Identification of novel peptide motifs in the serpin maspin that affect vascular smooth muscle cell function.

Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses.

Structural essentials for ß-N-acetylhexosaminidase inhibition by amides of prolines, pipecolic and azetidine carboxylic acids.

This paper explores the computer modelling aided design and synthesis of ß-N-acetylhexosaminidase inhibitors along with their applicability to human disease treatment through biological evaluation in both an enzymatic and cellular setting. We investigated the importance of individual stereocenters, variations in structure-activity relationships along with factors influencing cell penetration. To achieve these goals we modified nitrogen heterocycles in terms of ring size, side chains present and ring nitrogen derivatization. By reducing the inhibitor interactions with the active site down to the essentials we were able to determine that besides the established 2S,3R trans-relationship, the presence and stereochemistry of the CH2OH side chain is of crucial importance for activity. In terms of cellular penetration, N-butyl side chains favour cellar uptake, while hydroxy- and carboxy-group bearing sidechains on the ring nitrogen retarded cellular penetration. Furthermore we show an early proof of principle study that ß-N-acetylhexosaminidase inhibitors can be applicable to use in a potential anti-invasive anti-cancer strategy.

Impact of PTEN abnormalities on outcome in pediatric patients with T-cell acute lymphoblastic leukemia treated on the MRC UKALL2003 trial.

PTEN gene inactivation by mutation or deletion is common in pediatric T-cell acute lymphoblastic leukemia (T-ALL), but the impact on outcome is unclear, particularly in patients with NOTCH1/FBXW7 mutations. We screened samples from 145 patients treated on the MRC UKALL2003 trial for PTEN mutations using heteroduplex analysis and gene deletions using single nucleotide polymorphism arrays, and related genotype to response to therapy and long-term outcome. PTEN loss-of-function mutations/gene deletions were detected in 22% (PTEN(ABN)). Quantification of mutant level indicated that 67% of mutated cases harbored more than one mutant, with up to four mutants detected, consistent with the presence of multiple leukemic sub-clones. Overall, 41% of PTEN(ABN) cases were considered to have biallelic abnormalities (mutation and/or deletion) with complete loss of PTEN in a proportion of cells. In addition, 9% of cases had N- or K-RAS mutations. Neither PTEN nor RAS genotype significantly impacted on response to therapy or long-term outcome, irrespective of mutant level, and there was no evidence that they changed the highly favorable outcome of patients with double NOTCH1/FBXW7 mutations. These results indicate that, for pediatric patients treated according to current protocols, routine screening for PTEN or RAS abnormalities at diagnosis is not warranted to further refine risk stratification.

'It's something you have to put up with'--service users' experiences of in utero transfer: a qualitative study.

OBJECTIVE: The purpose of this study was to gain in-depth insight and enhance understanding of service users' experiences of the in utero transfer (IUT) process, in order to inform policy and improve the current service provision of maternal care. DESIGN: Qualitative descriptive study using semi-structured interviews. SETTING: Participant's home or hospital in the Midlands, UK. POPULATION: Fifteen women transferred in utero to a tertiary level maternity hospital; five male partners and two grandmothers. METHODS: Audio-recorded individual or paired semi-structured interviews transcribed verbatum and analysed thematically using nvivo 9. MAIN OUTCOME MEASURES: Facilitators and barriers of the IUT experience. RESULTS: Findings suggest that IUT is an emotional experience that financially disadvantages patients and their families. Male partners were perceived to be most negatively affected by the experience. The quality of the IUT experience was influenced by a range of factors, including the lack of proximity to home and the lack of information. Patients had little knowledge or awareness of IUT, and most felt unprepared for displacement. Despite this, there was resigned acceptance that IUT was a necessary rather than adverse experience. CONCLUSIONS: The experience of IUT for service users could be enhanced by ensuring that they are better informed about the process and the circumstances that necessitate displacement, that they are better informed about the hospital to which they are being transferred, and that they are transferred as close to home as possible. Efforts to minimise the emotional and socio-economic impact of IUT on women and their families also need to be considered.

Classical and Alternative Activation and Metalloproteinase Expression Occurs in Foam Cell Macrophages in Male and Female ApoE Null Mice in the Absence of T and B Lymphocytes.

BACKGROUND: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. METHODS AND RESULTS: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice. CONCLUSION: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.

The human ether-a'-go-go related gene (hERG) K+ channel blockade by the investigative selective-serotonin reuptake inhibitor CONA-437: limited dependence on S6 aromatic residues.

Diverse non-cardiac drugs adversely influence cardiac electrophysiology by inhibiting repolarising K(+) currents mediated by channels encoded by the human ether-a-go-go-related gene (hERG). In this study, pharmacological blockade of hERG K(+) channel current (I(hERG)) by a novel investigative serotonin-selective reuptake inhibitor (SSRI), CONA-437, was investigated. Whole-cell patch-clamp measurements of I(hERG) were made from human embryonic kidney (HEK 293) cells expressing wild-type (WT) or mutant forms of the hERG channel. With a step-ramp voltage-command, peak I(hERG) was inhibited with an IC(50) of 1.34 µM at 35 ±1°C; the IC(50) with the same protocol was not significantly different at room temperature. Voltage-command waveform selection had only a modest effect on the potency of I(hERG) block: the IC50 with a ventricular action potential command was 0.72 µM. I(hERG) blockade developed rapidly with time following membrane depolarisation and showed a weak dependence on voltage, accompanied by a shift of ≈ -5 mV in voltage-dependence of activation. There was no significant effect of CONA-437 on voltage-dependence of I(hERG) inactivation, though at some voltages an apparent acceleration of the time-course of inactivation was observed. Significantly, mutation of the S6 aromatic amino acid residues Y652 and F656 had only a modest effect on I(hERG) blockade by CONA-437 (a 3-4 fold shift in affinity). CONA-437 at up to 30 µM had no significant effect on either Nav1.5 sodium channels or L-type calcium channels. In conclusion, the novel SSRI CONA-437 is particularly notable as a gating-dependent hERG channel inhibitor for which neither S6 aromatic amino-acid constituent of the canonical drug binding site on the hERG channel appears obligatory for I(hERG) inhibition to occur.

Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways.

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development.

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra(-/-) mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTßR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.

PI3K/mTOR inhibition upregulates NOTCH-MYC signalling leading to an impaired cytotoxic response.

PI3K, mTOR and NOTCH pathways are frequently dysregulated in T-cell acute lymphoblastic leukaemia (T-ALL). Blockade of PI3K and mTOR with the dual inhibitor PI-103 decreased proliferation in all 15 T-ALL cell lines tested, inducing cell death in three. Combined PI3K/mTOR/NOTCH inhibition (with a γ-secretase inhibitor (GSI)) led to enhanced cell-cycle arrest and to subsequent cell death in 7/11 remaining NOTCH mutant cell lines. Commitment to cell death occurred within 48-72 h and was maximal when PI3K, mTOR and NOTCH activities were inhibited. PI-103 addition led to upregulation of c-MYC, which was blocked by coincubation with a GSI, indicating that PI3K/mTOR inhibition resulted in activation of the NOTCH-MYC pathway. Microarray studies showed a global increase in NOTCH target gene expression upon PI3K/mTOR inhibition. NOTCH-MYC-induced resistance to PI3K/mTOR inhibition was supported by synergistic cell death induction by PI-103 and a small molecule c-MYC inhibitor, and by reduction of the cytotoxic effect of PI-103+GSI by c-MYC overexpression. These results show that drugs targeting PI3K/mTOR can upregulate NOTCH-MYC activity, have implications for the use of PI3K inhibitors for the treatment of other malignancies with activated NOTCH, and provide a rational basis for the use of drug combinations that target both the pathways.

Impact of NOTCH1/FBXW7 mutations on outcome in pediatric T-cell acute lymphoblastic leukemia patients treated on the MRC UKALL 2003 trial.

Activating mutations in the NOTCH1 pathway are frequent in pediatric T-cell acute lymphoblastic leukemia (T-ALL) but their role in refining risk stratification is unclear. We screened 162 pediatric T-ALL patients treated on the MRC UKALL2003 trial for NOTCH1/FBXW7 gene mutations and related genotype to response to therapy and long-term outcome. Overall, 35% were wild-type (WT) for both genes (NOTCH1(WT)FBXW7(WT)), 38% single NOTCH1 mutant (NOTCH1(Single)FBXW7(WT)), 3% just FBXW7 mutant (NOTCH1(WT)FBXW7(MUT)) and 24% either double NOTCH1 mutant (NOTCH1(Double)FBXW7(WT)) or mutant in both genes (NOTCH1(MUT)FBXW7(MUT)), hereafter called as NOTCH1±FBXW7(Double). There was no difference between groups in early response to therapy, but NOTCH1±FBXW7(Double) patients were more likely to be associated with negative minimal residual disease (MRD) post-induction than NOTCH1(WT)FBXW7(WT) patients (71% versus 40%, P=0.004). Outcome improved according to the number of mutations, overall survival at 5 years 82%, 88% and 100% for NOTCH1(WT)FBXW7(WT), NOTCH1(Single)FBXW7(WT) and NOTCH1±FBXW7(Double) patients, respectively (log-rank P for trend=0.005). Although 14 NOTCH1±FBXW7(Double) patients were classified as high risk (slow response and/or MRD positive), only two had disease progression and all remain alive. Patients with double NOTCH1 and/or FBXW7 mutations have a very good outcome and should not be considered for more intensive therapy in first remission, even if slow early responders or MRD positive after induction therapy.

Methyl 2-(5,5-dimethyl-1,3,2-dioxa-borinan-2-yl)-4-nitro-benzoate.

The six-membered boronate ester ring of the title compound, C(13)H(16)BNO(6), adopts an envelope conformation with the C atom bearing the dimethyl substituents at the flap. The O-B-C-C torsion angles between the boronate group and the benzene ring are 72.5 (2) and 81.0 (2)°. The 4-nitro-benzoate unit adopts a slightly twisted conformation, with dihedral angles between the benzene ring and the nitrate and methyl ester groups of 17.5 (2) and 14.4 (3)°, respectively. In the crystal, inversion-related pairs of mol-ecules show weak π-π stacking inter-actions [centroid-centroid distance = 4.0585 (9) Šand inter-planar spacing = 3.6254 (7) Å].

Enhancement of tritium concentrations on uptake by marine biota: experience from UK coastal waters.

Concentrations of tritium in sea water and marine biota as reported over the last approximately 10 years from monitoring programmes carried out by this laboratory under contract to the UK Food Standards Agency are reviewed from three areas: near Cardiff; Sellafield; and Hartlepool. Near Cardiff, enhancement of concentration factors (CFs) above an a priori value of approximately 1 have already been studied, and attributed to compounds containing organically bound tritium in local radioactive waste discharges. Further data for Cardiff up to 2006 are reported in this note. Up to 2001, CFs increased to values of more than approximately 7000 in flounders and approximately 4000 in mussels, but have subsequently reduced; this variability could be due to changes in the organic constitution of compounds discharged. Near Sellafield and Hartlepool, enhancements to the tritium concentration factor are observed but they are relatively small compared with those near Cardiff. Near Sellafield, plaice and mussels appear to have a CF for tritium of approximately 10; in some cases concentrations of tritium in winkles are below detection limits and positively measured values indicate a CF of approximately 3. The variation could be due to mechanisms of uptake by the different organisms. Near Hartlepool there were only a few cases where tritium was positively measured. These data give a value of approximately 5 for the CF in plaice (on the basis of two samples); approximately 15 in winkles (eight samples); and > 45 in mussels (two samples). Any differences between the behaviours at Sellafield and Hartlepool would need to be confirmed by improved measurements. Possible causes are the organic composition of the effluent and differences in environmental behaviour and uptake by organisms near the two sites. These potential causes need further investigation. It is emphasised that results from tritium analyses are heavily method dependent; thus comparison with results from other programmes needs to take this into account. Further, the results for enhancement of CF will also depend on the definition of CF itself.

1-(1-Carboxy-methyl-1,4-anhydro-2,3-O-isopropyl-idene-α-d-erythrofuranos-yl)thymine.

X-Ray crystallography unequivocally determined the stereochemistry of the thymine base in the title compound, C(14)H(18)N(2)O(7). The absolute stereochemistry was determined from the use of d-ribose as the starting material. There are two independent mol-ecules in the asymmetric unit (Z' = 2) which exist as N-H⋯O hydrogen-bonded pairs in the crystal structure.

3,4-O-Isopropyl-idene-2-C-methyl-d-galactonolactone.

X-ray crystallography unequivocally confirmed the stereochemistry of the 2-C-methyl group in the title mol-ecule, C(10)H(16)O(6), in which the 1,5-lactone ring exists in a boat conformation. The use of d-galactose in the synthesis determined the absolute stereochemistry. The crystal exists as O-H⋯O hydrogen-bonded layers in the ab plane, with each mol-ecule acting as a donor and acceptor for two hydrogen bonds.

(1R,2R,3S,6aS,7R,8R,9S,12aS)-1,2,3,7,8,9-Hexahydroxy-perhydro-dipyrido[1,2-a:1',2'-d]pyrazine-6,12-dione.

The crystal structure of the title compound, C(12)H(18)N(2)O(8), exists as O-H⋯O hydrogen-bonded layers of mol-ecules running parallel to the ab plane. Each mol-ecule is a donor and acceptor for six hydrogen bonds. The absolute stereochemistry was determined by the use of d-glucuronolactone as the starting material.

6-Azido-3-O-benzyl-6-de-oxy-N,N-diethyl-1,2-O-isopropyl-idene-d-glycero-α-d-gluco-heptofuran-uronamide.

Reaction of 3-O-benzyl-1,2-O-isopropyl-idene-α-xylo-pentodialdo-1,4-furan-ose with N,N-diethyl-2-(dimethyl-sulfuranil-idene)acetamide gave stereoselectively an ep-oxy-amide, which was regioselectively opened by NaN(3) in dimethyl formamide to give the title compound, C(21)H(30)N(4)O(6). X-ray crystallography confirmed the relative stereochemistry of the title compound and the absolute configuration was determined by the use of d-glucose as the starting material. There are two mol-ecules in the asymmetric unit (Z' = 2). The crystal structure consists of two types of chains of O-H⋯O hydrogen-bonded mol-ecules running parallel to the b axis, with each mol-ecule acting as a donor and acceptor of one hydrogen bond.

Loss of CTL function among high-avidity tumor-specific CD8+ T cells following tumor infiltration.

A major problem in generating effective antitumor CTL responses is that most tumors express self-antigens to which the immune system is rendered unresponsive due to mechanisms of self-tolerance induction. CTL precursors (CTLp) expressing high-affinity T-cell receptors (TCR) are often functionally deleted from the repertoire, leaving a residual repertoire of CTLp having only low-affinity TCR. Furthermore, even when unique antigens are expressed, their presentation by dendritic cells (DC) may predispose to peripheral tolerance induction rather than the establishment of CTL responses that kill tumor cells. In this study, we examined both high-avidity (CL4) and low-avidity (CL1) CD8(+) T-cell responses to a murine renal carcinoma expressing, as a neoantigen, high and low levels of the hemagglutinin (HA) protein from influenza virus A/PR/8 H1N1 (PR8; RencaHA(high) and RencaHA(low)). Our data show that, following encounter with K(d)HA epitopes cross-presented by bone marrow-derived DC, low-avidity CL1 cells become tolerized within tumor-draining lymph nodes (TDLN), and in mice bearing either RencaHA(high) or RencaHA(low) tumors, very few form tumor-infiltrating lymphocytes (TIL). In marked contrast, high-avidity CL4 cells differentiate into effector CTL within the TDLN of mice bearing either RencaHA(high) or RencaHA(low) tumors, and although they form TIL in both tumors, they lose CTL effector function. Critically, these results show that anticancer therapies involving either adoptive transfer of high-avidity tumor-specific CTL populations or targeting of preexisting tumor antigen-specific memory CD8(+) T cells could fail due to the fact that CTL effector function is lost following tumor infiltration.

Expression of the transcription factor cKrox in peripheral CD8 T cells reveals substantial postthymic plasticity in CD4-CD8 lineage differentiation.

Most T cells belong to either of two lineages defined by the mutually exclusive expression of CD4 and CD8 coreceptors: CD4 T cells are major histocompatibility complex (MHC) II restricted and have helper function, whereas CD8 T cells are MHC I restricted and have cytotoxic function. The divergence between these two lineages occurs during intrathymic selection and is thought to be irreversible in mature T cells. It is, however, unclear whether the CD4-CD8 differentiation of postthymic T cells retains some level of plasticity or is stably maintained by mechanisms distinct from those that set lineage choice in the thymus. To address this issue, we examined if coreceptor or effector gene expression in mature CD8 T cells remains sensitive to the zinc finger transcription factor cKrox, which promotes CD4 and inhibits CD8 differentiation when expressed in thymocytes. We show that cKrox transduction into CD8 T cells inhibits their expression of CD8 and cytotoxic effector genes and impairs their cytotoxic activity, and that it promotes expression of helper-specific genes, although not of CD4 itself. These observations reveal a persistent degree of plasticity in CD4-CD8 differentiation in mature T cells.