Stargazin and AMPA receptor membrane expression is increased in the somatosensory cortex of Genetic Absence Epilepsy Rats from Strasbourg.

Absence-like seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model are believed to arise in hyperexcitable somatosensory cortical neurons, however the cellular basis of this increased excitability remains unknown. We have previously shown that expression of the Transmembrane AMPA receptor Regulatory Protein (TARP), stargazin, is elevated in the somatosensory cortex of GAERS. TARPs are critical regulators of the trafficking and function of AMPA receptors. Here we examine the developmental expression of stargazin and the impact this may have on AMPA receptor trafficking in the GAERS model. We show that elevated stargazin in GAERS is associated with an increase in AMPA receptor proteins, GluA1 and GluA2 in the somatosensory cortex plasma membrane of adult epileptic GAERS. Elevated stargazin expression is not seen in the epileptic WAG/Rij rat, which is a genetically distinct but phenotypically similar rat model also manifesting absence seizures, indicating that the changes seen in GAERS are unlikely to be a secondary consequence of the seizures. In juvenile (6 week old) GAERS, at the age when seizures are just starting to be expressed, there is elevated stargazin mRNA, but not protein expression for stargazin or the AMPA receptor subunits. In neonatal (7 day old) pre-epileptic GAERS there was no alteration in stargazin mRNA expression in any brain region examined. These data demonstrate that stargazin and AMPA receptor membrane targeting is altered in GAERS, potentially contributing to hyperexcitability in somatosensory cortex, with a developmental time course that would suggest a pathophysiological role in the epilepsy phenotype.

Heterogeneity at the HLA-DRB1 allelic variation locus does not influence multiple sclerosis disease severity, brain atrophy or cognition.

BACKGROUND: HLA-DRB1*1501 (DR15) and other HLA class II alleles increase the risk of developing multiple sclerosis (MS). However, the contribution of genetic heterogeneity to the clinical course of MS remains controversial. We examined the influence of DR15 and other common DRB1 alleles (DRB1*01 (DR1), DRB1*03 (DR3) and DRB1*04 (DR4) on MS severity in a large, Australian, population-based cohort. METHODS: We studied the association between common HLA-DRB1 alleles and genotypes and age of onset as well as three clinical disease severity descriptors: Multiple Sclerosis Severity Score, progression index), and the interval between the first and second attack in 978 patients with relapsing remitting MS and secondary progressive MS. We assessed cognition using the Symbol Digit Modalities Test in 811 patients and brain atrophy using the linear magnetic resonance imaging marker, the intercaudate ratio, in 745 patients. RESULTS: Carrying DR15 significantly decreased the age of MS onset by 3.2 years in homozygotes and 1.3 years in heterozygotes. Carrying the HLA-DR15, -DR1, -DR3 or -DR4 alone or in combination did not affect clinical disease severity, cognition or cerebral atrophy. CONCLUSIONS: This study confirms that heterogeneity of HLA-DRB1 does not influence disease outcome in relapsing MS patients, with the exception of a younger age of onset in HLA-DR15 carriers.

Apolipoprotein genotype does not influence MS severity, cognition, or brain atrophy.

BACKGROUND: The influence of APOE allelic heterogeneity on multiple sclerosis (MS) disease severity has been reported in multiple datasets with conflicting results. Several studies have reported an unfavorable association of APOE epsilon4 with more severe clinical disease course while, in contrast, APOE epsilon2 has been associated with a more benign disease course. In this study, we examine the influence of heterogeneity of the APOE gene on disease severity in a large, Australian, population-based MS cohort. METHODS: Associations between APOE allele status, 2 promoter region single nucleotide polymorphisms (-219 G/T and +113 C/G), and 4 measures of disease severity were tested in 1,006 patients with relapsing-remitting MS and secondary progressive MS: 1) Multiple Sclerosis Severity Score; 2) Progression Index (Expanded Disability Status Scale/disease duration); 3) age at first symptom; and 4) interval between the first and second attack. The Symbol Digit Modalities Test was used as a single cognitive marker in 889 patients. Brain atrophy was measured in 792 patients using the intercaudate ratio. APOE epsilon4 and epsilon3 carriers were stratified by -219 G/T or +113 C/G to investigate haplotypic heterogeneity in the APOE gene region. RESULTS: In this MS study, neither APOE allele status nor promoter region heterogeneity at positions -219 G/T or +113 C/G influenced the clinical disease severity, cognition, or cerebral atrophy. CONCLUSIONS: Allelic and haplotypic heterogeneity of the APOE gene region does not influence multiple sclerosis disease course in this well-defined Australian multiple sclerosis cohort.

HLA-DRB1 associations with disease susceptibility and clinical course in Australians with multiple sclerosis.

Human leucocyte antigen (HLA)-DRB1*1501 and other class II alleles influence susceptibility to multiple sclerosis (MS), but their contribution if any to the clinical course of MS remains uncertain. Here, we have investigated DRB1 alleles in a large sample of 1230 Australian MS cases, with some enrichment for subjects with primary progressive (PPMS) disease (n = 246) and 1210 healthy controls. Using logistic regression, we found that DRB1*1501 was strongly associated with risk (P = 7 x 10(-45)), as expected, and after adjusting for DRB1*1501, a predisposing effect was also observed for DRB1*03 (P = 5 x 10(-7)). Individuals homozygous for either DRB1*15 or DRB1*03 were considerably more at risk of MS than heterozygotes and non-carriers. Both the DRB1*04 and the DRB1*01/DRB1*15 genotype combination, respectively, protected against PPMS in comparison to subjects with relapsing disease. Together, these data provide further evidence of heterogeneity at the DRB1 locus and confirm the importance of HLA variants in the phenotypic expression of MS.

Replication of KIAA0350, IL2RA, RPL5 and CD58 as multiple sclerosis susceptibility genes in Australians.

A recent genome-wide association study (GWAS) conducted by the International Multiple Sclerosis Genetics Consortium (IMSGC) identified a number of putative MS susceptibility genes. Here we have performed a replication study in 1134 Australian MS cases and 1265 controls for 17 risk-associated single nucleotide polymorphisms (SNPs) reported by the IMSGC. Of 16 SNPs that passed quality control filters, four, each corresponding to a different non-human leukocyte antigen (HLA) gene, were associated with disease susceptibility: KIAA0350 (rs6498169) P=0.001, IL2RA (rs2104286) P=0.033, RPL5 (rs6604026) P=0.041 and CD58 (rs12044852) P=0.042. There was no association (P=0.58) between rs6897932 in the IL7R gene and the risk of MS. No interactions were detected between the replicated IMSGC SNPs and HLA-DRB1*15, gender, disease course, disease progression or age-at-onset. We used a novel Bayesian approach to estimate the extent to which our data increased or decreased evidence for association with the six most-associated IMSGC loci. These analyses indicated that even modest P-values, such as those reported here, can contribute markedly to the posterior probability of 'true' association in replication studies. In conclusion, these data provide support for the involvement of four non-HLA genes in the pathogenesis of MS, and combined with previous data, increase to genome-wide significance (P=3 x 10(-8)) evidence of an association between KIAA0350 and risk of disease.

Genetic absence epilepsy rats from Strasbourg have increased corticothalamic expression of stargazin.

Stargazin is membrane bound protein involved in trafficking, synapse anchoring and biophysical modulation of AMPA receptors. A quantitative trait locus in chromosome 7 containing the stargazin gene has been identified as controlling the frequency and duration of absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Furthermore, mutations in this gene result in the Stargazer mouse that displays an absence epilepsy phenotype. GAERS stargazin mRNA expression is increased 1.8 fold in the somatosensory cortex and by 1.3 fold in the thalamus. The changes were present before and after the onset of absence seizures indicating that increases are not a secondary consequence of the seizures. Stargazin protein expression was also significantly increased in the somatosensory cortex after the onset of spontaneous seizures. The results are of significant importance beyond the GAERS model, as they are the first to show that an increase in stargazin expression may be pro-epileptic.

SNP mapping and candidate gene sequencing in the class I region of the HLA complex: searching for multiple sclerosis susceptibility genes in Tasmanians.

This study is an extension to previously published work that has linked variation in the human leukocyte antigen (HLA) class I region with susceptibility to multiple sclerosis (MS) in Australians from the Island State of Tasmania. Single nucleotide polymorphism (SNP) mapping was performed on an 865-kb candidate region (D6S1683-D6S265) in 166 Tasmanian MS families, and seven candidate genes [ubiquitin D (UBD), olfactory receptor 2H3 (OR2H3), gamma-aminobutyric acid B receptor 1 (GABBR1), myelin oligodendrocyte glycoprotein (MOG), HLA-F, HLA complex group 4 (HCG4) and HLA-G] were resequenced. SNPs tagging the extended MS susceptibility haplotype were genotyped in an independent sample of 356 Australian MS trios and SNPs in the MOG gene were significantly over-transmitted to MS cases. We identified significant effects on MS susceptibility of HLA-A*2 (OR: 0.51; P = 0.05) and A*3 (OR: 2.85; P = 0.005), and two coding polymorphisms in the MOG gene (V145I: P = 0.01, OR: 2.2; V142L: P = 0.04, OR: 0.45) after full conditioning on HLA-DRB1. We have therefore identified plausible candidates for the causal MS susceptibility allele, and although not conclusive at this stage, our data provide suggestive evidence for multiple class I MS susceptibility genes.

Wound healing response is a major contributor to the severity of cutaneous leishmaniasis in the ear model of infection.

In the conventional mouse model for cutaneous leishmaniasis involving infection with stationary phase Leishmania major promastigotes at the base of the tail, mice congenic for leishmaniasis resistance loci designated lmr1,2,3 cured their lesions more rapidly and laid down more ordered collagen fibres than the susceptible parental BALB/c mice, while the opposite was the case for the congenic mice carrying the susceptibility loci on the resistant C57BL/6 background. In that model, we showed that wound healing and not T cell responses played a major role in determining the resolution of skin infection. Here, we show a similar disease phenotype in the mouse model that mimics more closely the situation in humans, that is, strictly intradermal infection in the ear pinna with small numbers of metacyclic promastigotes. The data show that at the site of infection the innate and adaptive immune responses act in concert to clear parasites, and induce tissue repair and wound healing. Importantly, the data show that the host responses controlled by the lmr loci, which act locally to control infection in the skin, are distinct from the host responses operating systemically in the draining lymph node.

MRI of the foetal brain.

Ultrasound examinations for foetal brain abnormalities have been a part of the routine antenatal screening programme in the UK for many years. In utero brain magnetic resonance imaging (MRI) is now being used increasingly successfully to clarify abnormal ultrasound findings, often resulting in a change of diagnosis or treatment plan. Interpretation requires an understanding of foetal brain development, malformations and acquired diseases. In this paper we will outline the technique of foetal MRI, relevant aspects of brain development and provide illustrated examples of foetal brain pathology.

The New Zealand Diabetes Passport Study: a randomized controlled trial of the impact of a diabetes passport on risk factors for diabetes-related complications.

AIMS: To assess the efficacy (change in HbA1c) of a patient-held communication, self-empowerment and educational device for people with diabetes (the New Zealand Diabetes Passport) in patients with poor glycaemic control. RESEARCH DESIGN AND METHODS: A 12-month, multicentre, general practice-based randomized controlled trial in urban, provincial and rural New Zealand involving 398 people with poorly controlled Type 1 or Type 2 diabetes. The intervention included a specifically designed and piloted New Zealand Diabetes Passport including information relating to diabetes knowledge, self-assessments, and guidance concerning how to engage with diabetes health professionals. The primary end point was change in HbA1c. Assessments were made at 0, 6 and 12 months. RESULTS: Two hundred and twenty-two patients received the Passport, 176 the control booklet, coming from 69 and 66 general practitioners, respectively. Use of the Passport was associated with a relative reduction in HbA1c of 0.4% (P = 0.017) and a relative increase in weight of 1.0 kg/m2 (P = 0.028), but no changes in diabetes knowledge, attitudes to diabetes or risk factors for diabetic tissue damage. CONCLUSIONS: The dissemination of the New Zealand Diabetes Passport, in isolation, was not associated with improvements in either diabetes knowledge or self-empowerment. While a small improvement in glycaemic control occurred, this was probably due to changes in insulin therapy in the intervention group. It is possible that linking the use of the Passport with other behavioural and educational interventions may make the Passport more useful. Further study is required to confirm the effect of such multifaceted interventions.

Dissociation of disease susceptibility, inflammation and cytokine profile in lmr1/2 congenic mice infected with Leishmania major.

Severity of disease caused by Leishmania major depends on the genetics of the host. Early induction of T helper cell type 1 (Th1)-type responses in resistant C57BL/6 mice and T helper cell type 2 (Th2) in susceptible BALB/c mice is thought to determine cure or disease respectively. We have mapped three loci that confer susceptibility or resistance upon congenic mice on the C57BL/6 or BALB/c backgrounds. Here we examine the histopathology and production of interleukin 4 (IL-4) and interferon gamma (IFN-gamma) in the skin and draining lymph nodes in the congenic and parental mice. We show an evolving granuloma with a staged infiltration of inflammatory cells, but no difference between the groups. As an indication of an early-polarised Th1/Th2 response we measured IFN-gamma and IL-4 in the lymph nodes and found no difference between any of the mice during the first 48 h. During infection, the level of IL-4 correlated with the lesion size, indicating that IL-4 reflects the disease severity rather than controls it. Considering this effect, B6.C(lmr1,lmr2) mice had similar cytokine levels to the parental C57BL/6 mice despite increased susceptibility and C.B6(lmr1,lmr2) were similar to BALB/c despite increased resistance. We conclude that the lmr loci affect disease severity by a mechanism independent of conventional helper T-cell responses.

Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway.

The severity of disease caused by infection with Leishmania major depends critically on the genetics of the host. Early induction of T helper (Th)1-type immune responses in the resistant C57BL/6 mice and Th2-type responses in the susceptible BALB/c mice are thought to determine cure or disease, respectively. We have previously mapped three host response loci in a genetic cross between C57BL/6 and BALB/c mice, and here we show definitively the involvement of these loci in disease severity using animals congenic for each of the loci. Surprisingly, in the late stage of infection when the difference in disease severity between congenic and parental mice was most pronounced, their cytokine profile correlated with the genetic background of the mice and not with the severity of disease. This indicates that the loci that we have mapped are acting by a mechanism independent of Th phenotype.

The hydraulic influence in androgen-related hair growth: implications in autoimmune disease.

Androgen-related changes in hair growth represent something of a mystery. Through the action of dihydrotestosterone (DHT), hair growth is increased in specific areas of the body. Elevated levels of DHT produce a general increase over the larger part of the body, often accompanied by hair loss in specific areas of the scalp. Because of this 'opposite' effect, a genetic difference in the hair follicles is proposed. This view is supported through the success of the 'plug graft' transplantation technique. However, this is unsatisfactory, because transplantation procedures that should work well according to this theory, ultimately fail. There is an alternative 'mechanism', that demonstrates its origins in the prime function of hair as an insulator. This simple mechanism makes sense of all the recognized effects of DHT in the dermal system, and throughout the body. In DHT-related hair growth it can be directly observed. The implication is that DHT achieves its effects through a primary physiological action that can be easily tested given the necessary expertise. Given existing knowledge, such a proven action of DHT would have serious implications for further understanding of female susceptibility to autoimmune disease.

Generating mouse models of retinal disease using ENU mutagenesis.

We used the chemical mutagen, N-ethyl-N-nitrosourea, to induce random point mutations in the germline of the mouse strain C57BL/6 in order to generate models of retinal diseases. 1163 mutagenised first generation mice produced using this approach were examined for eye abnormalities. Approximately one-third (412) presented with some form of ocular abnormality. Most changes were unilateral and confined to the anterior segment of the eye. Less than 10% (44) of identified changes affected the posterior segment of the eye. 21 mice with varying ocular abnormalities, including 17 with retinal changes, were bred to produce second generation mice to confirm genetic inheritance. Genetic inheritance was confirmed in several of these lines including three with retinal changes.

Rapid imaging of human melanoma xenografts using an scFv fragment of the human monoclonal antibody H11 labelled with 111In.

H11 is a human IgM monoclonal antibody which recognizes a novel tumour-associated antigen expressed on melanoma, glioma, breast cancer, colon cancer, prostate cancer, lung cancer and B-cell lymphoma. In this study, a recombinant single-chain Fv (scFv) fragment of H11 labelled with 111In was investigated for tumour imaging in athymic mice implanted subcutaneously with A-375 human melanoma xenografts. H11 scFv was derivatized with diethylenetriaminepentaacetic acid (DTPA) for labelling with 111In. The immunoreactivity of DTPA-H11 scFv against A-375 cells in vitro ranged from 23% to 36%. 111In-DTPA-H11 scFv was rapidly eliminated from the blood and most normal tissues (except the kidneys) reaching maximum tumour/blood ratios of 12:1 at 48 h post-injection. Tumours were imaged as early as 40 min after injection. The kidneys accumulated the highest concentration of radioactivity (up to 185% injected dose/g). Tumour uptake was 1-3% injected dose/g. The whole-body radiation absorbed dose predicted for administration of 185 MBq of 111In-DTPA-H11 scFv to humans was 37 mSv. The radiation absorbed dose estimates for the kidneys, spleen and intestines were 405 mSv, 698 mSv and 412 mSv, respectively. The results of this preclinical study and a concurrent phase I trial suggest a promising role for H11 scFv for tumour imaging.

Constructing contigs from large-insert clones.

This unit describes three approaches that are widely used to define alignments between overlapping clones bearing large-insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence-tagged site (STS) content mapping, repetitive-element hybridization fingerprinting, and Alu-PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive-element hybridization fingerprinting to detect overlaps and build contigs with full-genomic YAC libraries.

Disability profile and health care costs of Medicare beneficiaries under age sixty-five.

Medicare has more than five million beneficiaries with disabilities who are under age sixty-five, and enrollment is rising rapidly. This paper presents a profile of nonelderly beneficiaries in fee-for-service Medicare by major disability category, excluding those with end-stage renal disease. The profile is based on Medicare Current Beneficiary Survey (MCBS) Cost and Use data for 1994-1996. We estimate Medicare costs by service type and health care costs by payer type, and we discuss implications for Medicare reform and related federal disability policy issues.