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Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.

Glioma is one of the most refractory types of brain tumor. Accurate tumor boundary identification and complete resection of the tumor are essential for glioma removal during brain surgery. We present a method based on visible resonance Raman (VRR) spectroscopy to identify glioma margins and grades. A set of diagnostic spectral biomarkers features are presented based on tissue composition changes revealed by VRR. The Raman spectra include molecular vibrational fingerprints of carotenoids, tryptophan, amide I/II/III, proteins, and lipids. These basic in situ spectral biomarkers are used to identify the tissue from the interface between brain cancer and normal tissue and to evaluate glioma grades. The VRR spectra are also analyzed using principal component analysis for dimension reduction and feature detection and support vector machine for classification. The cross-validated sensitivity, specificity, and accuracy are found to be 100%, 96.3%, and 99.6% to distinguish glioma tissues from normal brain tissues, respectively. The area under the receiver operating characteristic curve for the classification is about 1.0. The accuracies to distinguish normal, low grade (grades I and II), and high grade (grades III and IV) gliomas are found to be 96.3%, 53.7%, and 84.1% for the three groups, respectively, along with a total accuracy of 75.1%. A set of criteria for differentiating normal human brain tissues from normal control tissues is proposed and used to identify brain cancer margins, yielding a diagnostic sensitivity of 100% and specificity of 71%. Our study demonstrates the potential of VRR as a label-free optical molecular histopathology method used for in situ boundary line judgment for brain surgery in the margins.

Background: N-terminally truncated and modified pyroglutamate-3 amyloid-β protein (pE3-Aβ) is present in most, if not all, cerebral plaque and vascular amyloid deposits in human Alzheimer’s disease (AD). pE3-Aβ deposition is also found in AD-like transgenic (tg) mouse brain, albeit in lesser quantities than general Aβ. pE3-Aβ resists degradation, is neurotoxic, and may act as a seed for Aβ aggregation. Objective: We sought to determine if pE3-Aβ removal by passive immunization with a highly specific monoclonal antibody (mAb) impacts pathogenesis in a mouse model of Alzheimer’s amyloidosis. Methods: APPswe/PS1ΔE9 tg mice were given weekly intraperitoneal injections of a new anti-pE3-Aβ mAb (mAb07/1) or PBS from 5.8 to 13.8 months of age (prevention) or from 23 to 24.7 months of age (therapeutic). Multiple forms of cerebral Aβ were quantified pathologically and biochemically. Gliosis and microhemorrhage were examined. Results: Chronic passive immunization with an anti-pE3-Aβ mAb significantly reduced total plaque deposition and appeared to lower gliosis in the hippocampus and cerebellum in both the prevention and therapeutic studies. Insoluble Aβ levels in hemibrain homogenates were not significantly different between immunized and control mice. Microhemorrhage was not observed with anti-pE3-Aβ immunotherapy. Conclusions: Selective removal of pE3-Aβ lowered general Aβ plaque deposition suggesting a pro-aggregation or seeding role for pE3-Aβ.

Background and Objective: Could a normal – but persistent – stress response to impeded axonal transport lead to late-onset Alzheimer’s disease (AD)? Our results offer an affirmative answer, suggesting a mechanism for the abnormal production of amyloid-β (Aβ), triggered by the slowed axonal transport at old age. We hypothesize that Aβ precursor protein (APP) is a sensor at the endoplasmic reticulum (ER) that detects, and signals to the nucleus, abnormalities in axonal transport. When persistently activated, due to chronically slowed-down transport, this signaling pathway leads to accumulation of Aβ within the ER. Methods and Results: We tested this hypothesis with the neuronal cell line CAD. We show that, normally, a fraction of APP is transported into neurites by recruiting kinesin-1 via the adaptor protein, Fe65. Under conditions that block kinesin-1-dependent transport, APP, Fe65 and kinesin-1 accumulate in the soma, and form a complex at the ER. This complex recruits active c-Jun N-terminal kinase (JNK), which phosphorylates APP at Thr668. This phosphorylation increases the cleavage of APP by the amyloidogenic pathway, which generates Aβ within the ER lumen, and releases Fe65 into the cytoplasm. Part of the released Fe65 translocates into the nucleus, likely to initiate a gene transcription response to arrested transport. Prolonged arrest of kinesin-1-dependent transport could thus lead to accumulation and oligomerization of Aβ in the ER. Conclusion: These results support a model where the APP:Fe65 complex is a sensor at the ER for detecting the increased level of kinesin-1 caused by halted transport, which signals to the nucleus, while concomitantly generating an oligomerization-prone pool of Aβ in the ER. Our hypothesis could thus explain a pathogenic mechanism in AD.

To establish whether the DNA expansion linked to spinocerebellar ataxia type 8 (SCA 8) is associated with ataxia in Scotland; to clarify the range of associated clinical phenotypes; and to compare the findings with previous reports.DNA was screened from 1190 anonymised controls, 137 subjects who had tested negative for Huntington's disease, 176 with schizophrenia, and 173 with undiagnosed ataxia. Five unrelated ataxic patients with the SCA 8 expansion and a sixth identified subsequently had clinical and psychometric assessment; the clinical features were available in a seventh. A systematic search for other reports of SCA 8 was undertaken.Over 98% of SCA 8 CTA/CTG repeat lengths fell between 14 and 40. Repeat lengths over 91 were observed in three healthy controls (0.12%), two patients with suspected Huntington's disease (0.73%), and six ataxic subjects (1.74%; p0.0005 v healthy controls). Repeat lengths over 100 occurred in five ataxic subjects but in only one control. All seven symptomatic subjects with the SCA 8 expansion had a cerebellar syndrome; four had upper motor neurone signs; and 5/6 assessed had cognitive complaints. There was personality change in two and mood disturbance in three. In published reports, SCA 8 repeat lengths over 91 occurred in approximately 0.5% of the healthy population but were over-represented among ataxic patients (3.4%; p0.0001). The predominant clinical phenotype was cerebellar, with pyramidal signs in 50%, and neuropsychiatric features in some cases.SCA 8 expansion is a risk factor for a cerebellar syndrome, often associated with upper motor neurone and neuropsychiatric features. The expansion occurs unexpectedly often in the general population.

Objective: To determine the rate and correlates of weight change in a large, well characterised sample of patients with Huntington’s disease followed at 44 sites by the Huntington Study Group. Participants and methods: Weight change was assessed in 927 adults with a definite diagnosis of Huntington’s disease who were followed prospectively for (mean (SD)) 3.4 (1.4) years. The unified Huntington’s disease rating scale was used to assess weight, motor dysfunction (including chorea and dystonia), depressive symptoms, and functional decline. Results: Random effects modelling determined that patients gained an average of 0.11 (1.7) kg/year and their chorea scores increased by 0.36 (0.78) points/year. There were significant but weak relations between weight loss and increasingly severe chorea ( r = −0.13), worse baseline motor performance ( r = −0.12), less severe baseline depressed mood ( r = 0.14), and poorer baseline independence ratings ( r = 0.07). Patients who were within 0 to 2 years of symptom onset at the time of the baseline visit gained more weight than those with longer disease duration. Conclusions: Weight loss following symptom onset is not a consistent feature of Huntington’s disease. The mechanisms contributing to weight change in this condition are unclear and probably multifactorial. Future studies examining asymptomatic carriers of the mutation could be helpful in identifying incipience of low body weight and may be better suited for identifying clinical correlates of weight loss than studies in symptomatic patients.

Objective: To determine whether polymorphic variations in the apolipoprotein E gene ( APOE ) are associated with increased risk of frontotemporal lobar degeneration (FTLD) when mutation in tau gene is absent. Methods: The APOE gene was genotyped by polymerase chain reaction from DNA routinely extracted from blood or brain tissues. The APOE e4 allele frequency in 198 patients with FTLD not associated with mutations in tau gene was compared with that of a control group of 756 normal individuals drawn from the same geographical region. Analyses were done according to clinical subtype or sex. Results: The APOE e4 allele frequency (19.4%) was increased (p = 0.01) in FTLD v the whole control group (14.1%), while the APOE e2 allele frequency in FTLD (6.5%) was slightly lower than in controls (8.0%) (NS). The APOE e4 allele frequency in men with FTLD (22.3%) was greater (p = 0.002) than in male controls (12.3%); the frequency in women (16.3%) was similar to that in female controls (14.8%) (NS). The APOE e2 allele frequency in men with FTLD was 4.9% while in male controls it was 9.5% (p = 0.06), but there was no difference in women (7.5% v 7.9%, NS). Neither the APOE e2 nor APOE e4 allele frequency varied significantly between any of the clinical subtypes. Conclusions: In FTLD not associated with mutations in tau gene, possession of APOE e4 allele in men roughly doubles the chances of developing disease, whereas this has no impact upon disease risk in women.

Spinal muscular atrophy (SMA) is a common, often fetal, autosomal recessively inherited disease leading to progressive muscle wasting and paralysis as a result of degeneration of anterior horn cells of the spinal cord. The SMA-determining gene, called the survival of motor neuron gene (SMN), is present on 5q13 in two nearly identical copies, telomeric SMN (SMN1) and centromeric SMN (SMN2). It has been established that SMA is caused by mutations in SMN1 whereas homozygous deletion of SMN2 has apparently no pathological consequences. The aim of this study is to develop an easy and inexpensive method for the isolation of high-quality template DNA from blood samples for SMA carrier screening by multiplex polymerase chain reaction. We have developed a protocol that optimizes detection of the SMN1 copy number in the human genome, producing a specific and sensitive assay using DNA extracted from a dried blood spot on IsoCode paper.

Objective: Spinocerebellar ataxia type 2 (SCA2) has been reported as the commonest dominant hereditary ataxia in India. However, India is an ethnically and religiously diverse population. Previous studies have not clearly indicated exact ethnic and religious origins, and must therefore be interpreted with caution. The purpose of this study was to determine the prevalence of different SCA mutations in a relatively homogeneous population from eastern India. Methods: We identified 28 families with autosomal dominant cerebellar ataxia from eastern India. Each underwent full clinical evaluation and were analysed for the presence of SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, SCA12, and SCA17 mutations. In addition, haplotype analysis was carried out in seven of the 16 families with SCA2. Results: Seven patients from four (14%) families were positive for an expansion in SCA1 and 26 patients from 16 (57%) families were positive for an expansion in SCA2. No mutations were detected in the remaining eight families (29%). Most of the SCA1 and SCA2 families were Hindu from the state of Bihar. Five out of 26 SCA2 patients in this study did not have slow saccades. In addition, four of seven SCA1 patients had slow saccades. We found an association between the SCA2 CAG repeat expansion and the 285 base pair (bp) allele of microsatellite marker D12S1672, and also data supportive of the association between the expansion and the 225 bp allele of D12S1333, which has been previously described. Conclusions: We conclude that (1) although slow ocular saccades are highly suggestive of SCA2, that they are not universal, nor are they exclusive to this disorder and (2) SCA2 is likely to be the commonest dominant ataxia in eastern India, with further evidence for a founder effect.

Background: Spinocerebellar ataxia type 1 (SCA1) is one of the autosomal dominant neurodegenerative disorders commonly linked to pathological expansion of the CAG repeat of the relevant gene. Nuclear inclusions and neurodegeneration are both triggered by this pathological expansion of the CAG/polyglutamine repeat on ataxin-1, but it remains to be determined whether or not nuclear inclusion formation is associated with accelerated neurodegeneration. Objective: To examine the influence of nuclear inclusions on nuclear size and deformity in human brains from patients suffering from SCA1. Material: Pontine sections of brains obtained at necropsy from seven patients with SCA1 and five controls. Methods: The size and deformity of each neuronal nucleus was quantified. Nuclei with and without inclusions were examined separately to assess the possible influence of nuclear inclusions on neurodegeneration. Results: Nuclear shrinkage and deformity were more marked in SCA1 brains than in controls. This shrinkage was attenuated in neurones containing nuclear inclusions. Conclusions: The existence of nuclear inclusions in SCA1 is presumably linked to a mechanism that attenuates rather than accelerates nuclear shrinkage. This in vivo finding may provide a clue to constructing a rational therapeutic strategy for combating neurodegeneration associated with nuclear inclusions.

1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.