1A and B, respectively)
1A and B, respectively). household contacts of known TB cases and were divided into two groups: PPD reactive (PPD+) (= 60; age range, 1 to 14 years; average age SD, 7.1 4.1 years) and non-PPD reactive (PPD?) (= 17; age range, 3 to 14 years; average age SD, 7.2 3.5 years). PPD+ subjects were treated with isoniazid only and followed the same schedule used for TB patients. Enzyme-linked immunosorbent assay plates (MaxiSorp; Nunc, Copenhagen, Denmark) were coated overnight at 37C with 50 l/well of 1-g/ml recombinant HSP16 (a gift from J. Ivanyi) in carbonate buffer (0.1 M sodium bicarbonate in distilled water [pH 8.2]). Plates were blocked with phosphate-buffered saline (PBS)-10% fetal calf serum for 1 h at 37C. Plates were incubated for 90 min at 37C with 50 l of serum samples diluted 1:50 in PBS. After four washes with PBS-Tween, 50 l of anti-human IgG or IgM (both peroxidase conjugated; Sigma-Aldrich, Milan, Italy) was added to each well, and the mixture was incubated for 2 h at 37C. The plates were washed six occasions with PBS-Tween and then colorimetrically designed with values of 0.05 were considered significant. To assess whether determination of HSP16-specific antibody responses could be useful for monitoring the efficacy of chemotherapy, we measured HSP16-specific IgG and IgM levels in sera from patients and healthy controls (PPD+) before and after therapy. The results are presented as percentages of anti-HSP16 IgG- and IgM-positive sera (Fig. ?(Fig.1)1) and as mean ODs before and after chemotherapy (Fig. ?(Fig.2).2). The percentages of HSP16-specific IgG and IgM responders decreased after chemotherapy both for TB patients (from 73.3 to 37.7% for IgG; from 73.3 to 20% for IgM) and for healthy PPD+ subjects (from 48.3 to 25% for IgG; from 58.33 to 15% for IgM) (Fig. 1A and B, respectively). Percentages of sera positive for HSP16-specific IgG or IgM were low for PPD? patients (Fig. ?(Fig.1C1C). Open in a separate windows FIG. 1. Frequencies of anti-HSP16 IgG or IgM responses before and after chemotherapy. Shown are percentages of sera with IgG Balsalazide disodium and IgM responses to HSP16 for TB patients (= 45) (A) and for healthy PPD+ (= 60) (B) and PPD? (= 17) (C) subjects. Frequencies of positive sera were obtained by dividing the number of positive sera for each group by the total number of sera tested for that group. Hatched bars, results obtained with sera after 4 months of therapy. Open in a separate windows FIG. 2. Comparison of mean ODs of anti-HSP16 IgG before and after chemotherapy. (A and B) Mean ODs for anti-HSP16 IgG were analyzed before (A) and after (B) chemotherapy. For panel A, statistical comparisons were performed between data obtained with sera from TB patients and those from healthy PPD+ subjects ( 0.05) and between data for healthy PPD+ versus PPD? subjects ( 0.05). For panel B, data from TB patients were compared with data from healthy PPD+ subjects ( 0.05). (C and D) Mean ODs for anti-HSP16 IgM were analyzed before (C) and after (D) chemotherapy. For panel C, statistical comparisons were performed between data for TB patients versus healthy Balsalazide disodium PPD+ subjects ( 0.05) and between data for healthy PPD+ versus PPD? subjects ( 0.05). Balsalazide disodium For panel D, data obtained from TB patients were compared with data for healthy PPD+ subjects ( 0.05). Further, anti-HSP16 IgG (Fig. 2A and B) and IgM (Fig. 2C and D) levels of TB patients and PPD+ subjects were compared before and after therapy. Before therapy, mean responses were higher for TB patients (ODs, 0.330 0.231 for IgG and 0.390 0.266 for IgM) than for healthy PPD+ (ODs, 0.167 0.147 for IgG and 0.237 0.213 for IgM) or PPD? (ODs, 0.109 0.135 for IgG EIF4EBP1 and 0.104 0.162 for IgM) subjects. After chemotherapy, IgG and IgM levels decreased more in TB patients (ODs, 0.190 0.174 and 0.120 0.085, respectively) than in healthy PPD+ individuals (ODs, 0.113 0.078 and 0.094 0.067, respectively) ( 0.05 for all those parameters). We found that levels of IgG and IgM against the 16-kDa antigen in infected children and in healthy contacts decrease.