Deaths by suicide and their relationship with general and psychiatric hospital discharge: 30-year record linkage study.

BACKGROUND: Studies have rarely explored suicides completed following discharge from both general and psychiatric hospital settings. Such research might identify additional opportunities for intervention. AIMS: To identify and summarise Scottish psychiatric and general hospital records for individuals who have died by suicide. METHOD: A linked data study of deaths by suicide, aged ≥15 years from 1981 to 2010. RESULTS: This study reports on a UK data-set of individuals who died by suicide (n = 16 411), of whom 66% (n = 10 907) had linkable previous hospital records. Those who died by suicide were 3.1 times more frequently last discharged from general than from psychiatric hospitals; 24% of deaths occurred within 3 months of hospital discharge (58% of these from a general hospital). Only 14% of those discharged from a general hospital had a recorded psychiatric diagnosis at last visit; an additional 19% were found to have a previous lifetime psychiatric diagnosis. Median time between last discharge and death was fourfold greater in those without a psychiatric history. Diagnoses also revealed that less than half of those last discharged from general hospital had had a main diagnosis of 'injury or poisoning'. CONCLUSIONS: Suicide prevention activity, including a better psychiatric evaluation of patients within general hospital settings deserves more attention. Improved information flow between secondary and primary care could be facilitated by exploiting electronic records of previous psychiatric diagnoses.

Evaluation of methods for one-dimensional spatial analysis of two-dimensional patterns in mouse chimaeras.

The relative extent of cell mixing in tissues of mouse chimaeras or mosaics can be studied by comparing the distributions of the two cell populations in the tissues. However, the mean patch size is misleading because it is affected by both the extent of cell mixing and the relative contributions of the two cell populations. Previous work suggested that effects attributable to differences in tissue composition among chimaeras can be factored out either by correcting the mean patch size or by using the median patch size for the minority cell population and restricting the analysis to grossly unbalanced chimaeras. In the present study, computer simulations of two-dimensional mosaic arrays of black and white squares (representing cells) were used to simulate chimaeric tissues. Random arrays simulated tissues with extensive cell mixing, arrays of cell clumps (representing coherent clones) simulated less mixed tissues, and striped arrays simulated tissues with elongated but fragmented descendent clones. The computer simulations predicted that (i) the median patch length (minority cell population) and the corrected mean patch length would both distinguish between random and clumped patterns and (ii) differences in the variation of the composition of two perpendicular series of one-dimensional transects would distinguished between stripes and randomly orientated patches. Both predictions were confirmed by analysis of histological sections of the retinal pigment epithelium from fetal and adult mouse chimaeras. This study demonstrates that two types of non-random two-dimensional variegated patterns (clumps and stripes) can be identified in chimaeras without two-dimensional reconstruction of serial sections.

Reduced risk of oestrogen receptor positive breast cancer among peri- and post-menopausal women in Scotland following a striking decrease in use of hormone replacement therapy.

Many countries report a decline in breast cancer incidence among peri- and post-menopausal women following a decline in HRT prescribing. To investigate recent Scottish incidence trends, European age-standardised incidence rates from 1997 to 2005 were stratified by method of first detection, ER status and age group. We developed change point models of the annual age-specific cases for the peri- and post-menopausal age groups and ER status using Poisson regression. In Scotland all HRT categories together show a 32.4% increase in the number of items dispensed in 1993-2000 followed by a striking 61.8% decline by 2007. The incidence rates of screen-detected tumours increased gradually in the 50-64 and 65-74 age groups. For the older age group this increase accelerated after 2003 corresponding to an extension of the age range of screening. For ER positive tumours in the 50-64 age group, age-standardised rates increased 31.5% from 1997 to 2000, followed by a statistically significant decrease of 11.2% by 2005 (change in slope=-0.0943, P<0.0001). We conclude that an overall incidence in the 50-64 age group declined since 2000 reflecting the sudden fall in HRT dispensed items and is largely accounted for by the decrease in ER positive tumour incidence. A longer term decline in ER negative tumours for this age group was pre-existing and is unaffected by the collapse in HRT prescribing.

Porphyrinuria in childhood autistic disorder is not associated with urinary creatinine deficiency.

BACKGROUND: Urinary metabolite measurements are often normalized to levels of the ubiquitous metabolite creatinine (CRT) to take account of variations in fluid export. Following CRT normalization, excesses of porphyrins and isoprostanes have been reported in the urines of children with neurodevelopmental disorders. It was suggested (Whiteley et al., 2006, Pediatr. Int. 2006; 48: 292-297) that urinary CRT levels may be depressed in children with autism spectrum disorders. This prompted re-evaluation of CRT levels in such children. METHODS: First matinal urinary CRT levels were compared between subjects in different diagnostic categories including autistic disorder, pervasive developmental disorder not otherwise specified (PDD-NOS) and hyperactivity, before and after correction for age and gender. A larger reference group, consisting of subjects with unrelated disorders and Asperger disorder, with no reported porphyrin excess, was also compared to the group with autistic disorder, both for CRT and for porphyrin (coproporphyrin, COPRO) excess. RESULTS: No significant difference in CRT was observed between any of the categories analyzed, also when corrected for age and gender. In contrast, urinary COPRO levels were significantly higher in autistic disorder versus reference groups, either when expressed as absolute values (independent of CRT levels) or when normalized to CRT. CONCLUSIONS: These data do not support a systematic reduction in urinary CRT levels in subjects with autism spectrum disorders including autistic disorder and PDD-NOS. Urinary COPRO excess in autistic disorder was not associated with or consequent upon urinary CRT deficiency. Differences between affected and control subjects in age and sampling time, as reported by Whiteley et al., may underlie the apparent CRT reduction.

Porphyrinuria in childhood autistic disorder: implications for environmental toxicity.

To address a possible environmental contribution to autism, we carried out a retrospective study on urinary porphyrin levels, a biomarker of environmental toxicity, in 269 children with neurodevelopmental and related disorders referred to a Paris clinic (2002-2004), including 106 with autistic disorder. Urinary porphyrin levels determined by high-performance liquid chromatography were compared between diagnostic groups including internal and external control groups. Coproporphyrin levels were elevated in children with autistic disorder relative to control groups. Elevation was maintained on normalization for age or to a control heme pathway metabolite (uroporphyrin) in the same samples. The elevation was significant (P < 0.001). Porphyrin levels were unchanged in Asperger's disorder, distinguishing it from autistic disorder. The atypical molecule precoproporphyrin, a specific indicator of heavy metal toxicity, was also elevated in autistic disorder (P < 0.001) but not significantly in Asperger's. A subgroup with autistic disorder was treated with oral dimercaptosuccinic acid (DMSA) with a view to heavy metal removal. Following DMSA there was a significant (P = 0.002) drop in urinary porphyrin excretion. These data implicate environmental toxicity in childhood autistic disorder.

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea.

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.

Bos taurus and Bos indicus (Sahiwal) calves respond differently to infection with Theileria annulata and produce markedly different levels of acute phase proteins.

Disease-resistant livestock could provide a potentially sustainable and environmentally sound method of controlling tick and tick-borne diseases of livestock in the developing world. Advances in the knowledge and science of genomics open up opportunities to identify selectable genes controlling disease resistance but first, breeds and individuals with distinguishable phenotypes need to be identified. The Bos indicus breed, Sahiwal, has been exploited in dairy breeding programmes, because it is resistant to ticks and has relatively good performance characteristics compared to other indigenous cattle breeds of tropical regions. The analyses reported here show that Sahiwal calves were also more resistant than European Bos taurus (Holstein) dairy breed calves to tick-borne tropical theileriosis (Theileria annulata infection). Following experimental infection with T. annulata sporozoites, a group of Sahiwal calves all survived without treatment, with significantly lower maximum temperatures (P<0.01) and lower rates of parasite multiplication (P<0.05) than a group of Holstein calves, which all had severe responses. Although the Sahiwals became as anaemic as the Holsteins, other measures of pathology, including enlargement of the draining lymph node and the acute phase proteins, alpha1 acid glycoprotein and haptoglobin, were significantly less in the Sahiwals than in the Holsteins (P<0.05). Additionally, the Sahiwals had significantly lower resting levels of alpha1 acid glycoprotein than the Holsteins (P<0.05). Production of a third acute phase proteins, serum amyloid A, had very similar kinetics in both breeds. Acute phase proteins are produced in response to systemic release of the kinds of pro-inflammatory cytokines that are thought to be responsible for the pyrexic, cachectic and anorexic responses characteristic of tropical theileriosis. The prolonged production of alpha1 acid glycoprotein in the Holsteins is indicative of chronic production of circulating pro-inflammatory cytokines. In contrast, Sahiwals appear able to overcome infection with T. annulata as well as limit pathology by preventing the over-stimulation of pathways involving these cytokines.

Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle.

The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

Mono-allelic expression of variegating transgene locus in the mouse.

We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation.

Improvement of an electrical activation protocol for porcine oocytes.

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

Multiple effects of genetic background on variegated transgene expression in mice.

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.