Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection.

BACKGROUND: Human cytomegalovirus (CMV) infection is an important risk factor in the post-transplant (Tx) recovery phase for both hematopoietic stem cell Tx (HSCT) and solid organ Tx (SOT) recipients. CMV infection may be prevented or controlled by simultaneously inducing both CMV-specific neutralizing antibody (nAb) and cellular immunity. Soluble (s) UL55 (surface glycoprotein), UL83 (tegument protein) and UL123/e4 (nuclear protein) are immunodominant in eliciting both CMV nAb and cellular immunity. An attenuated poxvirus, modified vaccinia Ankara (MVA) was selected to develop this vaccine strategy in Tx recipients, because of its clinical safety record, large foreign gene capacity, and capability to activate strong humoral and cellular immune responses against recombinant antigens. OBJECTIVES: A subunit vaccine that targets multiple CMV antigens will be used to gain maximal coverage and protective function against CMV infection. rMVA simultaneously expressing sUL55, UL83 and UL123/e4 will be generated, and humoral and cellular immunity it elicits will be characterized, after murine immunization and in vitro to amplify clinical recall responses. STUDY DESIGN: rMVA will be constructed in two steps using UL123/e4-pLW22 followed by sUL55-UL83-pLW51 transfer plasmids. Western blots will be used to characterize expression levels of each antigen. Primary immunity will be evaluated in mouse models, while recall responses to the virally expressed CMV antigens will be assessed in human peripheral blood. RESULTS: We generated CMV-MVA via homologous recombination, and demonstrated high expression levels of sUL55, UL83 and UL123/e4 by Western blot. CMV-MVA immunization potently induced both humoral and cellular immunity to sUL55, UL83 and UL123 after murine immunization, and cellular immunity to UL83 and UL123 by in vitro amplification of T cell recall responses in human PBMC. CONCLUSIONS: rMVA promotes high level expression of three immunodominant CMV antigens, which is reflected in results of immunization studies in which high titers of UL55-specific antibodies and CD4+ T-help are detected, as well as high levels of UL83-specific and moderate levels of UL123-specific CD8+ CTL.     

What should we do with donated embryos that may be genetically affected?

The ethical and legal issues arising from genetic screening in embryo donation are discussed in relation to two recent cases where embryos with uncertain genetic health were offered for donation.     

Actions of phencyclidine on rat locus coeruleus neurones in vitro

Intracellular recordings were made from neurones in the rat locus coeruleus in a brain slice maintained in vitro. Phencyclidine and related psychotomimetic drugs, applied in known concentrations in the fluid bathing the slice, depressed responses to N-methyl-D-aspartic acid noradrenaline (in the presence of the uptake inhibitor desmethylimipramine) and [D-Ala2,MePhe4,Gly-ol5]enkephalin and also prolonged the action potential. The sensitivities of these responses to depression by phencyclidine was N-methyl-D-aspartic acid (IC50 0.4 microM) greater than noradrenaline (IC50 3.9 microM) greater than [D-Ala2,MePhe4,Gly-ol5]enkephalin (IC50 8.5 microM) greater than prolongation of the action potential (41% increase by 30 microM). Stereoselectivity was observed only in the depression of responses to N-methyl-D-aspartic acid where (+)-1-(1-phenylcyclohexyl)-3-methyl piperidine was 3.3-fold more potent in suppressing N-methyl-D-aspartic acid depolarizations than its (-) isomer. The responses to N-methyl-D-aspartic acid were also depressed by the structurally unrelated psychotomimetic (+/-)-N-allyl-N-normetazocine (IC50 0.9 microM). All of the effects of the psychotomimetic drugs examined were slow in onset and difficult to reverse following washout. No effect of phencyclidine (0.03-100 microM) or related drugs was observed on membrane potential, input resistance or spontaneous action potential firing rate of locus coeruleus neurones. The depression of responses to N-methyl-D-aspartic acid by phencyclidine was the most potent and the only stereoselective effect of those studied. The importance of this effect and of those not showing stereoselectivity in relation to the phencyclidine behavioural syndrome is discussed.     

Diagnosis of adrenal cortical dysfunction by liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to measure 6 metabolic compounds of the adrenocorticosteroid pathway simultaneously on residual specimens from patients who had previously been previously diagnosed, on the basis of immunoassays, as having congenital adrenal hyperplasia (CAH), 11 beta-hydroxylase deficiency, 21-hydroxylase deficiency, or Addison disease (adrenal insufficiency). Two subjects with normal adrenal function had serum cortisol values of 13.6 and 8.9 micrograms/dL and serum cortisone values of 2.1 and 0.6 microgram/dL, but the rest of the compounds were undetectable. Two patients with 11 beta-hydroxylase deficiency had serum 11 beta-deoxycortisol values of 14.9 and 10.0 micrograms/dL and serum 11-deoxycorticosterone values of 3.9 and 1.0 microgram/dL, but their serum levels of cortisol and cortisone were diminished. A patient with 21-hydroxylase deficiency had a highly increased serum 17-hydroxyprogesterone concentration of 28.5 micrograms/dL (or 28,500 ng/dL, the traditional unit to report this assay) and a serum 21-deoxycortisol concentration of 6.9 ug/dL (this is a pathologic marker of 21-hydroxylase deficiency that is nondetectable in sera of healthy subjects). This patient also had diminished concentrations of serum cortisol and cortisone (0.9 and 0.3 microgram/dL, respectively). At 30 and 60 min after corticotropin (ACTH) stimulation, serum cortisol was the only compound that showed a dramatic increase in the normal subjects; the patient with 21-hydroxylase deficiency showed an increase of serum 17-hydroxyprogesterone level, but no increase of serum cortisol level; the patient with Addison disease showed no increase in the levels of serum cortisol or other compounds. Metyprapone, which blocks 11 beta-hydroxylase activity, increased the serum 11-deoxycorticosteroid levels and decreased the serum cortisol level. This pilot study demonstrates that it is feasible to use LC-MS/MS for the laboratory diagnosis of adrenal cortical dysfunction. The authors envision that LC-MS/MS may soon become an ideal analytical technique for the diagnosis of such endocrine diseases.     

A survey of bacterial diversity in ticks,lice and fleas from Australia

We isolated bacteria from ticks, lice and fleas. Partial small subunit rRNA sequences were obtained for each isolate and the closest matches in the FastA database were determined. These bacteria were mostly Gram-positive (Firmicutes), although representatives from the Proteobacteria (alpha, beta, gamma subdivisions) and CFB group were also isolated. Most of the isolates we found were from genera that were present in most of the ectoparasites studied, but a few genera were restricted to one species of ectoparasite. The most commonly isolated genera were Stenotrophomonas, Staphylococcus, Pseudomonas, Acinetobacter and Bacillus. Species of Bacillus and Proteus, which have biopesticide potential, were found in some of these ectoparasites. Overall, the communities of bacteria were similar to those found in other studies of parasitic arthropods.     

Overwhelmingly asynchronous firing of rat subthalamic nucleus neurones in brain slices provides little evidence for intrinsic interconnectivity

In Parkinson's disease the neurones of the subthalamic nucleus show increased synchrony and oscillatory burst discharge, thought to reflect a breakdown of parallel processing in basal ganglia circuitry. To understand better the mechanisms underlying this transition, we sought to mimic this change in firing pattern within sagittal slices of rat midbrain. The firing patterns of up to four simultaneously extracellularly recorded subthalamic nucleus (STN) neurones were analysed using burst and oscillation detection programs, and correlated activity between pairs of neurones assessed. In control conditions all but 11 of 488 (2%) neurones fired in a predominantly tonic pattern (with mean oscillation frequency >3 Hz), with no significantly cross-correlated activity in any of 393 pairs of neurones. The glutamate antagonists DL-2-amino-phosphonopentanoic acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-methyl-2-(phenylethynyl)pyridine (MPEP) did not change the firing rate or pattern of these cells, providing no evidence for a role of glutamatergic collaterals within the STN under these conditions. The GABA(A) receptor antagonist bicuculline and GABA(B) receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl]phenylmethyl phosphinic acid (CGP 55845) were also without effect on firing rate or pattern in these cells, suggesting that there was no active input from other GABAergic basal ganglia nuclei in this slice. The dopamine receptor antagonist haloperidol caused no significant change to firing rate or pattern of firing in these cells, suggesting that there was no active dopaminergic input in this slice. Excitations of STN neurones by muscarine, (+)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD), N-methyl-D-aspartic acid (NMDA) or dopamine were all unaccompanied by a change in firing pattern or any significant correlated activity between STN neurone pairs. Burst firing could be induced in STN neurones with either the potassium channel blocker tetraethylammonium (TEA; 10 mM; in 100/138 [72%] of cells) or with a combination of NMDA and the calcium-activated potassium channel blocker apamin (in 101/216 [47%] of cells). Burst firing in TEA was unchanged by CNOX and APV, MPEP, CGP55845, haloperidol, dopamine, and ACPD, although muscarine produced a significant increase in oscillation frequency. Burst firing in NMDA and apamin was unchanged by CNQX and APV, dopamine, muscarine and ACPD, although bicuculline caused a significant increase in oscillation frequency. Such burst firing was not accompanied by synchrony in any condition, either alone, or during application of excitatory agents or glutamate or GABA antagonists. As the bursting seen here was unaccompanied by the synchronous activity that has often been observed (pathologically) in vivo, it probably reflects solely intrinsic STN neuronal properties, rather than network activity. No functional role was found for glutamatergic collaterals within the STN, either when cells are firing tonically or burst firing. The circuitry needed to produce synchrony in the STN is most likely not intrinsic to the STN itself, but requires connections with other basal ganglia nuclei, and/or the cortex, which are not present in this preparation.     

Testing a policy for skull radiography (and admission) following mild head injury

There has been, and continues to be, conflicting advice regarding skull radiography following an apparently uncomplicated head injury. Policies on admission are often ill defined. In 1983, representatives of the Royal College of Radiologists met neurosurgeons at a seminar in Harrogate organized by the DHSS and laid down clear, but little publicized, joint recommendations: "The Harrogate Criteria". The survey described here evaluated an existing liberal policy for skull radiography (and a loose policy for admissions) against these recommendations, and it was concluded that the number of patients having skull radiography (74% of head injury attenders) was excessive. Measured against the "Harrogate Criteria", requests for 36% of the adult and 47% of children's skull radiographs did not fulfil these criteria. As a corollary, it was also concluded that loose admissions policies could usefully be altered to comply more closely with the Harrogate recommendations. This would not necessarily reduce the numbers admitted, but would provide casualty officers with a framework on which to base this crucial aspect of clinical decision making, where presently there is often very little structure at all. The authors suggest that all the options have not been fully explored, and that a simple but important alteration to the Harrogate guidelines would provide an even closer link between policy on skull radiography and patient admission. Specifically, most patients who are to be admitted do not need skull radiographs.