Absolute numbers of the studied cell subtypes.

<p>Absolute numbers of the indicated cell subtypes were analyzed as reported in Figure S1 and at the time points described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034493#pone-0034493-t001" target="_blank">Table 1</a>, of the patient who developed PML (pmlMS; black dots), natalizumab-treated patients (nMS; gray dots), and healthy donors (HD; white dots). Four samples were analyzed at the time point T4 in the pmlMS patient. ??? significant differences between cell subsets of the pmlMS patient in comparison to those of nMS patients; ??? significant differences between cell subsets of the pmlMS patient in comparison to those of HD. Horizontal lines indicate means and error bars indicate the 95% confidence intervals of the means.</p

Natalizumab-treated patient characteristics.

<p>Only the long-lasting and relevant therapies are reported. The interval between IFNβ 1a or GA therapies and natalizumab was at least of 30 days. plmMS received MTX from February 2004 to November 2006, nMS3 from February 2003 to March 2005 and nMS4 from July 2004 to February 2007.</p>**<p>There was no trend of EDSS up- or downwards.</p>***<p>Time point in which PML was diagnosed. Four different samples were obtained over a 1-month period. For the other time points and in all other patients, only one biological sample was analyzed.</p><p>F, female; M, male; y, years; w, weeks; IFN, interferon; MTX, mitoxantrone; GA, glatiramer acetate; na, not available.</p

Variations of CD34, DNTT and Vpreβ1 transcript expressions.

<p>Fold-change increase of transcripts for CD34, terminal deoxynucleotidyltransferase (DNTT), expressed by lymphoid precursors, and V pre-B lymphocyte gene 1 (Vpreβ1), a part of the pre-B cell receptor in the patient who developed PML (pmlMS; black circles) and natalizumab-treated patients (nMS; white circles) were represented at the indicated time points. Bars indicate medians and error bars indicate the 25^th and 75^th percentile. Data were calculated with the ΔΔCt method using the value obtained with pre-therapy samples (T0) as the calibrator for each patient.</p

T-cell repertoire heterogeneity.

<p>Percent usage of TCRBV and perturbations of TCR CDR3 length distributions at the indicated time points in the patient who developed PML (pmlMS) and patients treated with natalizumab (nMS1 to nMS5) were determined by spectratyping analysis. The TCRBV chains were considered overused when their percent usage was higher than mean+2SD (“x") or mean+3SD (“o") of the TCRBV chain usage found in 5 healthy donors (HD). Perturbations were obtained by calculating the generalized Hamming distance according to Gorochov et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034493#pone.0034493-Gorochov1" target="_blank">[26]</a> and were considered significant when their values were beyond the mean+2SD (light gray squares) or the mean+3SD (dark gray squares) of the CDR3 distribution found in HD. N*: number of perturbed TCRBV chains at each time point.</p

Levels of TRECs and KRECs.

<p>Levels of TRECs and KRECs in patient who developed PML (pmlMS; black dots), natalizumab-treated patients (nMS), untreated patients (uMS; white dots), and healthy donors (HD; white dots) were reported. The indicated time points (T0, T1, T2, T3, and T4) were those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034493#pone-0034493-t001" target="_blank">Table 1</a>. In the pmlMS patient, whose four samples were analyzed at the time point T4, TRECs were always significantly lower (p<0.01) in comparison to nMS patients, uMS patients, and HD. The levels of TRECs of uMS patients and HD were also significantly different (p<0.01). KRECs of the pmlMS patient were lower (p<0.01) than those of nMS and HD at T0 and T4, lower than uMS at T0, and higher than those of uMS and HD at T1, T2 and T3. Horizontal lines indicate means and error bars indicate the 99% confidence intervals of the means, calculated after log transformation of the data.</p

IgG, IgA, and IgM concentration.

<p>IgG, IgA, and IgM concentration was analyzed at the indicated time points in the sera of the patient who developed PML (pmlMS; black bars) and patients treated with natalizumab (nMS; white bars). Bar height represents the mean value and error bars represent the 95% confidence interval of the mean. * Measures of the pmlMS patient that were outside the 95% confidence interval of the means found in nMS. Dashed lines represent the lowest limit of the laboratory reference interval for healthy subjects.</p

Average weekly FLS prevalence in patients divided based on the IL-6 genotypes or on the presence of the C allele.

<p>Average weekly FLS prevalence in patients divided based on the IL-6 genotypes or on the presence of the C allele.</p

Average IL-6 levels in MS patients.

<p>(A) Average IL-6 levels in patients with the three indicated IL-6 genotypes. (B) Average IL-6 levels in patients bearing at least one allele with a C at position -174 of IL-6 gene and in those bearing only the allele with the G at the same position. 0 = samples collected just before the beginning of IFNβ-1a therapy; 1 = samples collected at about 12 hours after the first injection; 12 = samples collected at about 12 hours (h) from the injection performed at 12 weeks of therapy.</p

Consumption of anti-inflammatory/antipyretic drugs and its effects on FLS.

<p>(A) Comparison of mean number (no.) of anti-inflammatory/antipyretic drugs taken during the follow-up by patients bearing at least one C allele at position -174 of IL-6 gene vs. those bearing two G alleles at the same position. (B) Comparison of mean FLS severity score in patients taking the first dose of anti-inflammatory/antipyretic drugs in the peri-injection period vs. those that assumed the first dose hours later. (C) Effect of the peri-injection dose of antipyretic/anti-inflammatory drug on the probability of developing FLS in patients taking different total doses of antipyretic/anti-inflammatory drugs.</p

Characteristics of the 50 patients and transplant.

<p>Characteristics of the 50 patients and transplant.</p