Clinical correlates of a subset of anti-CENP-A antibodies cross-reacting with FOXE3p53-62 in systemic sclerosis

INTRODUCTION: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. METHODS: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. RESULTS: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009). CONCLUSIONS: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance

Clinical correlates of a subset of anti-CENP-A antibodies cross-reacting with FOXE3p53-62 in systemic sclerosis.

INTRODUCTION: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. METHODS: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. RESULTS: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009). CONCLUSIONS: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance

Screening of SSc sera for specificity to Ap^1-17.

<p>(<b>A.</b>) Sera from SSc patients (30 ACA⁺, 20 Scl70⁺; 7 ACA⁻/Scl70⁻) were screened for specificity to Ap^1-17 peptide in an indirect ELISA. Microtiter plates were coated with 5 µg/ml KLH-conjugated Ap^1-17 (filled circles) or KLH-conjugated Qp1-a (open circles). Wells were incubated for 4 h with serum samples (diluted 1∶100) from the three groups of patients and from 10 healthy blood donors (HBD); samples were tested in duplicate. Bound IgG was revealed with HRP-conjugated anti-human IgG (Fc portion) and <i>o</i>-phenylenediamine. Each data point is the mean of duplicate wells (SEM ≤8%). Horizontal lines indicate the mean for each group. O.D., optical density. *Mann-Whitney p<0.0001. (<b>B</b>) ROC analysis was performed by including the absorbance binding of each patient's serum to Ap^1-17 (minus the absorbance of the same sera to the unrelated peptide Qp1-a) as variable, and by comparing the ACA⁺ group to control ACA⁻ group.</p

Alignment of pt4 and pt14 deduced motifs with CENP-A protein sequence.

<p>Motif amino acid “S” shown in small letter could be used interchangeably with “T”.</p

Anti-Ap^1-17 Abs from different SSc patients recognize different motifs within the same peptide.

<p>Microtiter plates were coated with 10 µg/ml anti-Ap^1-17 Abs from 8 SSc patients or with IVIG as negative control. Supernatants from pt4 (Panel A) and pt14 (Panel B) phage clones were diluted 16-fold in PBS and added to the wells (100 µl/well). After a 4-h incubation, the Ab-phage clone interaction was detected with HRP-conjugated anti-M13 mAb and <i>o</i>-phenylenediamine. Each data point is the mean of duplicate wells. The data are representative of two experiments.</p

A Prospective, Randomized Comparative Study of Respiratory and Hemodynamic Monitoring during Colonoscopy using Remifentanyl Versus Propofol/Fentanyl

Objective: We hypothesized that remifentanil continuous infusion during colonoscopy in spontaneous respiration may give benefits in terms of quality of sedation and recovery compared to propofol, and that patients’ ventilatory drive and consciousness could be accurately evaluated by the continuous measurement of end-tidal CO2 (EtCO2), and of Bispectral Score (BIS) respectively. Methods: One-hundred and eighty patients scheduled for colonoscopy were randomized in two groups: 76 patients were included in Groupcontrol (propofol 0.5 mg/kg bolus plus infusion 1 mg/kg/h) and 78 patients in Gruopremi (0.5 mcg/Kg/1 min bolus plus infusion 0.08 mcg/kg/min, progressively reduced to 0.03 mcg/kg/min). Cardiovascular and respiratory variables were measured before induction and every 3 min throughout the procedure. Sedation level was estimated by BIS and Observer’s Assessment of Alertness/Sedation Scale (OAA/S). Respiratory function was evaluated by arterial oxygen saturation (SaO2) and EtCO2. Recovery from sedation and hospital discharge criteria were assessed by Modified Aldrete Score System (APRS) 30 min after colonoscopy completion. Results: Remifentanil was effective and well tolerated during colonoscopy. Hemodynamic parameters remained stable throughout the study steps in both groups. In Groupremi OAA/S and BIS score were higher (p<0.001), and EtCO2 (p<0.5) lower that in Groupcontrol. Recovery time was faster in the Groupremi (p<0.01). Conclusions: Our data show that analgosedation with remifentanil allowed to obtain a good quality colonoscopy without respiratory and hemodynamic impairment and with faster recovery than moderate sedation propofol/fentanyl. Moreover, BIS and EtCO2 monitoring proved to be well suited to evaluate the trend variations of patients’ sedation level and respiratory drive

Specific peptide inhibition of phage particle binding to anti-Ap^1-17 IgG demonstrates that phage insert sequences recognize the antigen-combining sites of anti-Ap^1-17 IgG.

<p>Microtiter plates were coated with affinity-purified anti-Ap^1-17 IgG from pt4 (A.) or pt14 (B.). After the blocking of free protein-binding sites with PBS-BSA, wells were incubated for 2 h with 50 µl PBS containing serial dilutions of either free (closed symbols) or KLH-conjugated (open symbols) Ap^1-17 (rhombus), Ap^17-30 (triangle), CBp^1-13 (circle) or Qp-1a (square). Then, without removing the inhibitor, 50 µl/well of an appropriate dilution of phage supernatant pc4.33 (A.) and pc14.49 (B.) was added and incubation prolonged for 2 h. Bound phage particles were detected by sequential addition of HRP-anti-M13 mAb and o-phenylenediamine solution. Results are expressed as percentage of binding inhibition. Each data point is the mean of duplicate wells. The data are representative of two experiments. The maximum amount of inhibitor (800 µg) corresponds to 382 nmol Ap^1-17, 558 nmol Ap^17-30, 486 nmol CBp^1-13 and 574 nmol Qp-1a.</p

Specificity of anti-Ap^1-17 IgG for CENP-A documented by human recombinant CENP-A inhibition of anti-Ap^1-17 IgG binding to KLH-Ap^1-17.

<p>Anti-Ap^1-17 Abs from pt4 (A) and pt14 (B) were diluted in PBS-T20 at the lowest concentration giving 80%–100% of maximal A₄₉₀ in binding assay, and pre-incubated with an equal volume of PBS containing 2-fold serial dilutions of CENP-A (○), CENP-B (•), and CENP-B-derived peptide CBp^1-13 (▪). Following a 2-h incubation, the mixture was added to microtiter plate wells coated with KLH-Ap^1-17. After a 4-h incubation and three washes, bound IgG was detected with HRP-conjugated anti-human IgG (Fc portion) and <i>o</i>-phenylenediamine. Inhibition by Ap^1-17 peptide (□) and by Qp-1a (X) were included as positive and negative controls, respectively. Results are expressed as percentage of binding inhibition. The data are representative of 2 experiments.</p

Numbers of human proteins expressing the pt4 and pt14 antigenic motifs, by organ system (apparatus) and specific tissue.

a)<p>The number of proteins expressing the pt4 and pt14 motifs were determined by searching in the Swiss-Prot database (<a href="http://prosite.expasy.org/scanprosite/" target="_blank">http://prosite.expasy.org/scanprosite/</a>), using human tissue expression (<u>Bgee</u> data) filters.</p