A phase I study of the combination of ro4929097 and cediranib in patients with advanced solid tumours (PJC-004/NCI 8503).

BACKGROUND: The Notch signalling pathway has been implicated in tumour initiation, progression, angiogenesis and development of resistance to vascular endothelial growth factor (VEGF) targeting, providing a rationale for the combination of RO4929097, a γ-secretase inhibitor, and cediranib, a VEGF receptor tyrosine kinase inhibitor. METHODS: Patients received escalating doses of RO4929097 (on a 3 days-on and 4 days-off schedule) in combination with cediranib (once daily). Cycle 1 was 42 days long with RO4929097 given alone for the first 3 weeks followed by the co-administration of both RO4929097 and cediranib starting from day 22. Cycle 2 and onwards were 21 days long. Soluble markers of angiogenesis were measured in plasma samples. Archival tumour specimens were assessed for expression of three different components of Notch signalling pathway and genotyping. RESULTS: In total, 20 patients were treated in three dose levels (DLs). The recommended phase II dose was defined as 20 mg for RO4929097 on 3 days-on and 4 days-off schedule and 30 mg daily for cediranib. The most frequent treatment-related adverse events (AEs) were diarrhoea, hypertension, fatigue and nausea. Eleven patients had a best response of stable disease and one patient achieved partial response. We did not detect any correlation between tested biomarkers of angiogenesis or the Notch pathway and treatment effect. There was no correlation between mutational status and time to treatment failure. CONCLUSION: RO4929097 in combination with cediranib is generally well tolerated at the DLs tested. Preliminary evidence of antitumour efficacy with prolonged disease stabilisation in some patients with progressive malignancies warrants further clinical investigation of this treatment strategy.

Particulate matter strongly associated with human Q fever in The Netherlands: an ecological study.

There are still questions about the importance of different animal reservoirs and environmental factors that played a role in the large Q fever epidemic in The Netherlands. We therefore investigated the spatial association between reported Q fever cases and different livestock and environmental factors at the national level. A spatial regression analysis was performed, with four-digit postal code areas as the unit of analysis. High level of particulate matter (< 24.5 µg/m³) with an aerodynamic diameter <10 µm (PM10) was by far the strongest risk factor for human Q fever with an odds ratio of 10.4 (95% confidence interval 7.0-15.6) using PM10 <24.5 µg/m³ as reference, in logistic regression analysis, controlling for differences in animal densities, vegetation and other risk factors. Particulate matter seems to play an important role in the transmission of Q fever from infected animals to humans and should be a focus for further studies on zoonotic infectious diseases and decision-making.

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.

The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.

Cellular stress induces TRB3/USP9x-dependent Notch activation in cancer.

Expression of the Notch ligand JAG1 and Notch pathway activation promote poor prognosis, basal-like breast cancer. We have recently shown that the pseudokinase Tribbles homolog 3 (TRB3) regulates JAG1 expression in this malignancy. TRB3 is a stress and metabolic sensor, and here we show that nutrient deprivation or endoplasmic reticulum stress markedly upregulate TRB3, which serves as a scaffold for the deubiquitinase USP9x. USP9x in turn stimulates JAG1 activity through two mechanisms: (1) through TRB3 deubiquitination and stabilization, and (2) through deubiquitination and activation of Mind Bomb 1, an E3 ligase required for JAG1 ubiquitination-mediated endocytosis and Notch activation. These USP9x activities are confined to the signal-sending cell of a cell pair undergoing Notch signaling. We demonstrate that USP9x is required for TRB3 upregulation and Notch activation in response to cellular stress in basal-like breast cancer cells. These data suggest that TRB3 functions as a sensor of tumor microenvironmental stress and together with USP9x induces the cell survival and tumor-promoting activities of Notch. These findings identify a novel mechanism by which cancer cells survive in their hostile environment and provide potential therapeutic targets in breast cancer.

CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein.

CaLB was originally observed as a conserved sequence motif in various calcium-responsive signalling proteins and also in p120 Ras GTPase activating protein (p120GAP) (Clark et al. Cell 65: 1043-1051, 1991). Here we show the 43 residue CaLB motif in p120GAP is a functional protein domain that when expressed as a fusion protein in vitro confers Ca(2+)-dependent interactions with cellular membranes and phosphatidylserine and phosphatidylinositol vesicles. p120GAP, but not a mutant lacking the CaLB domain, associates with the particulate fraction of cells in response to elevated intracellular Ca2+ suggesting that p120GAP may be regulated in part by calcium signals. Addition of the p120GAP CaLB domain was able to restore transforming activity and particulate localization to an otherwise transformation-defective and cytosolic mutant v-Sre tyrosine kinase. The CaLB domain appears to be a prevalent protein module that may affect the molecular interactions and subcellular localization of signalling proteins.

Replacing ritonavir by nelfinavir or nelfinavir/saquinavir as part of highly active antiretroviral therapy leads to an improvement of triglyceride levels.

In a randomized, parallel arm, open-label study, the effect of switching from ritonavir to either nelfinavir or nelfinavir plus saquinavir as part of a triple antiretroviral regimen was investigated in 16 patients with undetectable HIV-1 loads. Patients continued to use the same nucleoside reverse transcriptase inhibitors as before the switch of protease inhibitor. The period of follow-up was 48 weeks. In all patients HIV-1 load remained undetectable, whereas CD4 cell counts remained stable. Furthermore, lipid markers improved after the switch.

The value of Doppler echocardiography in the management of patients with valvular heart disease: analysis of one year of clinical practice.

Management recommendations based on Doppler echocardiographic examination and cardiac catheterization were compared in a prospective study in 100 consecutive patients who were admitted for evaluation and treatment of suspected valvular heart disease during 1988. Management recommendations were provided independently after both Doppler echocardiography and cardiac catheterization by different and blinded investigators. Criteria for severe (clinically significant) and moderate to mild (insignificant) valvular lesions and management recommendations were agreed on in advance. There was disagreement on the severity of aortic stenosis based on the aortic valve area and maximum instantaneous pressure gradient in 1 of 54 patients, which resulted in differing management recommendations. Mitral stenosis was severe (valve area less than or equal to 1 cm2) at Doppler echocardiography but not at cardiac catheterization in 5 of 14 patients. Because pulmonary artery pressure increase during exercise at cardiac catheterization also suggested severe obstruction, management recommendations were similar. There was a potentially significant disagreement on the severity of aortic regurgitation in 9 of 76 patients and of mitral regurgitation in 14 of 90 patients; however, this did not produce differing management recommendations because with most patients coexistent valvular lesions or an impaired ventricular function mainly determined the ultimate management decision. Although of good quality, Doppler echocardiographic examination was nonconclusive for clinical decision-making in 15% of the study population because of uncertainty about the severity of mitral regurgitation or aortic regurgitation or because of problems in assessing the degree of left ventricular dysfunction in patients with severe regurgitation.(ABSTRACT TRUNCATED AT 250 WORDS)

When do HIV-infected persons start with antiretroviral therapy? A retrospective analysis of patients' monitoring and treatment status in general practice, as compared with the 1991 Dutch HIV treatment guidelines.

OBJECTIVE: We aimed to compare, in a sample of Amsterdam general practices, the monitoring and treatment status of HIV-infected patients according to the 1991 Dutch consensus guidelines for antiretroviral treatment of HIV-infection, which advise that therapy be started at a peripheral blood CD4+ cell count of < or = 300 x 10(6)/l in asymptomatic patients, or < or = 400 x 10(6)/l in symptomatic patients. METHOD: In 1994, data were collected from the records of all 511 HIV-infected patients registered in 14 Amsterdam general practices (20 doctors). The main outcome measures were the antiretroviral treatment status of all patients who were eligible for treatment, and the disease stage and CD4+ cell counts at the onset of therapy for patients who started treatment after publication of the 1991 guidelines. RESULTS: For 472 patients, data were available on CD4+ cell measurement status and disease stage. For 15.9% of patients, CD4+ cells had never been measured; most of them were asymptomatic. In 84.1 % of patients, CD4+ cells had been measured. Of the 8.9% of patients whose results were not known to GPs, 93% were treated by a specialist and 76% were symptomatic. Of the remaining 355 (75.2%) patients whose CD4+ count and disease status were known, 201 (56.7%) met the guideline criteria for treatment. Of these, 53.7% received treatment, 27.4% were never treated and 18.9% had discontinued treatment. Of the 67 patients who started treatment after publication of the guidelines, 36.2% of asymptomatic patients and 92.8% of symptomatic patients started later than the guidelines advised. CONCLUSION: In the population studied, we found a discrepancy between the 1991 treatment guidelines and the actual situation. In a substantial proportion of eligible patients, antiretroviral treatment was either not administered at all or was administered at a (very) late disease stage. This can only be attributed to physicians' and/or patients' attitudes towards antiretroviral treatment. Other studies confirm that a number of psychological factors may influence treatment decisions. The new combination treatment of HIV-infection requires an early start and compliance with the guidelines. The degree to which doctors and patients are willing and able to comply with the guidelines is an important factor to be taken into account, both in research and in the development of guidelines.

Changing attitudes towards antiretroviral treatment of HIV infection: a prospective study in a sample of Dutch general practitioners.

This study investigated the attitude towards antiretroviral therapy (ART) for HIV infection over time, in a sample of Dutch general practitioners (GPs). Twenty-one GPs, of which 16 were practising in Amsterdam, completed multiple questionnaires on HIV-related topics between April 1995 and March 1997. In 1995, only 10% had a positive attitude towards treatment of asymptomatic persons with a CD4+ > 300 x 10(6)/l; at the end of the study 43% had. In 1995, 57% had a positive attitude towards treatment of asymptomatic persons with a CD4+ < or = 300 x 10(6)/l, and 52% towards treatment of symptomatic patients with a CD4+ < or = 400 x 10(6)/l; heterosexual GPs more often had a positive attitude as compared to homosexual GPs (p = 0.005 and p = 0.01, respectively). At the end of the study the proportions of GPs with a positive attitude had increased from 57 to 81% and 52 to 95%, respectively. The risk of adverse effects, strict dose regimens and medicalization were regarded as the main disadvantages of the current treatment strategy. The conclusion is that the attitude towards ART has become more positive since 1995. At the beginning of 1997, however, there still existed reservations about treatment of asymptomatic persons with CD4+ cell counts > 300 x 10(6)/l.

The c-fms gene complements the mitogenic defect in mast cells derived from mutant W mice but not mi (microphthalmia) mice.

Mutations at three loci in the mouse--W, Steel Sl), and microphthalmia (mi)--can lead to a deficiency in melanocytes and mast cells. As well, W and Sl mutants can be anemic and sterile, whereas mi mice are osteopetrotic due to a monocyte/macrophage defect. Recent data have shown that the c-kit receptor tyrosine kinase is the gene product of the W locus, whereas Sl encodes the ligand for this growth factor receptor. We show here that ectopic expression of c-fms, a gene that encodes a macrophage growth factor receptor that is closely related to the c-kit receptor, complements mutations at the W locus in an in vitro mast cell/fibroblast coculture system but is unable to reverse the inability of mi/mi mast cells to survive under these conditions. Furthermore, mast cells expressing the c-fms receptor survive on a monolayer of fibroblasts homozygous for the Sl mutation. These results suggest that ligand binding to the c-kit or c-fms receptor activates identical or overlapping signal transduction pathways. Furthermore, they suggest that mi encodes a protein necessary for transducing signals mediated by way of either the c-kit or c-fms receptor.

The unique insert of cellular and viral fms protein tyrosine kinase domains is dispensable for enzymatic and transforming activities.

The receptors for colony stimulating factor-1 (CSF-1), platelet derived growth factor and the c-kit protein tyrosine kinase (PTK) contain within their catalytic domains a stretch of 60-100 residues, largely unrelated in sequence, with no counterpart in other PTKs. Of the 64 amino acids within this kinase insert, 58 were deleted from the mouse CSF-1 receptor by oligonucleotide-directed mutagenesis. The mutant CSF-1 receptor was not markedly affected in its kinase activity, post-translational processing or its ability to induce autocrine transformation of NIH 3T3 mouse fibroblasts. Similarly, retention of kinase and transforming activities were observed following deletion of part or all of the kinase insert from the v-fms oncoprotein. The c- and v-fms kinase inserts were probed using monoclonal and polyclonal antibodies and were found to be highly antigenic. Two monoclonal antibodies raised to the v-fms cytoplasmic domain both recognized epitopes within the insert, and bound enzymatically active v-fms glycoproteins. These results indicate that the fms kinase insert is located on the surface of the protein and folds separately from the rest of the catalytic domain, but is not required for the biological activity of fms PTKs ectopically expressed in mouse fibroblasts. The insert may therefore play a specific function in cells such as monocytes and trophoblasts that normally express the CSF-1 receptor.

Bowel resection for intestinal endometriosis.

PURPOSE: The study contained herein was undertaken to evaluate which factors predict a good outcome following intestinal resection for endometriosis. METHODS: A retrospective analysis of all patients undergoing bowel resection for severe (American Fertility Society Stage IV) endometriosis at one institution between the years 1992 and 1996 was conducted using systematic chart review and follow-up by telephone interview. RESULTS: Twenty-nine patients were identified within the study period. The most frequent symptoms were pelvic pain, abdominal pain, rectal pain, and dysmenorrhea. Nearly all patients (93 percent) underwent low anterior resection of the rectum and distal sigmoid. Other intestinal procedures were appendectomy, terminal ileal resection, cecectomy, and sigmoid resection. Thirty-four percent of patients had simultaneous total abdominal hysterectomy and bilateral salpingooophorectomy. Complete follow-up was obtained on 26 patients (90 percent; mean follow-up 22.6 (range, 8-63) months). All patients (100 percent) reported subjective improvement. Forty-six percent of patients were "cured" according to the prospectively applied definition (resolution of symptoms without need for further medical or surgical therapy). The only variable analyzed that was associated with "cure" was concomitant total abdominal hysterectomy and bilateral salpingooophorectomy (odds ratio, 12; 95 percent confidence interval, 1.8-81.7). This association remained significant after correcting for age and the presence of gastrointestinal symptoms. CONCLUSION: Intestinal resection can be performed safely in most women with severe endometriosis and bowel involvement, although many of these patients experience persistent or recurrent symptoms. Total abdominal hysterectomy and bilateral salpingooophorectomy at the time of bowel resection correlates with improved outcome.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.

Liquid order at the interface of KDP crystals with water: evidence for icelike layers.

We present a surface x-ray diffraction study on the KDP-water interface in which the structure of both the crystalline and liquid part of the interface has been measured. We have been able to determine the ordering components in the liquid in both the perpendicular and parallel directions. We find interface-induced ordering in the first four layers of water molecules. The first two layers behave icelike and are strongly bound to the surface. The next two layers are more diffuse and show only minor lateral and perpendicular ordering. Subsequent layers are found to behave similar to a bulk liquid.

New (sqrt3 xsqrt 3)R30 degrees phase of Pb on Ge(111) and its consequence for the melting transition.

Depending on the preparation method, we find two different structures of the Pb/Ge(111) system at a nominal coverage of 4 / 3 monolayer that exhibit different melting points. One is the well studied beta phase that melts at 270 degrees C, but the other is a new and metastable phase that melts at 330 degrees C. Using surface x-ray diffraction the atomic structure of both phases is found to be surprisingly similar. The difference in melting points can be explained by the distribution of the excess Pb present on the surface, which has a direct effect on the vacancy density. We propose a modified phase diagram, in which the melting temperature of the beta phase depends strongly on coverage.

Safety, pharmacokinetics, and antiviral activity of A77003, a C2 symmetry-based human immunodeficiency virus protease inhibitor.

A77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, was administered to asymptomatic HIV-1-infected patients in a phase I trial. The drug was given by continuous intravenous infusion at dosages of 0.035, 0.07, 0.14, and 0.28 mg/kg of body weight per h. The drug was given first for 24 h and then for up to an additional 4 weeks in a second infusion period following at least a 6-day washout. Apart from reversible increases in hepatic transaminase levels in some patients, no systemic toxicities occurred during extended infusion of the drug. Dose-related local vein irritation, despite dilution of the infusate, however, caused severe infusion site phlebitis precluding dosage escalation beyond 0.28 mg/kg/h. Pharmacokinetic analysis demonstrated dose linear increases in mean steady-state concentrations. However, clearance of the drug from plasma was unexpectedly high, averaging 62 liters/h across all groups. The concentrations of A77003 in plasma achieved the in vitro 50% inhibitory concentration (0.16 microgram/ml) only in the 0.28-mg/kg/h dosage group, but it did not attain the 90% inhibitory concentration (0.48 micrograms/ml). No statistically significant effect on CD4 cell numbers occurred in any of the groups, and there was no evidence of antiviral activity, as determined by HIV-1 p24 antigen level, quantitative plasma and cell culture, and quantitation of viral RNA in plasma. In conclusion, A77003, as formulated in the present study, causes severe phlebitis, which prevents administration of the infusates necessary to achieve high concentrations of the drug in plasma. In conclusion, A77003, as formulated in the present study, causes severe phlebitis, which prevents administration of the infusates necessary to achieve high concentrations of the drug in plasma. The lack of antiviral activity observed in the study may be a consequence of the low concentrations in plasma in all groups.