Exome sequencing reveals a thrombopoietin ligand mutation in a Micronesian family with autosomal recessive aplastic anemia.

We recently identified 2 siblings afflicted with idiopathic, autosomal recessive aplastic anemia. Whole-exome sequencing identified a novel homozygous missense mutation in thrombopoietin (THPO, c.112C>T) in both affected siblings. This mutation encodes an arginine to cysteine substitution at residue 38 or residue 17 excluding the 21-amino acid signal peptide of THPO receptor binding domain (RBD). THPO has 4 conserved cysteines in its RBD that form 2 disulfide bonds. Our in silico modeling predicts that introduction of a fifth cysteine may disrupt normal disulfide bonding to cause poor receptor binding. In functional assays, the mutant-THPO-containing media shows two- to threefold reduced ability to sustain UT7-TPO cells, which require THPO for proliferation. Both parents and a sibling with heterozygous R17C change have reduced platelet counts, whereas a sibling with wild-type sequence has normal platelet count. Thus, the R17C partial loss-of-function allele results in aplastic anemia in the homozygous state and mild thrombocytopenia in the heterozygous state in our family. Together with the recent identification of THPO receptor (MPL) mutations and the effects of THPO agonists in aplastic anemia, our results have clinical implications in the diagnosis and treatment of patients with aplastic anemia and highlight a role for the THPO-MPL pathway in hematopoiesis in vivo.

Effective use of an audit tool devised to optimize the management of syphilis in an integrated sexual health clinic.

The objective of this study was to audit the management of syphilis in our integrated sexual health clinic according to the British Association for Sexual Health and HIV (BASHH) guideline using the 'Treponemal Infection Care' (TIC) audit tool devised by our clinic. The case notes of patients diagnosed with all stages of syphilis during an 18-month period were reviewed. At the time of diagnosis, the departmental TIC proforma was filled in: this proforma details BASHH auditable outcomes. The case notes of 83 patients diagnosed with syphilis during the audit period were reviewed. The majority of patients were men (76), men who had sex with men (69), HIV-negative (59) and were British (68). In line with current guidance all patients had a baseline Venereal Disease Research Laboratory (VDRL) titre at the start of treatment (target: 100%) and 97% of diagnosed patients completed treatment (target: 95%). A 'response to treatment' according to the decrease in VDRL was demonstrated in 50 (60%) patients with two (2%) patients failing to respond according to these criteria. However, 19 (23%) patients failed to return for their VDRL tests before demonstrating an adequate response to treatment, despite repeated attempts to contact them by letter and telephone. Fifty-four patients had at least 50% of their partners documented as traceable. Of those who were contactable, 100% attended for screening or treatment (target: 60%). In conclusion, our department performed well against BASHH auditable outcome targets. The introduction of the TIC proforma greatly facilitated the ease of audit and is a valuable tool within our clinic setting, which may have positively influenced our audit outcomes. Further action is required to highlight the importance of follow-up VDRLs to patients.

Energetics and longevity in birds.

The links between energy expenditure and ageing are different at different levels of enquiry. When studies have examined the relationships between different species within a given class the association is generally negative--animals with greater metabolism per gram of tissue live shorter lives. Within species, or between classes (e.g. between birds and mammals) the association is the opposite--animals with higher metabolic rates live longer. We have previously shown in mammals that the negative association between lifespan and metabolic rate is in fact an artefact of using resting rather than daily energy expenditure, and of failing to adequately take into account the confounding effects of body size and the lack of phylogenetic independence of species data. When these factors are accounted for, across species of mammals, the ones with higher metabolism also have the largest lifetime expenditures of energy-consistent with the inter-class and intra-specific data. A previous analysis in birds did not yield the same pattern, but this may have been due to a lack of sufficient power in the analysis. Here we present an analysis of a much enlarged data set (>300 species) for metabolic and longevity traits in birds. These data show very similar patterns to those in mammals. Larger individuals have longer lives and lower per-gram resting and daily energy expenditures, hence there is a strong negative relationship between longevity and mass-specific metabolism. This relationship disappears when the confounding effects of body mass and phylogeny are accounted for. Across species of birds, lifetime expenditure of energy per gram of tissue based on both daily and resting energy expenditure is positively related to metabolic intensity, mirroring these statistical relationships in mammals and synergizing with the positive associations of metabolism with lifespan within species and between vertebrate classes.

Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action.

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.

Analysis of gene and protein expression for drug mode of toxicity.

The rapid sequencing of the genomes of a number of organisms, including humans, has led to major changes in the drug industry. The abundance of genome data, and the reagents generated from these genomes, have enabled the study of changes in large numbers of genes and proteins in parallel, using methods such as DNA microarrays to examine gene expression changes, or 2D polyacrylamide electrophoresis (2D-PAGE) to observe changes in the expression of proteins. While these techniques have been in use for several years, their application has primarily focused on the target discovery phase, with some early work carried out on drug- or toxin-induced changes in proteins using 2D-PAGE. In the last two years, a slew of publications have appeared on the application of array technologies to the study of toxicology, and the aim of this review is to highlight some recent examples of these applications.

Expression databases--resources for pharmacogenomic R&D.

This review aims to outline the primary biological databases that are being generated to understand fundamental biology, identify new drug targets, and to look at compound profiling in a new light. We will give a brief overview of four of the main areas being studied in molecular biology: genomics, pharmacogenomics, pharmacogenetics and proteomics. Looking initially at each data set and some of its potential applications, we will go on to describe some of the potentially enormous advantages gained by fully integrating these data sets.

The pharmacology of apoptosis.

Apoptosis is an area of intense scientific interest, which encompasses the study of and triggers mechanisms involved in mediating the cell biology of programmed cell death. A number of low molecular weight compounds have been used to inhibit or enhance this fundamental cellular process and so apoptosis has now become amenable to pharmacological manipulation. In this review Ross Kinloch, Mark Treherne, Mike Furness and Iradj Hajimohamadreza will focus on the current literature describing the pharmacology of apoptosis, with particular reference to the therapeutic potential that could arise from the development of pro- and anti-apoptotic drugs. The pivotal role of apoptosis in such diverse pathological processes as tumour growth, the immune response and neurodegeneration suggests that an understanding of how apoptosis can be regulated by drugs will become increasingly important to the pharmaceutical industry.

Assessment of patients with memory problems using a nurse-administered instrument to detect early dementia and dementia subtypes.

BACKGROUND: With the development of pharmacological treatments for Alzheimer's disease there will be an increase in the numbers of patients requiring assessment from specialist services. Could the role of the specialist clinician be supported by other health professionals screening those who might benefit from treatment? METHOD: Sixty-four consecutive referrals to the Leicester University Memory Clinic were assessed at home by a community psychiatric nurse using a semi-structured interview. The nurse then reported her findings to a psychiatrist and a diagnosis was agreed. This diagnosis was then compared to the Memory Clinic diagnosis and a standardized (ICD-10) diagnosis recorded by another psychiatrist examining the clinic records. RESULTS: The nurse assessment procedure performed well in detecting dementia, with a kappa statistic (k) of 0.75 when compared to the standardized and Memory Clinic diagnoses. There was, however, only moderate concordance between the ICD-10 diagnosis and nurse (k = 0.46) and the Memory Clinic and nurse (k = 0.60) for Alzheimer's disease. The relatively low k value for Alzheimer's disease was principally a result of difficult in differentiating vascular dementia. CONCLUSIONS: A single supervised community psychiatric nurse, using a structured assessment instrument, can adequately detect early dementia in a sample of patients referred with memory problems. Subtypes of dementia are not, however, accurately differentiated.

Refined tuberculosis contact tracing in a low incidence area.

Our aim was to evaluate the efficacy of a revised tuberculosis (TB) contact tracing procedure in South Glamorgan whereby routine annual radiological surveillance was abandoned and contacts were either discharged or referred to chest clinic following their initial screening. We reviewed and evaluated data from the TB contact tracing clinic, the Public Health Service Mycobacterium Reference Unit, Cardiff and the Consultant in Communicable Diseases Control, South Glamorgan Health Authority and compared these results with those of our previous study. One hundred and three index cases and 732 contacts were identified. Seven hundred and seven contacts, 526 close and 181 casual, were screened, of whom 102 casuals should not have been. One hundred and sixty-one contacts were given BCG vaccination. Fifty-four contacts were referred to the chest clinic. Seven cases of TB were detected, all in young, unvaccinated, close contacts of pulmonary disease. Twenty-one contacts were given chemoprophylaxis, 20 of whom were close contacts of pulmonary TB and one of extrapulmonary disease. Five contacts who were screened and initially discharged developed TB later: in two the protocol had not been followed and three presented with extrapulmonary TB. Compared with the results of the previous protocol fewer contacts were unnecessarily screened and referrals to the chest clinic increased, as did the number given chemoprophylaxis. The case finding rate is similar to that found prior to the revision of the protocol. The yield from tracing casual contacts continues to be nil. It is very low in contacts of extrapulmonary disease. When the protocol was followed no case of pulmonary TB was missed. The revised protocol seems to be as effective as the previous, more complex protocol. In our area, one of low incidence of TB, screening of casual contacts and of contacts of extrapulmonary TB is not cost-effective. We will concentrate even more on screening close contacts of pulmonary TB.

Pharmacogenomics - it's not just pharmacogenetics.

New technologies in both combinatorial chemistry and combinatorial biology promise to unlock new opportunities for drug discovery and lead optimisation. Using such genome-based technologies to measure the dynamic properties of pharmacological systems, pharmacogenomics can now provide an objective measure of a drug's biological efficacy, including its potential adverse effects.

Cloning and pharmacological characterization of human alpha-1 adrenergic receptors: sequence corrections and direct comparison with other species homologues.

We have cloned cDNAs encoding three human alpha-1 adrenergic receptor (AR) subtypes and characterized pharmacological properties of the expressed receptor protein. A number of significant sequence corrections have been identified and compared with previously published data, at both nucleotide and amino acid levels; the most major differences occur for the human alpha-1a/dAR. Pharmacological characterization was performed simultaneously using six cloned alpha-1AR subtypes (human and rat alpha-1a/d, human and hamster alpha-1b, human and bovine alpha-1c) stably expressed in rat-1 fibroblasts at approximately equal receptor concentrations (1-2 pmol/mg of total protein). In general, human alpha-1AR subtypes have similar pharmacology compared to their rat, hamster and bovine homologs, although a few minor species differences important for alpha-1AR classification are noted. In addition, much lower inactivation (approximately 20%) by the alkylating agent chloroethylclonidine is noted in this study compared to previous reports for both human and bovine alpha-1cAR membrane preparations. All six alpha-1AR subtypes couple to phosphoinositide hydrolysis in a pertussis toxin-insensitive manner, including the cloned human alpha-1a/dAR which had not been expressed previously. In spite of significant sequence differences between human alpha-1ARs and their other species counterparts, previously established ligand selectivity remains fairly comparable. In summary, these data represent the first side-by-side comparison of pharmacological properties between species homologs of alpha-1AR subtypes and should facilitate the development of alpha-1AR subtype selective drugs for clinical use.

Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids.

The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that 'illness'-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found.

Illness-induced hyperalgesia is mediated by a spinal NMDA-nitric oxide cascade.

A variety of experimental manipulations produce enhanced pain responsivity. Recent work has demonstrated that activation of N-methyl-D-aspartate (NMDA) receptors in the spinal cord can produce persistent enhancement of pain via production of nitric oxide and/or prostaglandins. To date, the behavioral paradigms used to study NMDA mediated hyperalgesia have all involved direct excitation of spinal cord dorsal horn neurons via activation of primary nociceptive afferents. The present series of experiments examined whether the NMDA cascade would also be activated by events that do not produce direct pain input to the spinal cord dorsal horn. The hyperalgesia-inducing paradigm used was intraperitoneal lipopolysaccharide (LPS), which causes transient illness. Prior work has shown that LPS induces hyperalgesia via activation of hepatic vagal afferents to the brain, thereby activating a centrifugal pain facilitory circuit. The present study demonstrates that this centrifugal hyperalgesia is produced via activation of the NMDA-nitric oxide cascade at the level of the spinal cord.

Subcutaneous formalin produces centrifugal hyperalgesia at a non-injected site via the NMDA-nitric oxide cascade.

Previous work has demonstrated that pain facilitation can occur following injection of subcutaneous irritants, such as formalin. Such studies have focused on apparent pain facilitation induced at the site of irritant injection. Changes in processing of incoming pain information have typically been assumed to result from activation of neurocircuitry intrinsic to the spinal cord. The present series of studies have examined hyperalgesia exhibited at a site distant from the site of irritant injection and have begun to define the neurocircuitry and neuropharmacology underlying this pain enhancement. This work demonstrates that s.c. formalin injected into the dorsum of one hindpaw in rats produces prolonged hyperalgesia as measured by the tailflick test. Hyperalgesia is not mediated solely by circuitry intrinsic to the spinal cord, but rather involves activation of centrifugal pathways originating within the brain and descending to the spinal cord via pathway(s) outside of the dorsolateral funiculus. At the level of the spinal cord, this hyperalgesic state is mediated by an NMDA-nitric oxide cascade, since hyperalgesia can be abolished by administration of either an NMDA antagonist (APV) or a nitric oxide synthesis inhibitor (L-NAME).

Neurocircuitry of illness-induced hyperalgesia.

We have previously demonstrated that illness-inducing agents such as lithium chloride (LiCl) and the bacterial cell wall endotoxin lipopolysaccharide (LPS) produce hyperalgesia on diverse pain measures. The present series of studies attempted to identify the neurocircuitry mediating these effects. These studies have demonstrated that illness-inducing agents produce hyperalgesia by activating: (a) peripheral nerves rather than by generating a blood-borne mediator (Expt. 1); (b) vagal afferents, specifically afferents within the hepatic branch of the vagus (Expt. 2); (c) as yet unidentified brain site(s) rostral to the mid-mesencephalon (Expt. 6); (d) a centrifugal pathway that arises from the nucleus raphe magnus, and not from the adjacent nucleus reticularis paragigantocellularis pars alpha (Expts. 4 and 5); (e) a centrifugal pathway in the dorsolateral funiculus of the spinal cord (Expt. 3); and (f) the same centrifugal pathways for diverse illness inducing agents (Expts. 3, 7 and 8). These data call for the re-evaluation of a number of assumptions inherent in previous studies of hyperalgesia.