Functional characterization of ABCC2 promoter polymorphisms and allele-specific expression.

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele-specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 -24C>T both alone and as part of a common haplotype (-24C>T/-1019A>G/-1549G>A) increased promoter function 35% compared with the reference sequence (P<0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele-specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972C>T variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972C>T polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P<0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/untranslated region variants (-1549G>A, -1019A>G and -24C>T). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

Police officer anxiety after occupational blood and body fluid exposure.

BACKGROUND: In the course of their work, police staff are at risk of exposure to blood and body fluids (BBF) and potentially at risk of acquiring a blood-borne viral infection. AIMS: To examine levels of anxiety among Scottish police staff following an occupational exposure to BBF. METHODS: Police staff who reported an incident of exposure to their occupational health (OH) provider were invited to complete a postal questionnaire about their levels of self-reported anxiety after the incident and after contact with medical services (namely, OH and accident and emergency (A&E)). RESULTS: Seventy exposed individuals (66% of those invited to take part) completed a questionnaire. Participants' self-reported anxiety after the incident varied widely. Levels of anxiety reduced over time and following contact with medical services. A&E staff were more likely to be the first point of medical contact for the most anxious individuals. Pre-incident training was not associated with post-incident anxiety. CONCLUSIONS: The findings suggest that contact with medical services helps to alleviate post-exposure anxieties among police staff.

Effects of acute hypoxia on postural and kinetic tremor.

Human physiological tremor is a complex phenomenon that is modulated by numerous mechanical, neurophysiological, and environmental conditions. Researchers investigating tremor have suggested that acute hypoxia increases tremor amplitude. Based on the results of prior studies, we hypothesized that human participants exposed to a simulated altitude of 4,500 m would display an increased tremor amplitude within the 6-12 Hz frequency range. Postural and kinetic tremors were recorded with a laser system in 23 healthy male participants before, during, and after 1 h of altitude-induced hypoxia. A large panel of tremor characteristics was used to investigate the effect of hypoxia. Acute hypoxia increased tremor frequency content between 6 and 12 Hz during both postural and kinetic tremor tasks (P < 0.05, F = 6.142, Eta(2) = 0.24 and P < 0.05, F = 3.767 Eta(2) = 0.14, respectively). Although the physiological mechanisms underlying the observed changes in tremor are not completely elucidated yet, this study confirms that acute hypoxia increases tremor frequency in the 6-12 Hz range. Furthermore, this study indicates that changes in physiological tremor can be detected at lower hypoxemic levels than previously reported (blood saturation in oxygen = 80.9%). The effects of hypoxia mainly result from a cascade of events starting with the activation of the hypothalamic-pituitary-adrenal axis causing in turn an increase in catecholamine release, leading to an augmentation of tremor amplitude in the 6- to 12-Hz interval and heart rate increase.

Management of blood and body fluid exposures in police service staff.

BACKGROUND: Police service staff are at risk of occupational exposure to blood and body fluids with the consequent risk of blood-borne virus (BBV) infection. AIMS: To examine the types of occupational exposure incidents experienced by Scottish police service staff and to evaluate the post-incident management provided by their occupational health (OH) services. METHODS: Data were collected on the circumstances and the post-incident management of each incident reported to OH over 12 months. An expert panel reviewed the post-incident management provided by OH. RESULTS: The panel considered that the majority of cases of occupational exposure incurred little or no risk of BBV transmission. In general, the expert panel assessed the post-incident management provided by OH units serving the police as adequate and appropriate. However, some concerns were raised in relation to a small number of incorrect risk assessments and an inconsistent approach to hepatitis C virus (HCV) follow-up blood testing. CONCLUSIONS: The study findings suggest that most Scottish police OH departments were providing adequate post-incident management. There is, however, a need for more clarity around BBV risk assessment terminology and development of a standardized HCV testing protocol.

The transfer of unsterilized material from Mars to Phobos: Laboratory tests, modelling and statistical evaluation.

Sample return missions to Phobos are the subject of future exploration plans. Given the proximity of Phobos to Mars, Mars' potential to have supported life, and the possibility of material transfer from Mars to Phobos, careful consideration of planetary protection is required. If life exists, or ever existed, on Mars, there is a possibility that material carrying organisms could be present on Phobos and be collected by a sample return mission such as the Japanese Martian Moons eXplorer (MMX). Here we describe laboratory experiments, theoretical modelling and statistical analysis undertaken to quantify whether the likelihood of a sample from Phobos material containing unsterilized material transferred from Mars is less than 10-6, the threshold to transition between restricted and unrestricted sample return classification for planetary protection. We have created heat, impact and radiation sterilization models based on the Phobos environment, and through statistical analyses investigated the level of sterilization expected for martian material transferred to Phobos. These analyses indicate that radiation is the major sterilization factor, sterilizing the Phobos surface over timescales of millions of years. The specific events of most relevance in the Phobos sample return context are the 'young' cratering events on Mars that result in Zunil-sized craters, which can emplace a large mass of martian material on Phobos, in a short period of time, thus inhibiting the effects of radiation sterilization. Major unknowns that cannot yet be constrained accurately enough are found to drive the results - the most critical being the determination of exact crater ages to statistical certainty, and the initial biological loading on Mars prior to transfer. We find that, when taking a conservative perspective and assuming the best-case scenario for organism survival, for a 100 g sample of the Phobos regolith to be below the planetary protection requirement for unrestricted sample return, the initial biological loading on Mars must be <8.2 × 103cfu kg-1. For the planned MMX mission, a ∼10 g sample to be obtained from a 25-30 mm diameter core as planned would require an initial martian biological loading to be <1.6 × 104cfu kg-1, in order to remain compliant with the planetary protection threshold.

Differential cleavage of LexA and UmuD mediated by recA Pro67 mutants: implications for common LexA and UmuD binding sites on RecA.

In Escherichia coli, RecA-mediated cleavage of LexA repressor is a key regulatory event required for expression of SOS genes involved in the repair of DNA damage. RecA also mediates the cleavage of UmuD protein to UmuD, a form active in SOS mutagenesis. To determine whether LexA and UmuD have common binding determinants on RecA, we have compared the ability of several recA mutants to function in the cleavage of LexA versus UmuD in vivo. The data reveal that while some recA mutations at Pro67 have a similar effect on LexA and UmuD cleavage, others have striking differential effects. For example, a Pro67-->Trp mutation results in a high level of constitutive cleavage of both proteins. However, Pro67-->Asp and Glu mutations promote constitutive cleavage of LexA and reduce induction of UmuD cleavage to just 5 to 10% of wild-type activity. In contrast, Pro67-->Arg prevents LexA cleavage while allowing nearly 50% of wild-type induction of UmuD cleavage. These results are consistent with the idea that Pro67 is located at a site in the nucleoprotein filament where both LexA and UmuD contact RecA.

bcr-abl oncogene activation in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Tumor-specific alterations in oncogenes are thought to play a central role in the development of cancer. An example is the consistent fusion of the bcr gene to the c-abl oncogene on the Ph chromosome in CML. The Ph chromosome can also be observed in ALL. About 50% of Ph+ ALL cases, in contrast to CML, do not exhibit chromosomal breakpoints in the major cluster region or mcr (Ph+ mcr- ALL). These cases may have a novel bcr-abl fusion gene instead. We tested this hypothesis in eight Ph+ mcr- ALL patients by amplifying the putative hybrid part of the bcr-abl cDNA, using the polymerase chain reaction method. All cases examined showed the same joining of the first exon of the bcr gene to the c-abl oncogene. Thus, the novel bcr-abl fusion in Ph+ mcr- ALL is the result of a molecularly distinct Ph chromosome. This allows the definition of Ph+ leukemias by their respective bcr-abl oncogene activation. Moreover, the cDNA amplification method we use is a clinically useful tool to screen for bcr-abl oncogene activations in leukemia patients.

Molecular analysis of Philadelphia positive essential thrombocythemia.

Seven patients with Philadelphia (Ph) chromosome positive essential thrombocythemia (ET) were investigated for the presence of a rearrangement within the major breakpoint cluster region (M-bcr) using the Southern blot technique and, in six cases, for the presence of the hybrid bcr-abl mRNA using the polymerase chain reaction (PCR). The molecular studies showed rearrangement of M-bcr in all cases; there was evidence of the b2a2 mRNA junction in one case and of b3a2 junction in five cases. These findings are identical to what might have been expected in Ph-positive chronic myeloid leukemia. These features may explain the poor prognosis of Ph-positive ET in comparison with cytogenetically normal cases. Conversely, the differences in clinical presentation may be due to other genetic changes.

RAS mutations in myelodysplasia detected by amplification, oligonucleotide hybridization, and transformation.

Members of the RAS gene family have been implicated in many neoplasms with activating mutations around amino acid positions 12 and 61. We have assessed the mutational activation of H, K, and NRAS in myelodysplasia (MDS) by polymerase chain reaction and hybridization with synthetic oligonucleotide probes. Using this method, point mutations in codons 12/13 and 61 of these RAS genes were detected in 20 of 50 patients including two with refractory anemia with ringed sideroblasts (RARS). Ten normal individuals had no detectable RAS mutations. In 11 instances, DNA from patients with detectable RAS mutations were shown to register in either NIH3T3 focus-forming or nude mouse tumorigenicity assays. In addition, one patient (RARS) was shown to have an activated NRAS gene detected by a tumorigenicity assay and Southern blot analyses. Two MDS patients had mutations detected in two different RAS genes. DNA from one of these patients was observed to give rise to transformants with activated N and HRAS. Two patients with detectable NRAS mutations in the MDS stage progressed to AML and DNA from the AML stage registered positively in a transformation assay with NRAS activation. These results show that RAS mutations can occur at early, as well as late, stages of leukemic progression. The incidence of RAS mutations appears to be significantly higher in CMML than in the other subgroups (p = 0.02).

Preclinical development of AMG 139, a human antibody specifically targeting IL-23.

BACKGROUND AND PURPOSE: AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. EXPERIMENTAL APPROACH: The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. KEY RESULTS: AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4-13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg(-1) and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg(-1) s.c. or i.v.). CONCLUSIONS AND IMPLICATIONS: The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

Characterization of HIV-1 neutralization escape mutants.

Infection by molecularly cloned HIV-1, in the presence of a high-titre neutralizing monoclonal antibody (MAb), resulted in the selection of plaques in MT4 cells releasing HIV resistant to neutralization by the same MAb. The epitope recognized by the MAb was mapped to the V3 neutralization epitope at amino acids 305-321. The HIV-1 variants showed a reduced binding capacity for the selecting MAb as determined by immunofluorescence. Polymerase chain reaction (PCR) amplification of complementary DNA derived from viral RNA, cloning and sequencing identified a base pair (bp) change C----G at position 6663 in variant 110.5/1, predicting a change at amino acid 308 Arg----Gly. No other changes in the epitope were observed by sequencing three other variants. Differential hybridization of PCR amplified viral RNA and DNA, with oligonucleotides specific for the observed bp change or the 'wild type' sequence, indicated that the variants 110.5/1 and 110.5/7 were genotypically mixed for 308Gly/Arg. Subsequent screening of biologically 'recloned' variants 110.5/1 and 110.5/7 identified two subclones homozygous for the 308Gly change. The Arg----Gly change appears to affect the binding of the antibody to the epitope, since the linear peptide substituting 308Gly for 'wild type' 308Arg was 100 times less potent in blocking the neutralization of parental HIV. Amino-acid residue 308 thus appears to be crucial for antibody binding to the epitope. In addition, mutations distant from the monoclonal antibody binding site may also affect neutralization by antibodies recognizing the V3 loop.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Activation of Ha-ras in human chronic granulocytic and chronic myelomonocytic leukaemia.

DNAs from chronic granulocytic leukaemia (CGL) and chronic myelomonocytic leukaemia (CMML) were assayed for transforming genes by transfection into NIH 3T3 cells. Foci DNA was tagged with a geneticin-resistance cosmid, then followed through a drug selection and tumorigenicity assay. Activated Ha-ras genes, with point mutations at codon 12 (glycine to valine) were subsequently detected. The mutation was detected in the original samples by either MspI/HpaII digestion or polymerase chain reaction (PCR). Although mutations in the ras gene family may occur frequently in leukaemias, these are the first examples of Ha-ras mutations in CGL (blast crisis) and CMML.

Search for picornaviruses at onset of inflammatory myopathy.

Picornaviruses may not play a role as persistent agents in the inflammatory myopathies, but it is still thought likely that they may act as triggers of an autoimmune process. Forty one muscle biopsy specimens, taken from three weeks to six months (mean four months) after onset, were examined using three different picornaviral primers and PCR. Moderate to severe disease activity was evident in all specimens. The results were compared with those of 18 biopsy specimens examined later in the disease course, and with specimens from 27 patients with non-inflammatory myopathies. All results were negative. Thus, even as early as three weeks after clinical disease appears, picornaviruses are not detectable in these disorders.

Search for retrovirus in the chronic fatigue syndrome.

AIM: To examine peripheral blood and skeletal muscle from patients with chronic fatigue syndrome for exogenous retrovirus. METHODS: Blood samples from 30 patients and muscle biopsy specimens of 15 patients were examined for retroviral sequences by DNA extraction, polymerase chain reaction (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques. RESULTS: No differences between the patient and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV I/II). An endogenous gag band was observed in both the patient and control groups. All sera were negative for antibody to human foamy virus. CONCLUSION: The results indicate that there is no evidence of retroviral involvement in the chronic fatigue syndrome.

Proton magnetic resonance spectroscopy of basal ganglia in chronic fatigue syndrome.

Fatigue is a common symptom of neurological diseases that affect basal ganglia function. We used proton magnetic resonance spectroscopy ((1)H MRS) to study the metabolic functions of the basal ganglia in chronic fatigue syndrome (CFS) to test the hypothesis that fatigue in CFS may have a neurogenic component. (1)H MRS of left basal ganglia was carried out in eight non-psychiatric patients with CFS and their results were compared to age- and sex-matched healthy asymptomatic healthy controls. A highly significant increase in the spectra from choline-containing compounds was seen in the CFS patient group (p < 0.001). In the absence of regional structural or inflammatory pathology, increased choline resonance in CFS may be an indicator of higher cell membrane turnover due to gliosis or altered intramembrane signalling.

The effect of regular and enteric-coated aspirin on bleeding time, thromboxane, and prostacyclin.

We compared the effect of different aspirin schedules, dosages, and formulations on various bleeding time parameters including bleeding time, plasma and total blood volume, and levels of the stable metabolites of thromboxane A2 (TXA2) and prostacyclin (PGI2) (respectively, TXB2 and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha)) to determine the optimal dosage and formulation of aspirin to inhibit TXA2 production while sparing PGI2. In a randomized, parallel study, 52 healthy male volunteers (62 independent observations) with no history of bleeding disorders were given 80 mg or 325 mg of regular aspirin, or 325 mg of enteric-coated aspirin to ingest daily (14 pills) or every other day (7 pills) for a continuous 14 day period. Bleeding times were performed on day 1 before aspirin, 6 h after aspirin on day 1, and before aspirin on day 14. Bleeding times, plasma volume, and total volume increased significantly from before aspirin to after 6 h and 14 days (p < 0.0001 for all parameters) for all aspirin formulations. For day 1 before aspirin ingestion to 6 h later, both TX and PGI2 (p < 0.008) decreased significantly. 6 h after ingestion of aspirin on day 1 to day 14, both TX and PGI2 levels also significantly decreased (p < 0.0001). There was a highly significant decrease in PGI2 production on every other day aspirin schedules (p = 0.0001) particularly with 80 mg of aspirin, while the decrease in PGI2 production on daily aspirin was not significant (p = 0.10). The most favourable ratio of 6-keto-PGF1 alpha to TXB2 occurred with 80 mg daily.(ABSTRACT TRUNCATED AT 250 WORDS)

An examination of blood steroid and gonadotropin concentrations in relation to fertility status and testicular function in men.

Plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), testosterone (T), and 17 beta-estradiol (E2) have been measured in men complaining of infertility in comparison with men of proven fertility. Subgrouping of patients was achieved on the basis of the presence or absence of sperm in the ejaculate and further by the concentration of sperm or by testicular score. The levels of plasma LH, FSH, PRL, and T were found to be significantly different in the fertile men, compared with both infertile men with sperm in their ejaculates and azoospermic men. There were no significant differences between the groups for E2. There appeared to be an inverse relationship between LH concentrations and sperm count in both fertile and infertile men. FSH levels did not vary significantly in the fertile men in relation to sperm count grouping but were significantly less than those found for the infertile men with sperm. Azoospermic patients with high testicular scores had FSH levels indistinguishable from those of the fertile men. The results are discussed in terms of testicular abnormalities and on the interrelationship between the hormones examined.

PIP: Plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin (PRL), testosterone (T), and 17beta estradiol (E2) have been measured in men complaining of infertility in comparison with men with proven fertility. Subgrouping of patients was achieved on the basis of the presence or absence of sperm in the ejaculate and further by the concentration of sperm of by testicular score. The levels of plasma LH, FSH, PRL, and T were found to be significantly different in the fertile men, compared with both infertile men with sperm in their ejaculates and azoospermic men. There were no significant differences between groups for E2. There appeared to be an inverse relationship between LH concentrations and sperm count in both fertile and infertile men. FSH levels did not vary significantly in the fertile men in relation to sperm grouping count but were significantly less than those found for infertile men with sperm. Azoospermic men with high testicular scores had FSH levels indistinguishable which were from those of fertile men. The results are discussed in terms of testicular abnormalities and the interrelationship between the hormones examined.

Excimer photorefractive keratectomy after undercorrected radial keratotomy.

Radial keratotomy has been used for the treatment of myopia since 1979. Until recently, patients with undercorrected myopia had recourse only to repeated keratotomy. Now patients undercorrected after two or more radial keratotomy procedures can be treated with the excimer laser to reduce the residual myopia. Nineteen eyes of 17 patients with undercorrected myopia after repeated radial had excimer photoradial keratectomy performed. The mean residual spherical equivalent refractive error after radial keratotomy was -2.74 +/- 1.06 diopters (D). This was further reduced after PRK. Final uncorrected visual acuity ranged from 20/20 to 20/70. Excimer laser PRK offers a safe and more controlled method of treating residual after radial keratotomy.