Humoral Responses to Diverse Autoimmune Disease-Associated Antigens in Multiple Sclerosis

<div><p>To compare frequencies of autoreactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression.</p><p>Methods</p><p>The study analyzed 969 serum samples from 315 HC, 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114(Glc) and KIR4.1a and KIR4.1b and against 24 key endogenous antigens linked to diseases such as vasculitis, systemic sclerosis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, polymyositis, scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease and primary biliary cirrhosis. Associations with disability and MRI measures of lesional injury and neurodegeneration were assessed.</p><p>Results</p><p>The frequencies of anti-KIR4.1a and anti-KIR4.1b peptide IgG positivity were 9.8% and 11.4% in HC compared to 4.9% and 7.5% in RR-MS, 8.6% for both peptides in SP-MS and 6.1% for both peptides in PP-MS (<i>p</i> = 0.13 for KIR4.1a and <i>p</i> = 0.34 for KIR4.1b), respectively. Antibodies against CSF114(Glc), KIR4.1a and KIR4.1b peptides were not associated with MS compared to HC, or with MS disease progression. <i>HLA DRB1</i>*15:01 positivity and anti-Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies.</p><p>Conclusions</p><p>Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse autoreactive antibodies investigated are similar in MS and HC.</p></div

Frequency of positivity for different antibodies in healthy controls (HC), relapsing-remitting MS (RR-MS, includes CIS) and secondary progressive-MS (SP-MS includes secondary progressive MS, relapsing-remitting/secondary progressive MS), primary progressive MS (PP-MS includes primary relapsing MS) and Other Neurological Diseases (OND).

<p>* Pearson chi-square</p><p>Frequency of positivity for different antibodies in healthy controls (HC), relapsing-remitting MS (RR-MS, includes CIS) and secondary progressive-MS (SP-MS includes secondary progressive MS, relapsing-remitting/secondary progressive MS), primary progressive MS (PP-MS includes primary relapsing MS) and Other Neurological Diseases (OND).</p

Associations with MRI and clinical measures.

<p>EDSS: Expanded disability status scale; T2-LV: T2 lesion volume; T1-LV: T1 lesion volume; GMV: Normalized gray matter volume; WBV: Normalized whole brain volume. EDSS was analyzed with ordinal regression and the MRI variables were analyzed with linear regression. All analyses corrected for age, gender and type of MS. The Table entries summarize the unadjusted <i>p</i>-values from regression analyses. The <i>q</i>-values are shown in parentheses for cells with <i>p</i> ≤ 0.05</p><p>Associations with MRI and clinical measures.</p

Demographic, clinical, MRI characteristics and current disease-modifying therapies.

<p>* All continuous variables (age, disease duration, T2-LV, T1-LV) are mean ± standard deviation. For the ordinal EDSS, the median (inter-quartile range) are given.</p><p>^§ The remainder received other treatments.</p><p>Demographic, clinical, MRI characteristics and current disease-modifying therapies.</p

Associations with <i>HLA DR*15</i>:<i>01</i>, anti-EBV-EBNA-1 quartiles and anti-EBV-VCA quartiles and anti-CMV positivity.

<p>* Anti-DNA positivity status was analyzed with logistic regression and the remaining titer variables were analyzed with linear regression. All analyses corrected for age, gender and type of MS.</p><p>The Table entries summarize the unadjusted <i>p</i>-values from regression analyses. The <i>q</i>-values are shown for cells with <i>p</i> ≤ 0.05.</p><p>Associations with <i>HLA DR*15</i>:<i>01</i>, anti-EBV-EBNA-1 quartiles and anti-EBV-VCA quartiles and anti-CMV positivity.</p

Antigenic targets and disease associations of antibodies assessed.

<p>SS: Sjogren’s Syndrome, SLE: Systemic lupus erythematosus, LDL: Low-density lipoprotein, MCTD: Mixed Connective Tissue Disease (MCTD), PBC: Primary biliary cirrhosis.</p><p>Antigenic targets and disease associations of antibodies assessed.</p

Simultaneous Determination of Oxysterols, Cholesterol and 25-Hydroxy-Vitamin D3 in Human Plasma by LC-UV-MS

<div><p>Background</p><p>Oxysterols are promising biomarkers of neurodegenerative diseases that are linked with cholesterol and vitamin D metabolism. There is an unmet need for methods capable of sensitive, and simultaneous quantitation of multiple oxysterols, vitamin D and cholesterol pathway biomarkers.</p><p>Methods</p><p>A method for simultaneous determination of 5 major oxysterols, 25-hydroxy vitamin D3 and cholesterol in human plasma was developed. Total oxysterols were prepared by room temperature saponification followed by solid phase extraction from plasma spiked with deuterated internal standards. Oxysterols were resolved by reverse phase HPLC using a methanol/water/0.1% formic acid gradient. Oxysterols and 25-hydroxy vitamin D3 were detected with atmospheric pressure chemical ionization mass spectrometry in positive ion mode; in-series photodiode array detection at 204nm was used for cholesterol. Method validation studies were performed. Oxysterol levels in 220 plasma samples from healthy control subjects, multiple sclerosis and other neurological disorders patients were quantitated.</p><p>Results</p><p>Our method quantitated 5 oxysterols, cholesterol and 25-hydroxy vitamin D3 from 200 μL plasma in 35 minutes. Recoveries were >85% for all analytes and internal standards. The limits of detection were 3-10 ng/mL for oxysterols and 25-hydroxy vitamin D3 and 1 μg/mL for simultaneous detection of cholesterol. Analytical imprecision was <10 %CV for 24(S)-, 25-, 27-, 7α-hydroxycholesterol (HC) and cholesterol and ≤15 % for 7-keto-cholesterol. Multiple Sclerosis and other neurological disorder patients had lower 27-hydroxycholesterol levels compared to controls whereas 7α-hydroxycholesterol was lower specifically in Multiple Sclerosis.</p><p>Conclusion</p><p>The method is suitable for measuring plasma oxysterols levels in human health and disease. Analysis of human plasma indicates that the oxysterol, bile acid precursors 7α-hydroxycholesterol and 27-hydroxycholesterol are lower in Multiple Sclerosis and may serve as potential biomarkers of disease.</p></div

Comparison of the assay to external reference materials.

<p>The oxysterol reference materials were from Referenzinstitut für Bioanalytik and the vitamin D3 reference material was from The National Institute of Standards and Technology. 50pct; 50th percentile of the corresponding LC-MS sub-collective</p><p>Comparison of the assay to external reference materials.</p

LC-MS-PDA chromatogram of standards.

<p>The top panel shows the chromatogram of each selected ion-monitoring (SIM) channel with each analyte identified at the peak apex. MS Off/MS On indicated the activation points for the flow control valve. The bottom panel shows the PDA Chromatogram at 204 nm.</p

Side-chain oxygenated oxysterol levels in patient groups.

<p>Distribution of 24-hydroxy cholesterol (24HC; Fig 2A), 25-hydroxy cholesterol (25HC; Fig 2C) and 27-hydroxy cholesterol (27HC; Fig 2E) for healthy controls (HC), multiple sclerosis (MSC) and other neurological diseases (OND). The results in Fig 2B, 2D and 2F correspond to the oxysterols in Fig 2A, 2C and 2E, respectively, but are normalized to total cholesterol (TC) levels. The dot-plots of individual patients are superimposed on the box plots. The solid line in the box plots are the median, the box delineates upper and lower quartiles and the error bar represents the range. Panel E for 27HC and Panel F for 27HC-TC ratio contains the p-value from the Kruskal-Wallis test in the main body. The inset contains a bar graph and p-values from follow-up Mann-Whitney tests.</p