illicit trade in tobacco products; corporate political activity; smuggling, tobacco industry
Genetic manipulation of single-celled organisms such as the Leishmania parasite enables in depth analysis of the consequences of genotypic change on biological function. In probing the immune responses to infection, use of transgenic Leishmania has the potential to unravel both the contribution of the parasite to the infection process and the cellular interactions and mechanisms that characterize the innate and adaptive immune responses of the host. Here, we briefly review recent technical advances in parasite genetics and explore how these methods are being used to investigate parasite virulence factors, elucidate immune regulatory mechanisms and contribute to the development of novel therapeutics for the leishmaniases. Recent developments in imaging technology, such as bioluminescence and intravital imaging, combined with parasite transfection with fluorescent or enzyme-encoding marker genes, provides a rich opportunity for novel assessment of intimate, real-time host–parasite interactions at a previously unexplored level. Further advances in transgenic technology, such as the introduction of robust inducible gene cassettes for expression in intracellular parasite stages or the development of RNA interference methods for down-regulation of parasite gene expression in the host, will further advance our ability to probe host–parasite interactions and unravel disease-promoting mechanisms in the leishmaniases.
Nr2e1 encodes a stem cell fate determinant of the mouse forebrain and retina. Abnormal regulation of this gene results in retinal, brain, and behavioral abnormalities in mice. However, little is known about the functionality of human NR2E1. We investigated this functionality using a novel knock-in humanized-mouse strain carrying a single-copy bacterial artificial chromosome (BAC). We also documented, for the first time, the expression pattern of the human BAC, using an NR2E1-lacZ reporter strain. Unexpectedly, cerebrum and olfactory bulb hypoplasia, hallmarks of the Nr2e1-null phenotype, were not fully corrected in animals harboring one functional copy of human NR2E1. These results correlated with an absence of NR2E1-lacZ reporter expression in the dorsal pallium of embryos and proliferative cells of adult brains. Surprisingly, retinal histology and electroretinograms demonstrated complete correction of the retina-null phenotype. These results correlated with appropriate expression of the NR2E1-lacZ reporter in developing and adult retina. We conclude that the human BAC contained all the elements allowing correction of the mouse-null phenotype in the retina, while missing key regulatory regions important for proper spatiotemporal brain expression. This is the first time a separation of regulatory mechanisms governing NR2E1 has been demonstrated. Furthermore, candidate genomic regions controlling expression in proliferating cells during neurogenesis were identified.
Osetogenesis Imperfecta (OI) is a heritable disease, which results from an abnormal amount or structure of Type I collagen. Bisphosphonates, a class of synthetic antiresorptive drugs used in osteoporosis management, are also used to decrease fracture incidence and improve quality of life in children with OI. In this study we used the oim mouse to test the hypotheses that pamidronate treatment during active growth 1. produces larger, stronger, stiffer long bone diaphyses without altering bone material properties, and 2. negatively impacts longitudinal bone growth. Our results indicate that femoral cross-sectional moment of inertia in the distal metaphysis tended to increase with pamidronate treatment and that the treated bones are thicker and structurally stiffer, but shorter than their control-dose counterpar
osteogenesis imperfecta; bisphosphonate; pamidronate; oim mouse; bone
Bisphosphonates alleviate bone pain and fractures associated with osteogenesis imperfecta (OI). Using the oim mouse model to simulate variations in OI severity, the effect of pamidronate on bone growth was assessed. Homozygous (oim/oim) and heterozygous (oim/wt) mice from 4 to 12 weeks of age were given pamidronate at 0 mg/kg/wk (control), 1.25 mg/kg/wk (low) and 2.5 mg/kg/wk (high). Humerus and ulna lengths were reduced in oim/oim mice relative to those of the oim/wt. Further, the oim/oim genotype exhibited a 23.5% prevalence of fractures in these bones as compared to the 2.8% prevalence observed in the oim/wt mice. Pamidronate tended to reduce fracture prevalence in a dose dependent manner for the oim/oim genotype (p<0.08) but had no effect on the low fracture prevalence in oim/wtmice. The high dose of pamidronate reduced bone length in females of both genotypes but not males when compared to control (p<0.01). Pamidronate increased growth plate area (p<0.05) by increasing growth plate height, particularly the proliferative and hypertrophic zones, in both genotypes indicating reduced growth plate cell turnover. The increased area coincided with increased osteoclast numbers in the metaphyseal region (p<0.05) though when corrected for the greater mineralized surface area that accompanies bisphosphonate treatment, osteoclasts per surface area were reduced indicating reduced resorptive capacity. This study demonstrated that the effects of pamidronate were independent of the degree of collagen deficit and fracture prevalence was improved in the most severe OI model, the oim/oim genotype.
pamidronate; oim; growth plate; osteogenesis imperfecta; bisphosphonate
To assess whether macular dysfunction caused by unilateral subretinal neovascular membranes (SRNs) is associated with pupil “evasion” (that is, increased initial rate of re‐dilation following a brief light stimulus).
Comparative observational series. 20 eyes of 10 participants, all with unilateral SRNs and healthy fellow eyes. Dynamic infrared pupillography at seven stimulus intensities (duration 1100 ms, intensities over 2 log unit range). Pupil evasion ratio (PEVR; defined as the ratio of light response amplitude to amount of recovery at the mid‐time point of re‐dilation expressed as a percentage) was calculated for each stimulus intensity (mean of five recordings).
Inter‐eye PEVR is significantly reduced in eyes with SRN (that is, greater pupil evasion in SRN eyes: range p = 0.002 to p = 0.05 (paired t test)) and is most apparent at higher stimulus intensities.
PEVR is a novel parameter that is analogous to the pupil escape ratio, but measured following a short rather than a sustained light stimulus. PEVR is significantly altered by macular disease. Clinically PEVR may be used to detect occult unilateral or asymmetric maculopathy in situations such as ocular media opacities like cataract, when pupil reactions are unaffected or augmented, while other tests of retinal function are diminished. PEVR represents altered neuronal firing in cones and macular ganglion cells.
pupil evasion; pupil escape; pupil index; subretinal membrane; neovascular membrane
Phragmidium violaceum causes leaf rust on the European blackberry (Rubus fruticosus L. aggregate). Multiple strains of this pathogen have been introduced into southern Australia for the biological control of at least 15 taxa of European blackberry, a nonindigenous, invasive plant. In climates conducive to leaf rust, the intensity of disease varies within and among infestations of the genetically variable host. Genetic markers developed from the selective amplification of microsatellite polymorphic loci were used to assess the population genetic structure and reproductive biology of P. violaceum within and among four geographically isolated and diseased infestations of the European blackberry in Victoria, Australia. Despite the potential for long-distance aerial dispersal of urediniospores, there was significant genetic differentiation among all populations, which was not associated with geographic separation. An assessment of multilocus linkage disequilibrium revealed temporal and geographic variation in the occurrence of random mating among the four populations. The presence of sexual spore states and the results of genetic analyses indicated that recombination, and potentially random migration and genetic drift, played an important role in maintaining genotypic variation within populations. Recombination and genetic differentiation in P. violaceum, as well as the potential for metapopulation structure, suggest the need to release additional, genetically diverse strains of the biocontrol agent at numerous sites across the distribution of the Australian blackberry infestation for maximum establishment and persistence.
Bisphosphonates decrease chondrocyte turnover at the growth plate and impact bone growth. Likewise vascular endothelial growth factor (VEGF) plays an important role in endochondral bone elongation by influencing chondrocyte turnover at the growth plate. To investigate whether the action of bisphosphonate on the growth plate works through VEGF, VEGF protein expression and isoform transcription in endochondral chondrocytes isolated from growing mice and treated with a clinically used bisphosphonate, alendronate, were assessed. Alendronate at 10µM and 100µM concentrations decreased secreted VEGF protein expression but not cell associated protein. Bisphosphonates are known to inhibit the mevalonate intracellular signaling pathway used by VEGF. Addition of the mevalonate pathway intermediates farnesol (FOH) and geranylgeraniol (GGOH) interacted with the low concentration of alendronate to further decrease secreted VEGF protein whereas FOH partially restored VEGF protein secretion when combined with the high alendronate. Similar to the protein data, the addition of alendronate decreased VEGF mRNA isoforms. VEGF mRNA levels were rescued by the GGOH mevalonate pathway intermediate at the low alendronate dose whereas neither intermediate consistently restored the VEGF mRNA levels at the high alendronate dose. Thus, the bisphophonate alendronate impairs growth plate chondrocyte turnover by down-regulating the secreted forms of VEGF mRNA and protein by inhibiting the mevalonate pathway.
VEGF; chondrocyte; bisphosphonate.
Osteoclastogenesis is dependent on distinct stimuli that prime and activate osteoclast differentiation. One cytokine needed to prime monocytes for osteoclastogenesis is TGF-β, which enables and augments RANKL and TNF-α-induced osteoclast differentiation. However, the precise time-period during which this occurs and the molecular mechanism mediating this action are unknown. We report here TGF-β prime monocytes for osteoclast formation within 24 h by regulating expression of NFATc1, a key osteoclastic transcription factor. TGF-β directly induces cytoplasmic NFATc1 expression within 24 h, but is unable to stimulate NFATc1 nuclear translocation. Furthermore, RANKL-induced NFATc1 expression is dependent on the presence of TGF-β during the early stages of osteoclastogenesis. Similarly, TNF-α activates osteoclastogenesis by stimulating translocation of TGF-β-induced NFATc1. In light of these findings, it is apparent that osteoclast formation is dependent on coordinated interactions between TGF-β and RANKL/TNF-α that regulate the expression and intracellular distribution of NFATc1 during early stages of osteoclast differentiation.
Osteoclast; TGF-β; Priming; NFATc1; Monocyte differentiation
Bisphosphonates, used to treat diseases exhibiting increased osteoclast activity, reduce longitudinal bone growth through an as yet undefined mechanism. Pamidronate, an aminobisphosphonate, was given weekly to mice at 0, 1.25, or 2.50 mg/kg/wk beginning at 4 weeks of age. At 12 weeks of age, humeral length, growth plate area, regional chondrocyte cell numbers, chondrocyte apoptosis, TRAP stained osteoclast number, and osteoclast function assessed by cathepsin K immunohistochemistry were quantified. Humeral length was decreased in pamidronate treated mice compared to vehicle control mice, and correlated with greater growth plate areas reflecting greater proliferative and hypertrophic chondrocyte cell numbers with fewer hypertrophic cells undergoing apoptosis. Pamidronate treatment increased TRAP stained osteoclast numbers yet decreased cathepsin K indicating that pamidronate repressed osteoclast maturation and function. The data suggest that long term cyclic pamidronate treatment impairs bone growth by inhibition of osteoclast maturation thereby reducing cartilage-to-bone turnover within the growth plate.
Pamidronate; growth plate; bisphosphonates.
Congenital colobomata of the eye are important causes of childhood visual impairment and blindness. Ocular coloboma can be seen in isolation and in an impressive number of multisystem syndromes, where the eye phenotype is often seen in association with severe neurological or craniofacial anomalies or other systemic developmental defects. Several studies have shown that, in addition to inheritance, environmental influences may be causative factors. Through work to identify genes underlying inherited coloboma, significant inroads are being made into understanding the molecular events controlling closure of the optic fissure. In general, severity of disease can be linked to the temporal expression of the gene, but this is modified by factors such as tissue specificity of gene expression and genetic redundancy.
Peripheral immune activation can have profound physiological and behavioral effects including induction of fever and sickness behavior. One mechanism through which immune activation or immunomodulation may affect physiology and behavior is via actions on brainstem neuromodulatory systems, such as serotonergic systems. We have found that peripheral immune activation with antigens derived from the nonpathogenic, saprophytic bacterium, Mycobacterium vaccae, activated a specific subset of serotonergic neurons in the interfascicular part of the dorsal raphe nucleus (DRI) of mice, as measured by quantification of c-Fos expression following intratracheal (12 h) or s.c. (6 h) administration of heat-killed, ultrasonically disrupted M. vaccae, or heat-killed, intact M. vaccae, respectively. These effects were apparent after immune activation by M. vaccae or its components but not by ovalbumin, which induces a qualitatively different immune response. The effects of immune activation were associated with increases in serotonin metabolism within the ventromedial prefrontal cortex, consistent with an effect of immune activation on mesolimbocortical serotonergic systems. The effects of M. vaccae administration on serotonergic systems were temporally associated with reductions in immobility in the forced swim test, consistent with the hypothesis that the stimulation of mesolimbocortical serotonergic systems by peripheral immune activation alters stress-related emotional behavior. These findings suggest that the immune-responsive subpopulation of serotonergic neurons in the DRI is likely to play an important role in the neural mechanisms underlying regulation of the physiological and pathophysiological responses to both acute and chronic immune activation, including regulation of mood during health and disease states. Together with previous studies, these findings also raise the possibility that immune stimulation activates a functionally and anatomically distinct subset of serotonergic neurons, different from the subset of serotonergic neurons activated by anxiogenic stimuli or uncontrollable stressors. Consequently, selective activation of specific subsets of serotonergic neurons may have distinct behavioral outcomes.
depression; hippocampus; prefrontal cortex; raphe; 5-HT; vagus; ANOVA, analysis of variance; AP, area postrema; c-Fos-ir, c-Fos-like-immunoreactive; DR, dorsal raphe nucleus; DRC, dorsal raphe nucleus, caudal part; DRI, dorsal raphe nucleus, interfascicular part; ECG, electrocardiogram; EDTA, ethylenediaminetetraacetic acid; EMG, electromyogram; HPLC, high pressure liquid chromatography; IL-6, interleukin-6; IL-10, interleukin-10; i.t., intratracheal; LPS, lipopolysaccharide; LSD, least significant difference; mlf, medial longitudinal fasciculus; M. vaccae, Mycobacterium vaccae; Mv-NC, Mycobacterium vaccae antigen, M. vaccae coupled to nitrocellulose beads; NC, nitrocellulose beads; nTS, nucleus of the solitary tract; OVA, ovalbumin; OVA-NC, ovalbumin coupled to nitrocellulose beads; PBG, phenylbiguanide; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline containing 0.3% Triton X-100; RMg, raphe magnus; ROb, raphe obscurus; S.E.M., standard error of the mean; SolDL, dorsolateral part of the nucleus of the solitary tract; TGF-β, transforming growth factor-β; Th1, T helper cell 1; Th2, T helper cell 2; TNF-α, tumor necrosis factor-α; Treg, T regulatory cell; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin
Background/aims: To report the generation of a new mouse model for a genetically determined corneal abnormality that occurred in transgenesis experiments.
Methods: Transgenic mice expressing mutant forms of Rab27a, a GTPase that has been implicated in the pathogenesis of choroideremia, were generated.
Results: Only one transgenic line (T27aT15) exhibited an unexpected eye phenotype. T27aT15 mice developed corneal opacities, usually unilateral, and cataracts, resulting in some cases in phthisical eyes. Histologically, the corneal stroma was thickened and vacuolated, and both epithelium and endothelium were thinned. The posterior segment of the eye was also affected with abnormal pigmentation, vessel narrowing, and abnormal leakage of dye upon angiography but was histologically normal.
Conclusion: Eye abnormality in T27aT15 mice results from random insertional mutagenesis of the transgene as it was only observed in one line. The corneal lesion observed in T27aT15 mice most closely resembles posterior polymorphous corneal dystrophy and might result from the disruption of the equivalent mouse locus.
corneal dystrophy; mutagenesis; transgenic mice
Objective: To determine the effect of caesarean section on breast milk transfer (BMT) to the normal term infant over the first week of life.
Method: A sample of 88 healthy nursing mothers who had a normal vaginal delivery, and 97 mothers who had a caesarean section were recruited from a teaching hospital. Mothers and midwives were instructed to weigh the infants before and after each feed throughout the study period using calibrated portable electronic scales.
Results: The volume of milk transferred to infants born by caesarean section was significantly less than that transferred to infants born by normal vaginal delivery on days 2 to 5 (p < 0.05), but by day 6 there was no difference between the two groups (p = 0.08). The difference could not be explained by any of the maternal and infant variables measured. Birth weight was regained by day 6 in 40% of infants born vaginally compared with 20% in those born by caesarean section.
Conclusion: There is a lag in the profile of the daily volume of breast milk transferred to infants delivered by caesarean section compared with those born by normal vaginal delivery. This study also challenges the widely followed schedules of milk volumes considered to be suitable for the term infant, which appear to be excessive, at least for the first four to five days post partum.
Cetacean strandings elicit much community and scientific interest, but few quantitative analyses have successfully identified environmental correlates to these phenomena. Data spanning 1920–2002, involving a total of 639 stranding events and 39 taxa groups from southeast Australia, were found to demonstrate a clear 11–13- year periodicity in the number of events through time. These data positively correlated with the regional persistence of both zonal (westerly) and meridional (southerly) winds, reflecting general long-term and large-scale shifts in sea-level pressure gradients. Periods of persistent zonal and meridional winds result in colder and presumably nutrient-rich waters being driven closer to southern Australia, resulting in increased biological activity in the water column during the spring months. These observations suggest that large-scale climatic events provide a powerful distal influence on the propensity for whales to strand in this region. These patterns provide a powerful quantitative framework for testing hypotheses regarding environmental links to strandings and provide managers with a potential predictive tool to prepare for years of peak stranding activity.
cetacean strandings; southeast Australia; climate; meridional winds; zonal winds; sea-surface temperature
Sorsby’s fundus dystrophy; photodynamic therapy
Insect wings lack internal muscles, and the orderly, necessary deformations which they undergo in flight and folding are in part remotely controlled, in part encoded in their structure. This factor is crucial in understanding their complex, extremely varied morphology. Models have proved particularly useful in clarifying the facilitation and control of wing deformation. Their development has followed a logical sequence from conceptual models through physical and simple analytical to numerical models. All have value provided their limitations are realized and constant comparisons made with the properties and mechanical behaviour of real wings. Numerical modelling by the finite element method is by far the most time-consuming approach, but has real potential in analysing the adaptive significance of structural details and interpreting evolutionary trends. Published examples are used to review the strengths and weaknesses of each category of model, and a summary is given of new work using finite element modelling to investigate the vibration properties and response to impact of hawkmoth wings.
OBJECTIVE—Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W of EFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles of EFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA.
DESIGN—Molecular genetic analysis.
PARTICIPANTS—Ten unrelated families and 17 young drusen patients.
MAIN OUTCOME MEASURES—Exons 1 and 2 of EFEMP1 were characterised by 5' rapid amplification of cDNA ends and direct sequencing. Exons 1-12 of EFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1 R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data.
RESULTS—Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region.
CONCLUSIONS—EFEMP1 R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.
Keywords: autosomal dominant drusen; molecular genetics
out a pilot study to test the feasibility of health visitors (HVs)
performing neonatal otoacoustic emissions (OAE) hearing screening in
the community using Echoport ILO288 and to evaluate its acceptability
to parents and HVs.
SETTING—Local health centres and babies' homes in urban and
rural settings in West Gloucestershire.
HVs, 683 babies, and their parents.
MEASURES—Coverage rate, age at testing, referral
rate for formal audiology testing, and parental anxiety scores.
RESULTS—Of the 683 babies registered with the study HVs, 99% (675) were tested, with a
median age at first test of 18 days. Parental consent for the study was
refused for six of the eight not tested. Taking a unilateral pass as a
screening pass (for comparison with other studies), 4% (27/675) failed
the first OAE test, and 1.9% (13/675) failed a second OAE test
performed by the HV within a further two weeks and were referred for
formal audiology testing. One baby (0.15%) was found to have a
moderate sensorineural hearing loss on brain stem auditory evoked
responses, giving a false positive rate of 1.7% (12/675). Some 18%
(120/675) were tested at home, of which 80% (96/120) were combined
with another planned reason for HV contact. In all, 82% (555/675) of
tests were carried out in health centre clinics, of which 47%
(260/555) were combined purpose visits. Mean parental anxiety scores
(possible range 0-5) were 0.86, 2.27, and 3.45 before the first test,
first retest, and audiology test respectively. The median time taken
for one HV to complete testing was 12.2 minutes (range 3-65), compared with the 15 minutes currently allocated for two HVs to perform distraction testing. Based on the results of questionnaires, the test
was very well received by parents and HVs alike.
able to perform OAE testing in the neonatal period at home and in local
health centre clinics. They achieve high population coverage rates and
low false positive rates. Universal neonatal hearing screening by HVs
using OAE testing is feasible, well received, and could be less
demanding of HV time than the current distraction testing. This model
of universal neonatal hearing screening should be considered by the
National Screening Committee.
Recommendations exist for the optimal management of vascular surgical emergency patients. A telephone survey of on-call surgical registrars was performed to assess the current state of emergency vascular service provision across the Wessex and South West regions in the UK. Of the 24 hospitals surveyed, 10 had formal on-call arrangements for vascular surgical cover, 14 had informal arrangements where the general surgical consultant on-call provided cover and could contact a vascular surgeon if they were available and 3 hospitals had no such arrangements. No difficulties had been experienced by the on-call staff surveyed with any of the existing arrangements. Only 5 of the hospitals had formal on-call arrangements for interventional radiologists. We conclude that current emergency vascular service provision is suboptimal compared to national guidelines and patients may be subject to unequitable access to services. This may not be tenable in the new era of clinical governance.
The postpericardiotomy syndrome may occur as a complication of temporary and permanent pacing. Physicians involved in procedures which may be complicated by this condition therefore need to be aware of its diagnosis and management.
Keywords: postpericardiotomy syndrome; cardiac pacing