Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine

Molecular Histopathology Using Gold Nanorods and Optical Coherence Tomography

PURPOSE. To examine the novel application of a commercially available optical coherence tomography (OCT) system toward molecular histopathology using gold nanorod (GNR) linked antibodies as a functionalized contrast agent to evaluate ocular surface squamous neoplasia (OSSN). METHODS. GNRs were synthesized and covalently attached to anti–glucose transporter-1 (GLUT-1) antibodies via carbodiimide chemistry. Three specimens from each of three distinct categories of human conjunctival tissue were selected for analysis, including conjunctiva without epithelial atypia (controls); conjunctival intraepithelial neoplasia, carcinoma in situ (CIS); and conjunctival squamous cell carcinoma (SCC). Tissue sections were incubated initially with GNR tagged anti–GLUT-1 antibodies and then with a fluorescent-tagged secondary antibody. Immunofluorescence and OCT imaging of the tissue was performed and the results were correlated to the light microscopic findings on traditional hemotoxyin and eosin stained sections. RESULTS. No binding of the functionalized GNRs was observed within the epithelium of three normal conjunctiva controls. While immunofluorescence disclosed variable binding of the functionalized GNRs to atypical epithelial cells in all six cases of OSSN, the enhancement of the OCT signal in three cases of CIS was insufficient to distinguish these specimens from normal controls. In two of three cases of SCC, binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to atypical epithelial cells which stained intensely on immunofluorescence imaging. Binding of functionalized GNRs was sufficient to produce an increased scattering effect on OCT in areas correlating to regions of erythrocytes and hemorrhage which stained intensely on immunofluorescence imaging within all nine tested samples. CONCLUSIONS. We have demonstrated the use of OCT for molecular histopathology using functionalized gold nanorods in the setting of OSSN. Our results suggest a threshold concentration of functionalized GNRs within tissue is required to achieve a detectable enhancement in scattering of the OCT signal

The presence of T-lymphocyte subpopulations (CD4 and CD8) in pterygia: Evaluation of the inflammatory response

The aim of our study was to confirm the presence of inflammatory T-lymphocyte subpopulations (CD4 and CD8) in pterygium specimens with regards to clinical severity. Additionally, we examined the effect of topical anti-inflammatory agents on the presence of T-lymphocyte subpopulations.Pterygia from nineteen eyes of nineteen patients who underwent surgical excision at Duke University, North Carolina, were included in this study. Normal conjunctiva from one patient was included as a control. Pterygia were pre-operatively graded as mild, moderate or severe based on objective signs of inflammation. Immunohistochemical staining for both CD4 and CD8 subpopulations of T lymphocytes was performed. Distribution of lymphocytes within the epithelium and substantia propria was graded by a masked observer on the following scale: 0 (none/rare), 1+ (mild), 2+ (moderate), or 3+ severe. Statistical analysis was performed using the Fisher exact test and Chi-square test.A total of 16 (84%) pterygia specimens stained for T lymphocytes displayed approximately equal CD4 and CD8 infiltration of both the epithelium and the substantia propria. The majority of CD4 and CD8 lymphocytes were located in aggregates in the epithelium and upper substantia propria. The control specimen contained scant evidence of lymphocytic infiltration. There was no significant difference in the amount of lymphocytic infiltration between mild, moderate or severe pterygia. There was also no significant difference in lymphocytic infiltration between patients with (n=8) or without (n=11) a history of topical anti-inflammatory use.The presence of CD4 and CD8 lymphocytes was confirmed in pterygia. There was no significant difference in lymphocytic infiltrate in patients with or without prior topical anti-inflammatory use. Based on these findings, topical immunomodulators may have an adjunctive role in the treatment of pterygia

Single Step Nanoplasmonic Immunoassay for the Measurement of Protein Biomarkers

A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs) conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1). The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool

The Paternally Inherited Insulin Gene B Allele (1,428 FokI site) Confers Protection from Insulin-dependent Diabetes in Families

Several polymorphisms of the insulin gene and its flanking regions (INS region) are in linkage disequilibrium and confer susceptibility to insulin-dependent diabetes (IDDM). We have analysed INS AA and AB-BB genotypes at the 1,428 FokI site (3′ of the insulin gene) in 217 patients with IDDM, 402 non-diabetic first degree relatives negative for insulin (IAA) and islet cell autoantibodies (ICA), and 116 autoantibody positive (for ICA or IAA, or both) relatives of whom 39 became diabetic on follow-up. Most IDDM patients (83.4%, 181/217) had the AA genotype vs. 50% (25/50) of the controls ( P<10 -6). Only 16.6% (36/217) of IDDM patients carried the AB genotype and none was BB homozygous, suggesting a protective effect of the B allele. By segregation analysis of the B allele in the IDDM offspring of informative families (only one AB parent) from the United States, the maternal B allele was inherited by 19/35 (54.2%) of the IDDM offspring. In contrast, only 4/26 (15.3%) of the IDDM offspring inherited the paternal B allele ( P=0.001), suggesting maternal imprinting of the INS region. Therefore, the INS B allele may be protective only when paternally inherited. Among the 39 of 116 autoantibody positive relatives who developed IDDM on follow-up, only five of them had the B allele. The frequency of the B allele in this group was much lower (12.8%, 5/39) than that observed in non-diabetic autoantibody positive relatives (32.5%, 25/77, P=0.02). By lifetable analysis, autoantibody positive relatives with the B allele showed slower and lesser progression to IDDM than those without it (Log-rank test, P=0.04). In conclusion, we find a protective effect of the paternally inherited B allele in diabetic families from the United States and in their autoantibody positive first degree relatives

Vision-related quality of life in persons with unilateral branch retinal vein occlusion using the 25-item National Eye Institute Visual Function Questionnaire

AimTo evaluate vision-related quality of life in persons with branch retinal vein occlusion (BRVO) using the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25).DesignObservational, cross-sectional, interviewer-administered study.Methods46 patients with unilateral BRVO were included in this study. Scores on the VFQ-25 were analysed and converted to scaled scores per NEI VFQ-25 algorithms. Clinical data including age, gender, employment status, living arrangements, visual acuity, number of systemic diseases and duration of BRVO were also recorded. Subscale results were compared with previously published data, and subgroup analyses were performed.ResultsMean adjusted subscale responses among BRVO patients were higher (except for ocular pain) than known averages in patients with diabetic retinopathy, central retinal vein occlusion, age-related macular degeneration and low vision, but lower than known averages in a reference group of people without ocular disease. Subscale responses correlated significantly with visual acuity in the involved eye. This observation held true in eight of 12 subscales, even in patients who maintained vision of 20/25 or better in the uninvolved eye. The General Health subscale and number of systemic diseases correlated significantly with both the General Vision and Peripheral Vision subscale scores. There was no correlation between subscale responses and age.ConclusionsBRVO is a retinal vascular disease that is associated with a decrease in vision-related quality of life as determined by the VFQ-25. A decrease in VFQ-25 score is correlated with involved eye visual acuity, even when good visual acuity is maintained in the uninvolved eye

Visual Outcomes in Pediatric Optic Pathway Glioma After Conformal Radiation Therapy

To assess visual outcome prospectively after conformal radiation therapy (CRT) in children with optic pathway glioma. We used CRT to treat optic pathway glioma in 20 children (median age 9.3 years) between July 1997 and January 2002. We assessed changes in visual acuity using the logarithm of the minimal angle of resolution after CRT (54 Gy) with a median follow-up of 24 months. We included in the study children who underwent chemotherapy (8 patients) or resection (9 patients) before CRT. Surgery played a major role in determining baseline (pre-CRT) visual acuity (better eye: P=.0431; worse eye: P=.0032). The visual acuity in the worse eye was diminished at baseline (borderline significant) with administration of chemotherapy before CRT (P=.0726) and progression of disease prior to receiving CRT (P=.0220). In the worse eye, improvement in visual acuity was observed in patients who did not receive chemotherapy before CRT (P=.0289). Children with optic pathway glioma initially treated with chemotherapy prior to receiving radiation therapy have decreased visual acuity compared with those who receive primary radiation therapy. Limited surgery before radiation therapy may have a role in preserving visual acuity