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/ Timothy R. Sterling, MD a, Terezinha Martire, MD b,c, Alexandre Silva de Almeida, PhD b, Li Ding, MD d, David E. Greenberg, MD d, Lorena Alves Moreira, MS b, Houda Elloumi, PhD d, Angelica P.V. Torres, RN, BSN b, Clemax Couto Sant Anna, MD, PhD e, Eliane Calazans, MD f, Geraldo Paraguassu, MD g,h, Tebeb Gebretsadik, MPH i, Ayumi Shintani, PhD, MPH i, Kathleen Miller, RN, BSN a, Afranio Kritski, MD, PhD b, Jose Roberto Lapa e Silva, MD, PhD b and Steven M. Holland, MD d a Division of Infectious Diseases, Department of Medicine; b Academic Tuberculosis Program, Clementino Fraga Filho University Hospital; c Division of Pediatric Pneumology, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; d Laboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, Maryland; e Martagão Gesteira Pediatric Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; f Department of Pediatrics, Hospital San Sebastian, Rio de Janeiro, Brazil; g Department of Pediatric Neurosurgery, Hospital Sao Goncalo, Sao Goncalo, Brazil; h Department of Pediatric Neurosurgery, Hospital Municipal Jesus, Rio de Janeiro, Brazil; i Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, Tennessee 5 / (BCG) / 5 HIV / (PPD) (beadbased) + (IL) -12 +γ PPD) (IL-1β IL-4 IL-6 IL-8 IL-10 IL-12 γ -α ) Kruskal-Wallis 18 / 28 29 PPD 4.2 CD4 + (M tuberculosis); (tuberculosis); (tuberculosis meningitis); (miliary tuberculosis); (BCG vaccine); 1 (MCP-1) IL = interleukin MCP = monocyte chemoattractant protein PPD = purified protein derivative PBMC = peripheral blood mononuclear cell IFN = interferon TNF = tumor necrosis factor MANOVA = multivariate analysis 75

18 12 (67%) 28 26 (93%) 29 PPD 28 (97%) BCG / +γ PPD 1 / PPD 1 2518 G BCG / (12 ) IL-8 BCG (6 ) / BCG 1 1 of variance IQR = interquartile range Address correspondence to Timothy R. Sterling, MD, Division of Infectious Diseases, Vanderbilt University Medical Center, A2209 Medical Center North, 1161 21st Ave South, Nashville, TN 37232-2605. E-mail: timothy. sterling@vanderbilt.edu 10% [1] [2 5] HIV [2 4] [5] ( [2, 3, 6 8] ) [9, 10] / 5 [1, 11 13] / [14] HIV (IL)-8 HIV CD4 + [15] [16 18] (BCG) / [19 24] / BCG [25] BCG [26] BCG 1 (MCP-1 CCL-2) 76 STERLING et al

T MCP-1 [27] MCP-1 IL-12p40 MCP-1 AG GG [28] / / HIV HIV Clementino Fraga Filho (n =37) San Sebastian (n = 15) Gafree Guinle (n = 13) Sao Goncalo (n =5) Martagão Gesteira (n =5) / HIV / 5 ( ) CT MRI ( ) 2mg/dL HIV 5 5 HIV 5 (PPD) 10 mm (Clementino Fraga Filho San Sebastian Sao Goncalo Martagão Gesteira ) Gafree Guinle Vanderbilt (PBMC) 10 6 / ml RPMI 1640 (Mediatech Inc Herndon VA) [14] PBMC 37 5% CO 2 48 h 1% (Life Technologies Gaithersburg MD) 1 ng / ml +IL- 12p70 (R&D Systems Minneapolis MN) 200 ng / ml (Sigma St Louis MO) 1 000 U / ml + (IFN) -γ (Genentech South San Francisco CA) 48 h PBMC 10 mg / ml PPD 96 h [29] 96 h 70 77

IL-1β IL-4 IL-6 IL-10 IL-12p70 IFN-γ (TNF) -α MCP-1 Luminex (Bio-Rad Laboratories Hercules CA) Bio-Plex IL-8 (Meso Scale Discovery Gaithersburg MD) ( / ) MCP-1 MCP-1 ( 2518 G / A No. 1024611) (Foster City CA) Taq-Man (No. C_2590362_10) ABI 7500 15 5 - CCAGTATCTGGAATGCAGGC-3 ( ) 5 - ACAGGGAAGGTGAAGGGTAT-3 ( ) 4.2 (Gene Codes Ann Arbor MI) 2 80% α 0.05 ( / PPD ) Kruskal-Wallis χ 2 Fisher s ( ) 1 ( ) +1 5 48 h 2 96 h (MANOVA) [30] MANOVA MANOVA Box-Cox 1 M / M TB PulmTB PPD (n = 18) (n = 28) (n =29) P a (IQR) / y 4.4 (3.3 6.2) 4.0 (2.9 5.1) 4.2 (3.0 5.2) 0.52 / n (%) 10 (56) 15 (54) 14 (48) 0.87 / n (%) 10 (56) 20 (71) 23 (79) 0.31 BCG / n (%) 12 (67) 26 (93) 28 (97) 0.006 0.77 (IQR) / kg m -2 15.6 (14.8 20.0) 16.4 (13.8 18.2) 16.2 (15.5 18.0) n 17 26 26 CD4 (IQR) 1 578 (1 192 1 805) 1 239 (972 1 788) 1 190 (1 002 1 603) b 0.36 CD8 (IQR) 957 (659 1 546) 977(745 1 694) 885 (582 1 293) b 0.49 / n (%) 9 (50) 28 (100) NA NA NA M/M TB / PulmTB a Kruskal-Wallis χ 2 b n =27 78 STERLING et al

2 BCG BCG BCG PPD 1 28 29 2 26 28 4 1 5 1 4 5 + 1 4 5 + 0 3 3 Kruskal-Wallis Kruskal-Wallis posthoc χ 2 Hardy-Weinberg Fisher s / PPD MCP-1 χ 2 MCP-1 Kruskal-Wallis MCP-1 0.05 Stata 8.2 (Stata Corp College Station TX) SAS 9.1 (SAS Institution Cary NC) R 2.1.0 (R Project for Statistical Computing Vienna Austria) (IQR 13.7 24.5 ) / 47.2 mo (IQR 30.7 57.1 mo) 25.0 mo (IQR 17.6 37.3 mo) CD4 + CD8 + 18 / 12 (67%) 28 26 (93%) 29 PPD 28 (97%) BCG (P = 0.006) 2 / 75 48 h PPD 4 96 h 48 96 h INF-γ INF-γ INF-γ INF-γ IL-12 IL-12 48 h 3 / MCP-1 +INF-γ / 18 28 29 1 75 4.2 (IQR) 3.0 5.3 / 5.4 (IQR 3.6 10.2 ) 17.5 96 h PPD MCP-1 ( 3) 3 ( ) 79

3 a 48 h 48 h 48 h 48 h 48 h 96 h 96 h PHA PHA+IL-12 LPS LPS+INF-γ PPD IL-4 M/M TB 0.6 55 40 7 15 0.7 5 PulmTB 0.7 55 56 11 17 0.6 6 PPD 1.3 31 30 12 18 1.4 9 IL-6 M/M TB 63 15 723 18 462 103 951 182 152 38 16 782 PulmTB 80 15 224 15 859 155 871 225 081 77 37 472 PPD 70 15 171 15 232 90 468 14 2503 52 37 018 IL-10 M/M TB 0.5 223 194 263 127 0.7 19 PulmTB 0.9 196 156 460 225 1.2 31 PPD 0.7 206 167 306 149 1.2 17 INF-γ M/M TB 0.2 14 946 12 961 530 0.2 1 655 PulmTB 0.2 12 393 11 455 666 0.2 3 110 PPD 0.7 9 625 12 904 474 0.2 1 340 P NS NS NS NS NS NS TNF-α M/M TB 0.1 1 600 1 536 86 951 0.2 61 PulmTB 0.2 1 704 1 556 208 1 385 0.2 60 PPD 0.3 1 497 1 297 165 1 160 0.4 101 P NS NS NS NS NS 0.02 b 0.33 IL-1β M/M TB 0.3 25 21 334 872 0.1 5 PulmTB 0.3 26 24 631 1 114 0.2 10 PPD 0.2 22 17 433 783 0.3 11 IL-12 M/M TB 0.7 37 5 243 1.3 3 PulmTB 0.8 18 5 139 0.8 3 PPD 0.3 30 4 231 0.4 2 P NS NS NS NS NS NS MCP-1 M/M TB 1 181 19 100 13 755 12 843 11 202 3 417 57 202 PulmTB 1 703 26 286 30 850 18 363 17 671 4 392 86 803 PPD 570 18 053 14 186 10 226 6 787 2 284 35 129 P 0.002 b 0.07 0.10 0.02 b 0.02 b 0.07 0.001 4 b IL-8 M/M TB 1 184 61 559 55 226 74 219 74 123 1 630 48 845 80 STERLING et al

PulmTB 1 520 70 888 72 290 86 334 85 503 2 306 68 574 PPD 1 244 65 876 62 235 78 784 82 322 2 247 74 148 48 96 h PBMC pg / ml P Kruskal-Wallis MANOVA 5 48 h 2 96 h MANOVA (P 0.05) Kruskal-Wallis P P (NS) M/M TB / ( IL-8 17 ) PulmTB PPD ( 48 h IL-8 27 96 h IL-8 24 ) PHA LPS a PHA 1% IL-12 1ng/mL LPS 200 ng / ml IFN-γ 1 000 U / ml PPD 10 µg /ml b P 0.05 ( 10 ) 8 (P =0.01) +INF-γ (P =0.03) MCP-1 ( ) BCG / ( 4) IL-8 IL-8 BCG (P =0.01) +INF-γ (P = 0.01) PPD (P = 0.03) MCP-1 2518 / PPD AG GG G ( 5 6) 2518 Hardy-Weinberg (P = 0.20) AA AG GG MCP-1 ( 7) / PPD CD4 HIV CD4 [15] [16 18, 31 34] IFN-γ-IL-12-IL-23 [35] IFNγ-IL-12-IL-23 [36] HIV 81

4 BCG BCG / 48 h 48 h 48 h 48 h 48 h 96 h 96 h PHA PHA IL-12 LPS LPS INF-γ PPD IL-4 BCG 0.5 59 64 7 21 0.5 9 BCG 0.6 55 35 8 15 0.7 5 IL-6 BCG 61 35 812 25 952 216 804 408 367 59 88 949 BCG 64 14 166 13 078 86 118 145 537 32 12 793 IL-10 BCG 1.0 232 209 261 124 0.9 38 BCG 0.5 211 170 290 139 0.7 14 INF-γ BCG 0.2 25 383 19 784 570 0.2 4 058 BCG 0.2 14 946 7 523 467 0.2 1 214 P NS NS NS NS NS NS TNF-α BCG 0.1 2 364 1 716 103 1 899 0.2 137 BCG 0.2 1 476 1 141 66 886 0.2 61 IL-1β BCG 0.2 31 21 461 1 307 0.1 10 BCG 0.3 25 21 243 581 0.1 4 IL-12 BCG 0.4 49 5 306 0.5 3.8 BCG 1.2 32 5 202 2.0 1.8 P NS NS NS NS NS NS MCP-1 BCG 1 375 29 483 40 341 11 817 14 256 3 333 109 001 BCG 995 15 113 7 389 13 225 8 175 4 592 46 513 IL-8 BCG 1 571 101 105 115 729 104 468 125 455 2 643 118 972 BCG 1 112 52 987 37 726 61 051 59 491 1 450 35 900 P 0.42 0.07 0.06 0.01 a 0.01 a 0.16 0.03 a 48 96 h PBMC pg / ml P Kruskal-Wallis 12 BCG 6 MANOVA 5 48 h 2 96 h MANOVA (P 0.05) Kruskal-Wallis P P (NS) a P 0.05 82 STERLING et al

5 MCP-1 MCP-1 PulmTB M/M TB PPD (n =26) (n = 17) (n =29) AA 14 (0.54) 10 (0.59) 19 (0.66) AG 10 (0.38) 6 (0.35) 7 (0.24) GG 2(0.08) 1(0.06) 3(0.10) ( ) Fisher s P 0.83 72 DNA PulmTB M/ M TB ( ) PPD [37] CD4 TNF-γ INF-γ [38] CD4 MCP-1 ( 3) Hasan [27] PPD BCG MCP-1 Flores-Villanueva [28] PPD MCP-1 ( 2518A / G) 2518G MCP-1 IL-12p40 / MCP-1 / ( ) MCP-1 Flores-Villanueva [28] MCP-1-2518G MCP-1 GG ( ) GG-2518 AA AG MCP-1 IL-12 ( MCP-1 IL-12 MCP-1 7) 93% BCG BCG MCP-1 BCG 6 MCP-1 MCP-1 PulmTB M / M TB PPD PulmTB vs PulmT vs (n =52) (n =34) (n = 58) M/M TB PPD P P A 38 (0.73) 26 (0.76) 45 (0.78) 0.92 0.74 G 14 (0.27) 8 (0.24) 13 (0.22) ( ) PulmTB M/M TB ( ) PPD 83

7 MCP-1 MCP-1 IQR 48 h AA 960 535 3 036 AG 1 010 380 2 602 GG 997 570 1 428 P 0.96 PHA AA 22 807 10 834 49 056 AG 18 830 8 379 36 756 GG 16 701 12 213 18 917 P 0.29 PHA+IL-12 AA 22 825 11 811 54 291 AG 20 598 7 474 42 036 GG 3 342 664 23 223 P 0.08 LPS AA 13 771 7 444 19 262 AG 9 262 5 739 31 988 GG 19 583 17 349 21 745 P 0.53 LPS+INF-γ AA 14 067 6 067 25 899 AG 8 337 3 602 21 152 GG 13 650 4 600 17 529 P 0.66 96 h AA 3 798 2 051 6 762 AG 2 976 1 240 7 048 GG 3 029 2 284 5 969 P 0.81 PPD AA 67 239 32 564 107 609 AG 40 365 22 310 79 888 GG 43 471 32 526 62 052 P 0.18 MCP-1 PBMC MCP-1 AA 43 AG 23 GG 6 pg / ml P Kruskal-Wallis PHA LPS MCP-1 MCP-1 BCG MCP-1 97% BCG PPD MCP-1 BCG / BCG 18 / 12 BCG BCG ( 4) BCG IL-8 INF-γ PPD IL-8 BCG IL-8 BCG / BCG IL-8 Moreau BCG Pasteur MANOVA 84 STERLING et al

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