60 kD Ro and nRNP A Frequently Initiate Human Lupus Autoimmunity

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, humoral autoimmune disorder. The unifying feature among SLE patients is the production of large quantities of autoantibodies. Serum samples from 129 patients collected before the onset of SLE and while in the United States military were evaluated for early pre-clinical serologic events. The first available positive serum sample frequently already contained multiple autoantibody specificities (65%). However, in 34 SLE patients the earliest pre-clinical serum sample positive for any detectable common autoantibody bound only a single autoantigen, most commonly 60 kD Ro (29%), nRNP A (24%), anti-phospholipids (18%) or rheumatoid factor (15%). We identified several recurrent patterns of autoantibody onset using these pre-diagnostic samples. In the serum samples available, anti-nRNP A appeared before or simultaneously with anti-nRNP 70 K in 96% of the patients who had both autoantibodies at diagnosis. Anti-60 kD Ro antibodies appeared before or simultaneously with anti-La (98%) or anti-52 kD Ro (95%). The autoantibody response in SLE patients begins simply, often binding a single specific autoantigen years before disease onset, followed by epitope spreading to additional autoantigenic specificities that are accrued in recurring patterns

Clostridium difficile 027/BI/NAP1 encodes a hypertoxic and antigenically variable form of TcdB.

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD

Comparative survival curves of mice injected with TcdB₀₀₃ and TcdB_027.

<p>Kaplan-Meier graphs showing the time to death of BALB/c mice that were injected intravenously with TcdB. (A) Survival time of mice (n = 4) injected with 50 ug/kg, 25 ug/kg, 5 ug/kg, and 2.5 ug/kg of TcdB₀₀₃. (B) Survival time of mice (n = 4) injected with 10 ug/kg, 5 ug/kg, 2.5 ug/kg, 1.25 ug/kg, and 625 ng/kg of TcdB₀₂₇. (C) Kaplan-Meier graph comparing the time to death of mice injected with 5 ug/kg of TcdB₀₀₃ or TcdB₀₂₇. The difference between the curves is indicated by the p value determined from a log-rank analysis. (D) Kaplan-Meier graph comparing the time to death of mice injected with 2.5 ug/kg of TcdB₀₀₃ or TcdB₀₂₇. The difference between the curves is indicated by the p value determined from a log-rank analysis.</p

In vivo pathologies of TcdB₀₀₃ and TcdB₀₂₇.

<p>(A) Top- Liver pathologies from BALB/c mice injected with (from left to right) 2.5 µg/kg, 50 µg/kg, 25 µg/kg, and 5 µg/kg of TcdB₀₀₃. Bottom- Liver pathologies from BALB/c mice injected with (from left to right) 625 ng/kg, 10 µg/kg, 5 µg/kg, or 2.5 µg/kg of TcdB₀₂₇. All photos are a 20× magnification of H&E stained sections and are listed by survival time. (B) Pathologies of the cerebrum and cerebellum with arrows pointing to areas of hemorrhaging. Representative photos (20×) of H&E stained sections from BALB/c mice injected with 5 µg/kg TcdB₀₀₃ (top) or 2.5 µg/kg of TcdB₀₂₇ (bottom).</p

Identification of unique and shared epitopes between TcdB₀₀₃ and TcdB₀₂₇ using synthetic peptide ELISAs.

<p>Solid phase epitope mapping of αCTD₀₀₃ (black) and αCTD₀₂₇ (red) rabbit sera binding to overlapping decapeptides of the TcdB₀₀₃ CTD. (a) Peptides from the CTD₀₀₃ were constructed spanning amino acid 1651 through 2366, and the bars indicate the magnitude of reactivity of the sera to overlapping peptide sequences from the CTD of TcdB₀₀₃. Reactivity is shown for αCTD₀₀₃ (black) and αCTD₀₂₇ (red), and represents an average of sera from 2 rabbits per group. (B) The peaks were numbered and identified as either unique to αCTD₀₀₃ (left), unique to αCTD₀₂₇ (middle), or overlapping/shared between αCTD₀₀₃ and αCTD₀₂₇ (right). The amino acid location of each epitope is indicated, as well as the sequence of the peptides in TcdB₀₀₃, with amino acids that vary in TcdB₀₂₇ identified in red.</p

Protection against TcdB in vivo and in vitro after immunization with ToxoidB₀₂₇.

<p>(A) Percent viability of CHO cells treated for 24 hrs with TcdB₀₀₃ (black) or TcdB₀₂₇ (gray) alone or after preincubation for 30 minutes with αToxoidB₀₀₃ antiserum or αToxoidB₀₂₇ antiserum. Cell viability was determined by WST-8 staining and the error bars represent the standard deviation from the mean of three samples. ***p<0.001, *p<0.05 (B–C) Kaplan-Meier graphs showing the time to death of C57Bl/6 mice that were injected intravenously with a 2×LD₁₀₀ of TcdB₀₀₃ (A) or TcdB₀₂₇ (B) after immunization with ToxoidB₀₀₃ (red), ToxoidB₀₂₇ (dashed), or control peptide (black) (n = 9). Log-rank analysis performed using GraphPad Prism, *** p<0.001, ** p<0.01.</p

Potential mechanism for development of autoantibodies through a common environmental etiology.

<p>While potential structural mimics are known with EBNA-1 for Ro, Sm B, Sm D, nRNP A, nRNP C and nRNP 70 K, no such structural relationship is known for a heteroimmune response operating to generate anti-dsDNA, anti-chromatin or anti-ribosomal P.</p

Autoantibodies precede lupus classification and occur in linked subsets.

<p>Kaplan-Maier survival curves for the onset of each autoantibody specific as measured by a solid phase, bead-based assay are presented. Anti-60 kD Ro, anti-La and anti-52 kD Ro are among the earliest specificities detected by this method. In contrast, anti-68 kD nRNP and nRNP A specificities are frequently detected closer to the time of lupus classification.</p

Prevalence and time of onset of autoantibodies as detected by the BioPlex 2200 ANA Screen kit and demographics of patients positive for each autoantibody.

<p>Antibodies to nRNP A, Sm, and 60 kD Ro are significantly more common in African-Americans.</p><p>EA =  European-Americans.</p><p>AA = African-Americans.</p><p>*p<0.008.</p