Gabriel Martin

Hyperspectral computed tomographic imaging spectroscopy of vascular oxygen gradients in the rabbit retina in vivo.

Diagnosis of retinal vascular diseases depends on ophthalmoscopic findings that most often occur after severe visual loss (as in vein occlusions) or chronic changes that are irreversible (as in diabetic retinopathy). Despite recent advances, diagnostic imaging currently reveals very little about the vascular function and local oxygen delivery. One potentially useful measure of vascular function is measurement of hemoglobin oxygen content. In this paper, we demonstrate a novel method of accurately, rapidly and easily measuring oxygen saturation within retinal vessels using in vivo imaging spectroscopy. This method uses a commercially available fundus camera coupled to two-dimensional diffracting optics that scatter the incident light onto a focal plane array in a calibrated pattern. Computed tomographic algorithms are used to reconstruct the diffracted spectral patterns into wavelength components of the original image. In this paper the spectral components of oxy- and deoxyhemoglobin are analyzed from the vessels within the image. Up to 76 spectral measurements can be made in only a few milliseconds and used to quantify the oxygen saturation within the retinal vessels over a 10–15 degree field. The method described here can acquire 10-fold more spectral data in much less time than conventional oximetry systems (while utilizing the commonly accepted fundus camera platform). Application of this method to animal models of retinal vascular disease and clinical subjects will provide useful and novel information about retinal vascular disease and physiology.

Noninvasive assessment of retinal vascular oxygen content among normal and diabetic human subjects: a study using hyperspectral computed tomographic imaging spectroscopy.

Purpose: This pilot study was aimed to demonstrate the clinical feasibility of using hyperspectral computed tomographic spectroscopy to measure blood oxygen content in human retinal vessels. Methods: All procedures were performed under a University of Southern California Institutional Review Board–approved protocol and after obtaining informed consent. Fifty-seven subjects with and without diabetic retinopathy were dilated for standard fundus photography. Fundus photographs and retinal vascular oxygen measurements (oximetry) were made using a custom-made hyperspectral computed tomographic imaging spectrometer coupled to a standard fundus camera. Oximetry measurements were made along arteries (Aox) and veins (Vox) within vessel segments that were 1 to 2 disk diameters from the optic disk. Results: For all control subjects (n = 45), mean Aox and Vox were 93 ± 7% and 65 ± 5% (P = 0.001), respectively. For all diabetic subjects (n = 12), mean Aox and Vox were 90 ± 7% and 68 ± 5% (P = 0.001), respectively. In subjects with proliferative diabetic retinopathy, Aox was significantly lower, and Vox was significantly higher than other groups (85 ± 4% and 71 ± 4%, respectively; P = 0.04, analysis of variance). There was a highly significant difference in the arteriovenous difference between subjects with proliferative diabetic retinopathy and those in the control group (14 vs. 26%, P = 0.003). Conclusion: Hyperspectral computed tomographic spectroscopy is a clinically feasible method for measurement and analysis of vascular oxygen content in retinal health and disease. This study uses the techniques relevant to oximetry; however, the breadth of spectral data available through this method may be applicable to study other anatomical and functional features of the retina in health and disease.

Acute Variations in Retinal Vascular Oxygen Content in a Rabbit Model of Retinal Venous Occlusion

Purpose To study the variation in intravascular oxygen saturation (oximetry) during an acute retinal vein occlusion (RVO) using hyperspectral computed tomographic spectroscopy based oximetry measurements. Methods Thirty rabbits were dilated and anesthetized for experiments. Baseline oximetry measurements were made using a custom-made hyperspectral computed tomographic imaging spectrometer coupled to a fundus camera. RVO were induced using argon green laser following an intravenous injection of Rose Bengal. RVO induction was confirmed by fluorescein angiography. Retinal oximetry measurements were repeated in arterial and venous branches one hour after RVO induction and up to 4 weeks afterwards. Comparison of retinal oximetry before and after vein occlusion was made using the Student T-test. Results One hour after RVO induction, we observed statistically significant reductions in the intravascular oxygen saturation in temporal retinal arteries (85.1±6.1% vs. 80.6±6.6%; p<0.0001) and veins (71.4±5.5% vs. 64.0±4.7%; p<0.0001). This decrease was reversible in animals that spontaneously recannulated the vein occlusion. There were no statistically significant differences in oxygen saturation in the nasal control arteries and veins before and after temporal vein RVO induction. Conclusions We demonstrate, for the first time, acute changes in the intravascular oxygen content of retinal vessels 1 hour after RVO. These changes are reversible upon spontaneous recannulation of retinal vessels. This study demonstrates that hyperspectral computer tomographic spectroscopy based oximetry can detect physiological variations in intravascular retinal oxygen saturation. The study also provides the first qualitative and quantitative evidence of the variation in retinal vascular oxygen content directly attributable to an acute retinal vein occlusion.

Evaluation of X Chromosome Inactivation with Respect to HLA Genetic Susceptibility in Rheumatoid Arthritis and Systemic Sclerosis

Background Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. Methods Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. Results 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the “shared epitope” correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. Conclusion Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity.

A6.40 Copy number increase of TLR7 and TLR8 genes in men with rheumatoid arthritis

Background and objectives Men are less often affected by Rheumatoid arthritis (RA) than women. In mice studies, it was shown that a duplication in Toll-like receptor 7 (Tlr7) gene is sufficient to potentiate autoimmunity in males. We therefore propose to investigate whether an increase in copy number (CN) of TLR7, and its neighbouring paralog TLR8 on X chromosome, could contribute to the pathogenesis of RA in men. Materials and methods In a pilot analysis, we had first tested our hypothesis by real-time quantitative PCR assay to assess CN of TLR7 gene and TLR8 compared to Beta-Globin gene (HBB), using the relative standard curve calculation method, on DNA from PBMCs of 49 men with RA and 42 healthy controls. TLR7/8 copy numbers significantly increased in PBMCs from men with RA when compared to healthy men. Nevertheless, because men with RA were significantly older than healthy donors, the increased TLR7/8 CN could be age-dependent. In the current study, we have tested whether TLR7/8 CN varies with age on a large number of controls from birth to 82 years old (160 men and 180 women). We also tested an additional autosomal RPP30 gene to further validate our results. DNA samples from peripheral blood of 49 men with RA versus 160 healthy men and 44 women with RA versus 180 healthy women were tested for TLR7/8 CN compared to mean CN values of HBB and RPP30. Similarly, DNA samples from PBMCs of 53 men with RA versus 42 healthy men and 40 women with RA versus 41 healthy women were analysed. Results TLR7 or TLR8 CN does not vary with age in healthy individuals (women or men). Similar findings were observed in patients with RA. Nevertheless, we confirmed that men with RA had an increased TLR7 and TLR8 CN in peripheral blood compared to healthy men (p < 0.0001 and p = 0.002 respectively). A rather opposite trend was observed in women with RA. Similar results were observed in PBMCs. Conclusions We have presented evidence that copy number of X-linked TLR7 and TLR8 genes increases in men with RA. This increase is not only disease dependent but also sex-dependent. We are currently validating our results by digital droplet polymerase chain reaction to confirm this genetic heterogeneity between men and women with RA.

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus.

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10−4) and 60% versus 28% (P = 3.10−8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10−10) and 82% (P = 5.10−13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3’-5’ exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.