Identification of BRaf-Sparing Amino-Thienopyrimidines with Potent IRE1α Inhibitory Activity

Amino-quinazoline BRaf kinase inhibitor 2 was identified from a library screen as a modest inhibitor of the unfolded protein response (UPR) regulating potential anticancer target IRE1α. A combination of crystallographic and conformational considerations were used to guide structure-based attenuation of BRaf activity and optimization of IRE1α potency. Quinazoline 6-position modifications were found to provide up to 100-fold improvement in IRE1α cellular potency but were ineffective at reducing BRaf activity. A salt bridge contact with Glu651 in IRE1α was then targeted to build in selectivity over BRaf which instead possesses a histidine in this position (His539). Torsional angle analysis revealed that the quinazoline hinge binder core was ill-suited to accommodate the required conformation to effectively reach Glu651, prompting a change to the thienopyrimidine hinge binder. Resulting analogues such as 25 demonstrated good IRE1α cellular potency and imparted more than 1000-fold decrease in BRaf activity.     

Histone methyltransferase Suv39h1 deficiency prevents Myc‐induced chromosomal instability in murine myeloid leukemias

Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild‐type and histone methyltransferase Suv39h1‐deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc‐overexpressing wild‐type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end‐to‐end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc‐transduced Suv39h1‐deficient cells showed less critically short telomeres (P < 0.05) compared with Myc‐transduced wild‐type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc. © 2013 Wiley Periodicals, Inc.     

Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor [see comments]

Peripheral blood stem cells (PBSCs) are widely used in autologous transplantation because of ease of collection and rapid hematopoietic reconstitution. However, PBSCs have rarely been used for allogeneic transplantation because of concerns about donor toxicities from cytokine administration and the theoretical increased risk of graft- versus-host-disease (GVHD) from the large number of T cells infused. Eight patients with advanced malignancies received allogeneic PBSC transplants from genotypically HLA-identical sibling donors. All donors received 5 days of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 16 micrograms/kg/day) subcutaneously and were leukapheresed for 2 days. After treatment of the patient with total body irradiation and cyclophosphamide (n = 7) or etoposide, thiotepa, and cyclophosphamide (n = 1), PBSCs were infused immediately after collection and without modification. All patients received cyclosporine and either methotrexate (n = 6) or prednisone (n = 2) for GVHD prophylaxis, rhG-CSF was well tolerated with mild bone pain requiring acetaminophen occurring in two donors. All patients engrafted and in seven hematopoietic recovery was rapid, with 500 neutrophils/microL achieved by day 18 and 20,000 platelets/microL by day 12. Complete donor engraftment was documented by Y chromosome analysis in all four sex-mismatched donor-recipient pairs tested and by DNA analysis in two sex-matched pairs. One patient died on day 18 of veno-occlusive disease of the liver with engraftment but before chromosome analysis could be performed (results are pending in 1 patient). A second patient died of fungal infection 78 days after transplant. Grade 2 acute GVHD occurred in two patients and grade 3 GVHD occurred in one patient. One patient is 301 days from transplant in remission with chronic GVHD; the remaining five patients are alive and disease free 67 to 112 days after transplantation. Preliminary results indicate that allogeneic PBSCs mobilized by rhG-CSF can provide rapid hematologic recovery without an appreciably greater incidence of acute GVHD than would be expected with marrow. Further follow-up is required to determine the incidence of chronic GVHD and any potential beneficial effects on relapse after transplant.     

Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation

Allogeneic peripheral blood stem cell (PBSC) transplants from HLA- identical siblings were performed in 37 patients with advanced hematologic malignancies. Outcomes were compared to a historical group of 37 similar patients with advanced hematologic malignancies receiving bone marrow (BM) transplants from HLA-identical donors. The PBSC group and historical BM group were well matched for diagnosis, disease stage, age, and graft-versus-host disease (GVHD) prophylaxis. Patients received PBSC transplants between 1993 to 1995 while BM patients were treated between 1989 to 1994. Engraftment, measured by the time to reach a peripheral neutrophil count > 500/L and platelet count > 20,000/microL without transfusions, occurred on days 14 and 11 in the patients transplanted with PBSC compared to days 16 and 15 in the patients receiving BM (P = .00063, .00014). The PBSC group required a median of 8 U of red blood cells and 24 U of platelets compared to 17 U of red blood cells and 118 U of platelets for BM transplant recipients (P = .0005, .0001). The estimated risks of developing grades 2 to 4 acute GVHD were 37% for the PBSC group and 56% for the BM group (P = .18), while the estimated risks of grades 3 to 4 acute GVHD were 14% for the PBSC group and 33% for the BM group, P = .05). Chronic GVHD occurred in 7 of 18 evaluable patients receiving PBSC and 6 of 23 evaluable patients receiving BM, P = .5. The estimated risks of transplant-related mortality at 200 days were 27% versus 45% (P = .33) relapse were 70% versus 53% (P = .27) and of overall survival were 50% and 41% (P = .39) for patients transplanted with PBSC or BM, respectively. This retrospective comparison suggests that compared to marrow transplantation from HLA-identical donors, allogeneic PBSC transplantation from HLA-identical donors is associated with faster engraftment, fewer transfusions, and no greater incidence of acute or chronic GVHD.     

Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines

Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.     

Community-onset methicillin-resistant Staphylococcus aureus associated with antibiotic use and the cytotoxin Panton-Valentine leukocidin during a furunculosis outbreak in rural Alaska

BACKGROUND: Community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) reports are increasing, and infections often involve soft tissue. During a CO-MRSA skin infection outbreak in Alaska, we assessed risk factors for disease and whether a virulence factor, Panton-Valentine leukocidin (PVL), could account for the high rates of MRSA skin infection in this region. METHODS: We conducted S. aureus surveillance in the outbreak region and a case-control study in 1 community, comparing 34 case patients with MRSA skin infection with 94 control subjects. An assessment of traditional saunas was performed. S. aureus isolates from regional surveillance were screened for PVL genes by use of polymerase chain reaction, and isolate relatedness was determined by use of pulsed-field gel electrophoresis (PFGE). RESULTS: Case patients received more antibiotic courses during the 12 months before the outbreak than did control subjects (median, 4 vs. 2 courses; P=.01) and were more likely to use MRSA-colonized saunas than were control subjects (44% vs. 13%; age-adjusted odds ratio, 4.6; 95% confidence interval, 1.7-12). The PVL genes were present in 110 (97%) of 113 MRSA isolates, compared with 0 of 81 methicillin-susceptible S. aureus isolates (P<.001). The majority of MRSA isolates were closely related by PFGE. CONCLUSION: Selective antibiotic pressure for drug-resistant strains carrying PVL may have led to the emergence and spread of CO-MRSA in rural Alaska.     

Pathophysiologic and diagnostic aspects of heart failure

Ventricular dysfunction due to an abnormality of the heart which is associated with typical hemodynamic, renal and hormonal reactions, characterizes the clinical syndrome heart failure. The traditional definition of heart failure as the inability to pump an amount of blood sufficient to cover the metabolic needs of the body in the presence of adequate venous return, emphasizes mainly the reduction in cardiac output but not the increase in intracardiac pressures. Pressure or volume overload, decreased contractility, loss of muscle mass or restricted filling represent the most important pathological processes leading to heart failure. The disturbance of systolic ventricular function due to pressure or volume overload or diminished contractility is characterized by a decrease in the ejection fraction, the disturbance in diastolic ventricular function associated with restricted filling is characterized by elevated chamber stiffness. Decreased contractility is most commonly responsible for the development of heart failure. Impairment of diastolic ventricular function can only be regarded as the dominant mechanism leading to heart failure in the presence of a small noncompliant ventricle. Impairment of diastolic ventricular function in an enlarged heart is always associated with an impairment of systolic ventricular function and is, then, relegated to a subordinate role. Common causes of heart failure are coronary artery disease, hypertension, cardiomyopathies, valvular heart diseases and congenital heart diseases, for the incidence of which coronary artery disease is most frequently responsible. Most of these diseases lead to heart failure not via a single, but rather several of the specified pathophysiological processes. Possible mechanisms for loss of contractility include structural changes as well as alterations in excitation-contraction coupling. Possible mechanisms responsible for impaired diastolic ventricular function encompass, in addition to altered calcium flux, structural changes such as fibrosis and hypertrophy and factors such as asynchrony and abnormal loading conditions. With increasing derangement of cardiac function, there is recruitment of the compensatory mechanisms: hypertrophy of the cardiac muscle, Frank-Starling mechanism, activation of the sympathetic nervous system, the renin-angiotensin-aldosterone system and the arginine-vasopressin system. The goal is maintenance of adequate blood pressure and cardiac output whereby blood flow is redistributed in favor of the heart and brain and away from the skin, musculature and visceral organs. Activation of the neurohumoral system can lead to excessive vasoconstriction as well as sodium and water retention resulting in an undesired elevation of preload and afterload which, in turn, leads to further worsening of the heart failure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Die Diagnose des Vorhofseptumdefektes aus phonokardiographischen und auskultatorischen Befunden

Ohne ZusammenfassungMit 20 Abbildungen in 22 Einzeldarstellungen und 1 Tabelle     

K-ras mutations in the bile of patients with primary sclerosing cholangitis

BACKGROUND AND AIMS: The development of cholangiocarcinoma (CCC) is a complication of primary sclerosing cholangitis (PSC). To date, no reliable factors have been described which can define those PSC patients at high risk for the development of CCC and the clinical diagnosis of CCC in PSC patients is difficult. Therefore, molecular markers of cholangiocarcinogenesis, such as K-ras mutations, may improve the early diagnosis of CCC or the timing of liver transplantation. METHODS: K-ras mutations were analysed by enriched polymerase chain reaction/restriction fragment length polymorphism in the bile fluid of 56 PSC patients and 20 patients with other cholestatic diseases. To assess the value of K-ras mutations as a risk factor for cholangiocarcinogenesis, patients were prospectively investigated over a mean period of 31.5 months. RESULTS: In contrast with the control group, 17 (30%) patients with PSC revealed K-ras mutations in bile fluid. The mean Mayo score was not significantly different between PSC patients with (mean score 0.70) and without (mean score 0.13; p=0.2) K-ras mutations. In contrast with the group of PSC patients without K-ras mutations, four CCCs and two dysplasia were diagnosed in the group of patients with K-ras mutations during the follow up investigation (p<0.001). CONCLUSIONS: Our results indicate that K-ras mutations in bile fluid of PSC patients represent frequent early events during cholangiocarcinogenesis. However, most of the PSC patients with K-ras mutations remained tumour free after a long follow up investigation which is in agreement with the fact that these mutations are not specific for malignancy but may also occur in normal bile duct mucosa or in dysplasias. Therefore, analysis of K-ras mutations in bile should not be used for diagnosis of CCC in PSC patients. However, the results of our prospective follow up investigation indicate that K-ras mutations in bile fluid of PSC patients have to be considered as risk factors for the development of CCC which may have implications for the timing of liver transplantation.