Disseminated tuberculosis, CMV viraemia & haemophagocytic-lymphohistiocystosis syndrome in an adult patient with anti- IFNγ autoantibodies - case report and brief review.

We report a case of an adult female with disseminated tuberculosis, cytomegalovirus viraemia and haemophagocytic-lymphohistiocystosis syndrome associated with neutralizing anti- interferon gamma (IFNγ) autoantibodies demonstrated by absent IFNγ stimulated STAT1 phosphorylation in the presence of patient sera. A brief review of immunodeficiency caused by anti-IFNγ autoantibodies is also described.

Deep seam and minesoil carbon sequestration potential of the South Wales Coalfield, UK.

Combustion of coal for energy generation has been a significant contributor to increased concentrations of atmospheric carbon dioxide. It is of interest to evaluate the potential of former coalfields for mitigating these increases by carbon sequestration and to compare different options to achieving this end. Here, carbon sequestration in residual coal seams and through reclamation of spoil tips is compared, and their carbon dioxide storage potential in the South Wales Coalfield estimated. Coal seam sequestration estimates come from an established methodology and consider the total unmined coal resource below 500 m deep with potential for carbon sequestration. The most likely effective deep seam storage capacity is 104.9 Mt carbon dioxide, taking account of reservoir conditions and engineering factors. Whilst many spoil tips in South Wales have been reclaimed, the focus has not been on carbon sequestration potential. Estimates of minesoil restoration sequestration capacity were based on a survey of restored minesoil and vegetation carbon stocks, mainly on sites 20-30 years after restoration; data from this survey were then extrapolated to the coalfield as a whole. Minesoil storage is estimated at 1.5 or 2.5 Mt (+2.2 Mt in tree biomass) carbon dioxide based on average grassland or woodland measurements, respectively; modelled data predicted equilibrium values of 2.9 and 2.6 Mt carbon dioxide respectively in grassland or woodland minesoils. If all sites achieved close to the maximum capacity in their land use class, minesoil storage capacity would increase to 2.1 or 3.9 Mt carbon dioxide, respectively. Combining the best woodland minesoil and standing biomass values, sequestration capacity increases to 7.2 Mt carbon dioxide. The wider social, economic, environmental and regulatory constraints to achieving this sequestration for each approach are discussed. Coal seam sequestration has a much higher capacity but sequestration in mine sites is less costly and has fewer regulatory constraints. Findings indicate a significant combined potential for carbon sequestration in the South Wales Coalfield and highlight challenges in achieving this potential. On a global scale, ex-coalfield sequestration could contribute to broader efforts to mitigate emissions.

Effects of the mu-opioid receptor antagonist GSK1521498 on hedonic and consummatory eating behaviour: a proof of mechanism study in binge-eating obese subjects.

The opioid system is implicated in the hedonic and motivational processing of food, and in binge eating, a behaviour strongly linked to obesity. The aim of this study was to evaluate the effects of 4 weeks of treatment with the mu-opioid receptor antagonist GSK1521498 on eating behaviour in binge-eating obese subjects. Adults with body mass index ≥ 30 kg m(-2) and binge eating scale scores ≥ 19 received 1-week single-blind placebo run-in, and were then randomized to 28 days with either 2 mg day(-1) GSK1521498, 5 mg day(-1) GSK1521498 or placebo (N=21 per arm) in a double-blind parallel group design. The outcome measures were body weight, fat mass, hedonic and consummatory eating behaviour during inpatient food challenges, safety and pharmacokinetics. The primary analysis was the comparison of change scores in the higher-dose treatment group versus placebo using analysis of covariance at each relevant time point. GSK1521498 (2 mg and 5 mg) was not different from placebo in its effects on weight, fat mass and binge eating scores. However, compared with placebo, GSK1521498 5 mg day(-1) caused a significant reduction in hedonic responses to sweetened dairy products and reduced calorific intake, particularly of high-fat foods during ad libitum buffet meals, with some of these effects correlating with systemic exposure of GSK1521498. There were no significant effects of GSK1521498 2 mg day(-1) on eating behaviour, indicating dose dependency of pharmacodynamics. GSK1521498 was generally well tolerated and no previously unidentified safety signals were detected. The potential for these findings to translate into clinically significant effects in the context of binge eating and weight regain prevention requires further investigation.

Polymorphisms in the endothelin-1 (EDN1) are associated with asthma in two populations.

Endothelin-1 (EDN1) has been reported to be implicated in the pathophysiology of asthma. Literature results on the genetic association of EDN1 in asthma are inconsistent. Eleven single nucleotide polymorphisms in EDN1 were genotyped in 342 and 100 families from UK and Norway, respectively. Asthma, bronchial hyperreactivity (BHR) and atopic asthma phenotypes were analyzed for the family-based association. Five single nucleotide polymorphisms (SNPs) were associated with asthma (0.0017

The optimal measure of linkage disequilibrium reduces error in association mapping of affection status.

We have developed a simple yet powerful approach for disease gene association mapping by linkage disequilibrium (LD). This method is unique because it applies a model with evolutionary theory that incorporates a parameter for the location of the causal polymorphism. The method exploits LD maps, which assign a location in LD units (LDU) for each marker. This approach is based on single marker tests within a composite likelihood framework, which avoids the heavy Bonferroni correction through multiple testing. As a proof of principle, we tested an 890 kb region flanking the CYP2D6 gene associated with poor drug-metabolizing activity in order to refine the localization of a causal mutation. Previous LD mapping studies using single markers and haplotypes have identified a 390 kb significant region associated with the poor drug-metabolizing phenotype on chromosome 22. None of the 27 Single nucleotide polymorphisms was within the gene. Using a metric LDU map, the commonest functional polymorphism within the gene was located at 14.9 kb from its true location, surrounded within a 95% confidence interval of 172 kb. The kb map had a relative efficiency of 33% compared with the LDU map. Our findings indicate that the support interval and location error are smaller than any published results. Despite the low resolution and the strong LD in the region, our results provide evidence of the substantial utility of LDU maps for disease gene association mapping. These tests are robust to large numbers of markers and are applicable to haplotypes, diplotypes, whole-genome association or candidate region studies.

Linkage disequilibrium mapping identifies a 390 kb region associated with CYP2D6 poor drug metabolising activity.

The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 x 10(-4) to 4.54 x 10(-35)). Haplotype analysis revealed more significant levels of association (P = 3.54 x 10(-56)). Strong (D' > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

Polymorphisms in P21CIP1/WAF1 are not correlated with TP53 status in sporadic ovarian tumours.

In breast cancers and sarcomas, a variant polymorphism in the cell cycle inhibitor P21CIP1/WAF1 is under-represented in those individuals whose tumours contain mutated TP53. The aim of this study was to determine whether this variant polymorphism was also under-represented in those with ovarian carcinoma and TP53 mutations. We studied lymphocyte DNA from 104 women with ovarian carcinoma, 15 with borderline tumours and 16 with benign tumours, using a previously-reported PCR-RFLP technique. 96 of the ovarian carcinoma cases had been previously examined for mutations in TP53 and/or for overexpression of the TP53 protein. The variant allele was seen in 11 out of 104 women (10.6%) with ovarian carcinoma. There was no significant difference in the distribution of the variant allele in the women whose tumours had (7/47) or did not have (4/49) TP53 mutations (P = 0.523). It does not appear that the presence of this variant allele of P21CIP1/WAF1 has any aetiological role in ovarian carcinomas. Studies in other tumours support this finding.

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide.

Mechanisms of resistance to VP-16 were monitored in a series of sublines of the human testicular teratoma cell line (SuSa) derived following exposure either to fractionated X-irradiation (DXR-10) or to VP-16 using pulsed 24-hr exposures (VP10) or continuous exposure conditions (VPC2, VPC3 and VPC4). Orders of resistance expressed (ranging from 3- to 33-fold based on IC50 values derived from colony forming assays) were comparable with those likely to be encountered clinically. All of these resistant sublines showed some cross-resistance to VCR, and the 3 drug-selected sublines tested also proved cross-resistant to ADR. Resistance was not associated with modified 3H-VP-16 accumulation. However, decreased VP-16-induced SSBs were detectable in all the resistant sublines and a strong positive correlation was noted between the extent of SSB formation and VP-16 resistance by linear regression analysis. Topo II alpha protein content, as judged by Western blotting, was significantly decreased only in the sublines derived by continuous exposure to VP-16, but this was not progressive with increasing levels of resistance expressed. RNase protection assays also showed no significant differences in Topo II alpha expression in the low-level resistant DXR-10 and VP10 sublines, contrasting with the 2-fold decreases identified in the VPC2, VPC3 and VPC4 sublines. Significantly, however, mRNA levels of two alternately spliced Topo II beta mRNAs were markedly decreased (2- to 9-fold) in all the drug-selected resistant sublines. No mutations in consensus ATP-binding sequences or in the DNA-binding region of Topo II alpha were detected by single strand conformational polymorphism analysis. Significant Pgp overexpression was only identified in the most highly resistant sublines VPC3 and VPC4, which both showed 4-fold cross-resistance to VCR. Decreased 3H-VCR accumulation and partial reversal of resistance by VPM (6.6 microM) addition was also identified, consistent with a functional Pgp being overexpressed in these sublines. Modifications of Topo II expression therefore appear to precede Pgp overexpression in this series of sequentially derived VP-16 resistant sublines and to represent the predominant mechanism underlying low level (< 10-fold) resistance.

Characterisation of the unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines of the SuSa testicular teratoma cell line.

Three etoposide-selected resistant sublines of the SuSa testicular teratoma cell line expressing 9-, 21- and 33-fold levels of resistance, proved increasingly cross resistant to cisplatin with levels approximating to 3-, 4- and 6-fold in sublines VPC2, VPC3 and VPC4, respectively. Cisplatin resistance was not associated with any significant modifications in levels of total glutathione or associated enzyme activities. Decreased platinum (Pt) accumulation was detected, although this did not correlate either with total platination levels judged immunochemically or with peak induction of interstrand crosslinks (ISC) determined by alkaline elution. Following exposure to cisplatin in the least resistant subline, VPC2, total platination levels were markedly decreased (3-fold) relative to those of the parental cells, whilst peak ISC levels were markedly increased (4-fold). In the most highly resistant subline, VPC4, peak levels of ISCs were even higher (9-fold), although total platination levels remained comparable with those in parental cells. Both VPC2 and VPC4 cells appeared highly proficient in removing ISCs, unlike the parental cells. However, whilst VPC2 cells appeared to share deficient removal of the intrastrand platinated lesions with parental cells, VPC4 cells proved proficient in removing specific adducts in the sequence pApG. This unusual expression of cross resistance to cisplatin in a series of etoposide-selected resistant sublines derived from an inherently repair deficient parental cell line, SuSa, therefore appears to be associated with enhanced removal of the specific intrastrand crosslinks in the sequence pApG and/or of DNA-DNA ISCs. Similar mechanisms have been implicated in two other cisplatin resistant SuSa sublines selected following in vitro exposure to the drug itself or to fractionated X-irradiation.

Differential effectiveness of a range of novel drug-resistance modulators, relative to verapamil, in influencing vinblastine or teniposide cytotoxicity in human lymphoblastoid CCRF-CEM sublines expressing classic or atypical multidrug resistance.

A series of five potential modulators of resistance were tested for their relative ability, as compared with verapamil, to sensitize CEM lymphoblastoid leukemia drug-resistant tumor sublines expressing either the classic or the atypical multidrug-resistance (MDR) phenotype to vinblastine or teniposide. Maximal non-cytotoxic concentrations of each modulator were tested and sensitization induces (SIs) were derived by comparing the drug concentration required to inhibit growth by 50% in their presence or absence. Like verapamil (10 microM) itself, three of the other modulators tested, namely, S9788 (4 microM), flunarizine (20 microM) and quinidine (30 microM), resulted in 2- to 3-fold sensitization of vinblastine against the parental CEM cells, and comparable effects were noted in the CEM/VM-1 cells, which were not cross-resistant to vinblastine. In contrast, cyclosporin A (0.5 microM) and B859-35 (2 microM) did not enhance vinblastine growth inhibition in these lines. However, the greatest sensitization with all the modulators was noted in the classic MDR VBL1000 cells, with SIs ranging from 40- to 350-fold, except for cyclosporin A, which proved ineffective at the concentration tested (SI, 2.6). The greatest extent of differential sensitization of these VBL1000 tumor cells occurred with quinidine or B859-35, which proved significantly more effective than verapamil alone. Combinations of modulators resulted in additive effects, with B859-35 plus cyclosporin A proving superior to B859-35 plus verapamil. In contrast, none of these compounds proved effective as a sensitizer to teniposide. The growth-inhibitory effects of this drug were not modified significantly in either the 92-fold teniposide-resistant VM-1 cells or in the parental cells. Addition of verapamil itself also failed to modulate teniposide growth inhibition in the VBL1000 cells, which express significant cross-resistance to this drug (36-fold). However, SI values of 3- to 5-fold were obtained using quinidine or B859-35. These results serve (a) to emphasise the need to monitor the effects of modulators not only on drug-resistant cells but also on their drug-sensitive counterparts so as to ensure differential sensitization such that normal sensitive tissues are not likely to be adversely influenced and (b) to highlight the observation that the extent of modulation differs depending not only on the antitumor drug used but also on the mechanism of drug resistance expressed. This in vitro model system appears to provide a useful screening system for resistance modulators and certainly could be used in attempts to identify alternative agents that may influence teniposide sensitivity in these drug-resistant sublines.

Differential cytotoxic effects of docetaxel in a range of mammalian tumor cell lines and certain drug resistant sublines in vitro.

The in vitro cytotoxic effects of docetaxel (Taxotere; RP56976, NSC688503) proved both time and concentration dependent. Amongst thirteen human cell lines from various tumor types, exposure to increasing concentrations of docetaxel over 24 hrs resulted in a plateau-shaped dose response curve, suggesting that increased cell kill becomes more dependent on increased exposure duration than on concentration. IC50 concentrations (reducing survival by 50%) ranged from 0.13-3.3 ng/ml, with three neuroblastoma lines proving most sensitive and three breast and two colon carcinoma lines showing least sensitivity. There was significant cross-resistance to docetaxel in the classic multidrug resistant (MDR) Chinese hamster ovarian (CHO) CHRC5 line and the human lymphoblastoid CCRF-CEMVLB1000 line, as well as in two vincristine (VCR)-selected MDR MCF-7 sublines. All four of these MDR sublines overexpress P-glycoprotein (Pgp), as did a 6-fold docetaxel-selected resistant CHO subline. As an apparent corollary, in two human teratoma lines selected for etoposide resistance and showing some cross-resistance to VCR and in two CHO sublines expressing low levels of VCR resistance, yet all proving Pgp positive, no docetaxel cross-resistance was identified. Verapamil modulated docetaxel resistance only in sublines expressing resistance to the drug and overexpressing Pgp. Four other human tumor sublines selected for resistance to 5-fluorouracil, cisplatin or teniposide, showed a lack of cross-resistance to docetaxel. Furthermore, cross-resistance to docetaxel was not apparant in four epipodophyllotoxin-selected resistant sublines with alterations in topoisomerase II, indicating its effectiveness against tumor cells expressing the topoisomerase II-related MDR phenotype. Our observation that docetaxel cross-resistance was not automatically expressed by classic MDR tumour cells appears of interest and of potential clinical relevance.

Evaluation of S9788 as a potential modulator of drug resistance against human tumour sublines expressing differing resistance mechanisms in vitro.

Significant activity has been identified using S9788, a triazineaminopiperidine derivative, as a new modulator of multi-drug resistance against a series of drug-resistant human tumour-cell lines in vitro. Maximal non-cytotoxic concentrations (i.e., those resulting in < or = 10% cytotoxicity) of S9788 or verapamil were tested in combination with vinblastine, Adriamycin or vincristine and cytotoxicity was evaluated using a clonogenic assay, or the metabolic dye reduction MTT assay, or by monitoring growth inhibition. Under these conditions, the extent of resistance modulation by verapamil and by S9788 was comparable in the various tumour cell lines tested, although a definite concentration-dependent modulation was noted with both compounds. The highest dose-modification factors were noted in the highly vinblastine-resistant classic multi-drug-resistant subline CEM/VLB100, although resistance reversal was only partial. Resistance modulation by both verapamil and S9788 was noted in 4 drug-selected resistant sublines and 4 "intrinsically" resistant human tumour cell lines, which all exhibited significant P-glycoprotein expression. In contrast, in 2 drug-resistant human tumour sublines (GLC4/ADR and CEM/VM-1) characterized by altered topoisomerase-II activity and proving to be P-glycoprotein-negative, no resistance modulation relative to parental cells was observed. These data are consistent with the proposal that resistance modulation is mediated by interaction between S9788 and P-glycoprotein and support its clinical evaluation in patients with P-glycoprotein-positive tumours.

Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation.

Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody. Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi. The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

Expression of P-glycoprotein-mediated drug resistance in CHO cells surviving a single X-ray dose of 30 Gy.

We reported previously that Chinese hamster ovary (CHO) cells surviving exposure to repeated doses of 9 Gy of X-irradiation in vitro expressed a multiple drug resistance phenotype characterized by cross-resistance to epipodophyllotoxins and to Vinca alkaloids, and by P-glycoprotein (Pgp) overexpression (Hill et al. 1990). We have now shown that exposure of these CHO cells to a single 30-Gy X-ray dose similarly resulted in the survivors expressing resistance to vincristine and to etoposide and overexpressing Pgp. In agreement with data obtained on cells which received repeated X-ray exposures, this Pgp overexpression occurred in the absence of any significant elevation of Pgp mRNA. However, the reduced ability to accumulate rhodamine 123 identified in these sublines, and the ability of verapamil to reverse this accumulation defect, implies that the Pgp which was overexpressed was functional. These findings indicate that a series of X-ray exposures is not necessary for expression of this distinctive multiple drug resistance phenotype, suggesting that this results not from a general 'stress-type' response but rather more specifically from the radiation exposure itself, with both single-dose and repeated X-irradiation selecting for similar genetic mutants.