils. The disturbed Rac and PI-3K activation observed in MDS neutrophils did not appear to reflect a general GM-CSF or fMLP receptor-signaling defect, as fMLP-triggered Ras activation could be primed by GM-CSF in MDS and healthy neutrophils. Moreover, fMLP-induced activation of the GTPase Ral was also normal in neutrophils from MDS patients. Taken together, our data suggest that in neutrophils from MDS patients, a defect in priming of the PI-3K-Rac signaling pathway, located at the level of PI-3K, results in a decreased GM-CSF priming of ROS production.     

HLA–D and –DR antigens on human amniotic fluid cells

Human amniotic fluid cells, known to express HLA-A, -B, and -C antigens, were tested for the presence of lymphocyte-stimulating antigens (LD or HLA-D) using modifications of the mixed lymphocyte culture (MLC) and primed lymphocyte typing (PLT) tests. Peripheral blood lymphocytes were co-cultured with various concentrations of allogeneic amniotic fluid cells, either growing as a monolayer culture in microtiter plates or suspended in medium following treatment with trypsin. The kinetics of such mixed lymphocyte amniotic fluid cell culture (MLAC) reactions were followed during days 3 to 8. Under none of these conditions did amniotic fluid cells significantly stimulate allogeneic lymphocytes, even after lymphocytes were specifically primed in the PLT assay to the HLA-D antigens segregating in the family of the amniotic fluid cell donor. Furthermore, in three-cell experiments, amniotic fluid cells failed to inhibit an ongoing MLC reaction, indicating that the absence of proliferative response to amniotic fluid cells is not due to active suppression. Taken together, these data strongly suggest that amniotic fluid cells either do not express HLA-D antigens or do not express them in a form that is detectable in either primary or secondary MLC.     

Olanzapine treatment during breast feeding: a case report

Postpartum psychosis constitutes a severe complication that entails risk for both mother and child. Little is known about the use of olanzapine in the treatment of postpartum psychosis. In previous studies, it has been reported on mothers receiving relatively low doses of olanzapine. We report a 38-year-old patient who was admitted to the hospital for an acute psychotic exacerbation. She was breast feeding her 5-month-old child, and she wished to continue breast feeding. Olanzapine treatment was started with a daily dosage of 15 mg. The weight-corrected maternal dose was 270 mug/kg. The olanzapine concentration in the mother's plasma was 24 ng/mL. The analysis of olanzapine in breast milk applying two different high-performance liquid chromatography procedures revealed similar results: 12.2 ng/g without and 11.5 ng/g with additional hydrochloric acid extraction, respectively. In addition, breast milk of an unmedicated mother was used for establishing the analytical procedure so that the validity of the results was better confirmed. The milk-plasma ratio arising from our data was 0.5, and the relative infant dose was 0.3%. The olanzapine concentration was below the limit of detection (<5 ng/mL) in the infant's plasma sample. No adverse effects were noticed, and the mother experienced a rapid improvement in her psychopathology during her hospital stay. In future studies, long-term follow-up of both mother and child would be useful.     

Are residents of high-walkable areas satisfied with their neighbourhood?

Aim

While the association between walkability and walking for transport has been well established, less is known about the association between walkability and neighbourhood satisfaction. This study aims to examine the direction and strength of the association between objective measures of residential walkability and neighbourhood satisfaction, as well as the differences by sex.

Subjects and methods

Using a cross-sectional study design, outcome data were derived from the representative cross-sectional survey (n?=?843) ‘Bicycle-friendly City’ of adults in the city of Graz (Austria). Walkability was measured as gross population density, household unit density, entropy index, proportion of mixed land use, three-way intersection density, four-way intersection density and walkability indices. The outcomes were measured as general neighbourhood satisfaction and neighbourhood satisfaction with the general socio-environmental quality, social cohesion and local infrastructure. Logistic regression analyses were conducted, including age, socio-economic status and place of residence.

Results

Walkability was negatively associated with general neighbourhood satisfaction, neighbourhood satisfaction with general socio-environmental quality and social cohesion. It was positively associated with neighbourhood satisfaction with local infrastructure. Connectivity and the entropy index showed the weakest or no association with the outcomes. The strongest association was between walkability and neighbourhood satisfaction with socio-environmental quality. There were no differences by sex.

Conclusion

These results contribute to the current limited understanding of the association between walkability and neighbourhood satisfaction, especially in a European context. More comparable, longitudinal research would be helpful to determine what impact walkability has on neighbourhood satisfaction and to identify the important mediating factors.