INT J TUBERC LUNG DIS 18(6):671–675 Q 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0831

Cathelicidin and human b-defensin 2 in bronchoalveolar lavage fluid of children with pulmonary tuberculosis E. Cakir,* E. Torun,† A. H. Gedik,* T. Umutoglu,‡ E. C. Aktas,§ U. Topuz,‡ G. Deniz§ *Department of Paediatric Pulmonology, †Department of Paediatrics, ‡Department of Anaesthesiology, Bezmialem Vakif University Medical Faculty, Istanbul, §Department of Immunology, Institute of Experimental Medicine, Istanbul University, Istanbul, Turkey SUMMARY BACKGROUND:

The antimicrobial peptide cathelicidin LL-37/hCAP-18 and human b-defensins (hBD) are key factors in innate immune responses of the respiratory tract. O B J E C T I V E : To determine LL-37 and hBD-2 concentrations in the bronchoalveolar lavage (BAL) fluid of paediatric patients (aged ,16 years) with pulmonary tuberculosis (TB) and to compare these with concentrations in healthy children. M E T H O D S : We measured peptide concentrations using an immunosorbent assay (ELISA). R E S U LT S : Forty TB patients and 40 healthy controls were enrolled in the study (mean age 9.2 6 4.7 and 8.3 6 4.2 years, respectively, P ¼ 0.97). The two groups exhibited no statistically significant difference in terms of sex, body mass index, relative weight or 25-

hydroxyvitamin D levels. The mean BAL LL-37 level of the TB group was significantly higher than that of the control group (0.95 6 standard deviation [SD] 1.33 vs. 0.35 6 SD 0.51 ng/ml, P ¼ 0.01, t ¼ 2.54). The hBD-2 level was also higher in the TB group; however, the difference was not statistically significant (0.30 6 SD 0.58 vs. 0.14 6 SD 0.30 ng/ml, P ¼ 0.11). There was no correlation between LL-37, hBD-2 and 25-hydroxyvitamin D levels. C O N C L U S ˙IO N S : Our data suggest that LL-37 and hBD2 may play an important role in TB pathogenesis in children. To our knowledge, this is the first study on BAL LL-37 and hBD-2 concentrations in children with pulmonary TB. K E Y W O R D S : child; antimicrobial peptide; cathelicidin; defensin; tuberculosis

CATHELICIDINS AND b-DEFENSINS are the two principal antimicrobial peptide (AMP) families expressed in the epithelium of the human lung. These peptides include the cathelicidin LL-37/hCAP-18 and human b defensins 1 to 4 (hBD-1 to hBD-4).1–4 LL37/hCAP-18 is expressed as a pre-pro-peptide. The propeptide is called hCAP-18 and is stored in cells. After secretion, the propiece is cleaved off and the Cterminal peptide is called LL-37. LL-37 is the active peptide with antimicrobial and other activities. AMPs contribute to innate immunity by direct antimicrobial activity. LL-37 and defensins have multiple functions, such as the activation of inflammatory cells and the regulation of adaptive immunity.1–6 The role of AMPs in the pathogenesis of Mycobacterium tuberculosis has been examined in in vitro studies7–10 and some in vivo studies.11,12 However, there are no published reports on LL-37 and hBD-2 concentrations in bronchoalveolar lavage (BAL) fluid in childhood pulmonary tuberculosis (PTB). The aim of the present study was to measure LL-37 and hBD-2

concentrations in the BAL fluid of paediatric patients with PTB and to compare them with those of healthy children.

MATERIAL AND METHODS Pulmonary tuberculosis patients BAL samples were obtained from paediatric PTB patients (aged ,16 years) undergoing fiberoptic flexible bronchoscopy (FB) for clinical reasons. In our institution, FB is routinely performed among TB cases with clinical or radiological abnormalities and suspected airway involvement such as brassy cough, localised wheezing, appearance of large lymphadenopathy with consolidation or atelectasis, hyperinflation and haemoptysis. FB is also performed among patients with clinical or radiological resistance despite appropriate anti-tuberculosis treatment.13 Forty children who underwent FB were enrolled in the study. TB disease was diagnosed in accordance with the

Correspondance to: Erkan Cakir, Department of Paediatric Pulmonology, Bezmialem Vakif University, Adnan Menderes Avenue, PK 34093 Fatih, Istanbul, Turkey. Tel: (þ90) 212 453 1700. Fax: (þ90) 212 621 7580. e-mail: erkancakir1@yahoo. com Article submitted 17 November 2013. Final version accepted 29 January 2014.

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World Health Organization standard TB case definitions.14 Diagnosis was based on respiratory symptoms, radiological findings, tuberculin skin test (TST) positivity, history of contact with an active TB case, and acid-fast bacilli and M. tuberculosis culture positivity. Adequate criteria for a strong suspicion of TB was fulfilled in all the children. TST results were interpreted according to the recommendations of the American Thoracic Society (New York, NY, USA) and the US Centers for Disease Control and Prevention (Atlanta, GA, USA). Among the patients, 68% had a history of household contact and 82% were TST-positive. The most frequent symptoms were cough (80%), weakness and anorexia (50%), weight loss (45%) and night sweats (42.5%). All of the patients had radiological findings consistent with TB unresponsive to non-specific antibiotherapy. Forty-two per cent of the patients were M. tuberculosis culture-positive, 67% (n ¼ 31) had primary TB (primary infiltration with/without lymphadenopathy), 8% (n ¼ 3) had progressive primary TB (excessive, disseminated pulmonary infiltration) and 15% (n ¼ 6) had adult-type secondary TB (adult-type cavitary lesions). All of the children responded to anti-tuberculosis treatment. Control group Non-TB healthy controls (n ¼ 40) were selected from among children without lung disease who had been undergoing elective non-pulmonary surgical procedures such as inguinal hernia, cryptorchidism, etc. Neither the TB nor the control group had a history of immunosuppressive drug use, immune deficiency, chronic diseases or malnutrition. Malnutrition was assessed using body mass index (BMI) and relative weight. Standing height was measured to the nearest 0.1 cm with a Harpenden fixed stadiometer (Tartı Medikal, Istanbul, Turkey). Body weight (kg) was measured on a SECA balance scale (SECA, Hamburg, Germany) to the nearest 0.1 kg, with each subject dressed in a light T-shirt and shorts. BMI was calculated as kg/m2. Relative weight was calculated as weight/midpoint of the weight of other children of the same height x 100. Each patient’s serum biochemical markers, including albumin, total protein, 25-hydroxyvitamin D [25(OH)D] and complete blood count, were measured. Serum 25(OH)D levels were determined using the electrochemiluminescence enzyme immunassay method (ECLIA) (ADVIA Centaur; USADPC Co, Los Angeles, CA, USA), albumin and total protein were measured using the colorimetric technique (ROCHE Cobas 8000, Roche Diagnostics, Basel, Switzerland) and complete blood count was measured using the empedance technique (Sysmex XT 1800I, Sysmex Corp, Kobe, Japan).

Flexible bronchoscopy and BAL samples All FBs were performed on TB patients in the procedure room using one of the following: Pentax EB-1570K 4.9 mm (Hoya Corporation, Tokyo, Japan), Pentax EB-1170K, 3.7 mm (Hoya Corporation) or Fujinon FB-120P, 2.8 mm (Fujifilm Corporation, Tokyo, Japan) fibroscope. In the TB group, FB was performed using a laryngeal airway mask or transnasally. During the procedure, all patients were given oxygen and monitored for oxygen saturation and cardiac rhythm. Patients received midazolam, pethidine hydrochloride (meperidine), ketamine or propofol as premedication and lidocaine was used as topical anaesthetic, applied locally at a maximum dose of 4 mg/kg. FB (Fujinon FB-120P, 2.8 mm) was performed in the control group using an endotracheal tube under general anesthesia in the operation room before surgery. Bronchial washing was performed on the right middle lobe and left lingular lobe by wedging the tip of the bronchoscope in a subsegmental bronchus. Among TB and control subjects, 3 cc/kg of saline was infused into the lungs (mean 47 cc and 43 cc, respectively). Approximately 60–70% was recovered (31 cc and 28 cc, respectively). The mean number of cells in the BAL fluid was 180 000/ml in the TB group and 200 000/ml in the control group. In the TB group, the mean percentage of macrophages was 83% (range 78–89), of lymphocytes 9% (range 6–13), of neutrophils 1.4% (range 1–3), of eosinophils 0.3% and that of alveolar macrophages was 0.04%. In the control group, the mean percentage of macrophages was 81% (range 77–86), for lymphocytes it was 7% (range 6–11), neutrophils 2.1% (range 1–5), eosinophils 0.4% and that of alveolar macrophages 0.03%. Immunosorbent assays (ELISAs) for beta defensin and LL-37 BAL samples were centrifuged at 3000 rpm for 10 min and stored at 808C. LL-37 and hBD-2 levels in BAL samples were assessed using hDEFb2 (BMASSAY, Beijing, China) and hLL-37 (Hycult Biotechnology, Uden, The Netherlands) sandwich and immunosorbent assay (ELISA) kits. Samples were diluted to obtain the appropriate concentrations, and ELISA was performed according to the manufacturer’s instructions. Supplied standards were used to generate the standard curves. The samples and standards were added to the wells. Unbound protein was removed by washing, and then with biotinconjugate, followed by horseradish peroxidase-conjugated streptavidin added in a step-wise manner. After colour reaction with substrate, the optical density was recorded using an automated ELISA reader at a wavelength of 450 nm. Concentrations were calculated by plotting the graph between

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concentrations and corresponding absorbance of standards. The absorbance at 450 nm was converted to ng/ml for hDEFb2 and hLL-37. The minimal detection limits were 0.1 ng/ml for LL-37 and ,0.05 ng/ml for hDEFb2. Statistical analysis Statistical analysis was performed using IBM SPSS 19 (IBM, Armonk, NY, USA). Mean, median, standard deviation (SD) and interquartile ranges were used for metric variables. The Mann-Whitney U-test was used to calculate the difference of two parameters in groups; the Kruskall Wallis test was used to compare more than two independent parameters. Multiple comparisons were made using the Dunn test. Categorical data were evaluated using the v2 test, and P , 0.05 was accepted as statistically significant. The study was approved by the Bezmialem Vakif University ethics committee (B.30.2.BAV.0.05.05/ 133). Written informed consent was provided by the parents of participating children.

RESULTS Forty TB patients and 40 controls were enrolled in the study. The mean age of the children was respectively 9.2 years (SD 4.7, range 1–16) and 8.3 years (SD 4.2, range 2–16) (P ¼ 0.97). The male:female ratios of the two groups were 18:22 and 20:20, respectively. The difference in BMI SD and relative weight of the two groups was not statistically significant (0.62 6 1.2 vs. 0.3 6 0.8, P ¼ 0.33 and 98.2 6 13.1 vs. 102.2 6 11.5, respectively, P ¼ 0.81) (Table). The difference in 25(OH)D levels (20.9 6 5.8 ng/ml and 21.1 6 5.1 ng/ ml, respectively, P ¼ 0.78) and the levels of other biochemical markers between the two groups was not statistically different. The mean (6 SD) BAL LL-37 level of the TB group was significantly higher than that of the control group

Figure 1 LL-37 levels in children with tuberculosis and in healthy children (P ¼ 0.01).

(0.95 6 1.33 vs. 0.35 6 0.51 ng/ml, P ¼ 0.01, t ¼ 2.54) (Figure 1). The hBD-2 level was higher in the TB group than in healthy controls, but not to a statistically significant level (0.30 6 0.58 ng/ml vs. 0.14 6 0.30 ng/ml, P ¼ 0.11) (Figure 2). There was no correlation between LL-37, hBD-2, 25(OH)D levels, age, sex and BMI.

DISCUSSION The aim of this study was to analyse AMPs in the airways of children with PTB. We determined LL-37 and hBD-2 concentrations in the BAL fluid of TB patients and in healthy controls. Our results show that compared with the control group, BAL LL-37 levels in children with TB were significantly higher, and that although hBD-2 levels were also higher, the difference was not statistically significant. Innate immunity is the first line of defense against invading microorganisms, and triggers an antigenspecific adaptive immune response. AMPs are thought to be significant effectors of innate immunity due to their immunomodulatory activity and direct

Table Demographic features of the study population TB group n (%)

Control group n (%)

Sex Male Female

18 (45) 22 (55)

20 (50) 20 (50)

.0.05

TB sub-group Primary TB Progressive primary TB Secondary TB

31 (67) 3 (8) 6 (15) 9.2 6 4.7

8.3 6 4.2

.0.05

0.62 6 1.2

0.3 6 0.8

.0.05

Age, years, mean 6 SD Body mass index, kg/m2, mean 6 SD

P value

Relative weight, %, mean 6 SD 25-hydroxyvitamin D, ng/ml, mean 6 SD

98.2 6 13.1 20.9 6 5.8

102.2 6 11.5 21.1 6 5.1

.0.05 .0.05

LL-37, ng/ml, mean 6 SD

0.95 6 1.33

0.35 6 0.51

0.01

hBD-2, ng/ml, mean 6 SD

0.30 6 0.58

0.14 6 0.30

0.11

TB ¼ tuberculosis; SD ¼ standard deviation.

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Figure 2 hBD-2 levels in children with tuberculosis and in healthy children (P ¼ 0.11).

killing of microorganisms. AMPs are divided into two families, cathelicidins and defensins, both of which have been involved in the immunopathogenesis of several infectious diseases, including TB. The results of studies on the role of AMPs related to TB immunity have been primarily based on in vitro studies.2,4,7,9,10,15,16 The important role played by the cathelicidin LL37 in innate immune response against M. tuberculosis has been demonstrated; M. tuberculosis infection induces LL-37 gene expression and protein secretion in several human cell types. It is well known that alveolar macrophages are the main effector cell types involved in M. tuberculosis destruction in the lung, and that when bacilli enter the airways, the first cells that encounter the bacteria are alveolar macrophages and epithelial cells. In this regard, Rivas-Santiago et al. reported that alveolar macrophages infected with M. tuberculosis showed a significant percentage of LL-37 immunostained cells in a dose-dependent manner.7 They also demonstrated that when lung epithelial cells were infected with M. tuberculosis, high levels of LL-37 were produced, generally after 18 h and in a dose-dependent manner.7 In addition, they showed that hBD-2 is expressed and associated with M. tuberculosis during the infection of human alveolar epithelial cells.9,17 M´endez-Samperio et al. showed expression and secretion of cathelicidin LL-37 in human epithelial cells after infection with M. bovis bacille CalmetteGu´erin (BCG).10 They also demonstrated an induction of hBD-2 mRNA in epithelial A549 cells after infection with M. tuberculosis and M. bovis BCG.15,16 A more recent study has reported significant production of mRNA and protein expression of LL37 when epidermal keratinocytes, which function as the host’s primary defense against mycobacterial infection, were exposed to M. ulcerans.18 During experimental TB, cathelecidins and defensins are

abundantly produced from diverse cellular sources and could significantly participate in its pathogenesis.19 Although in vitro studies have reported on the role played by AMPs in M. tuberculosis, few in vivo studies are available in the literature.11,12 The first was conducted in our clinic among children with PTB;11 in that study we aimed to evaluate intracellular cytokine secretion, including LL-37, from monocytes and neutrophils in 15 children with PTB and compare it to secretion in 15 healthy children. The expression of LL-37, tumour necrosis factoralpha, interferon-gamma and interleukin 8 in CD14þ monocytes and CD15þ neutrophils were analysed using the flow cytometry method. We demonstrated that the expression of LL-37 and IL-8 from CD14þ monocytes was significantly higher in the TB group than in the control group.11 We also determined LL-37 and hBD-2 concentrations in the BAL fluid of TB patients and healthy controls and showed that pulmonary M. tuberculosis was associated with significantly higher concentrations of LL-37 and hBD-2. To our knowledge, this is the first report on BAL LL- 37 and hBD-2 concentrations in children with PTB. In vitro studies have demonstrated that cathelicidins are required for the 1,25D3-triggered antimicrobial activity against intracellular M. tuberculosis. It has been shown that the human monocytic cell line Thelper-1 expresses cathelicidin when incubated with 1,25D3, and that cathelicidin is required for 1,25D3mediated antimicrobial activity against intracellular M. tuberculosis in human monocytes.20 Selvaraj et al. reported that the addition of 1,25 D3 might lead to increased expression of cathelicidin, which could enhance immunity against M. tuberculosis.21 The single in vivo study in adult PTB found that the serum vitamin D status of the study subjects did not correlate with serum LL-37 concentrations.12 We also did not find any correlation between serum vitamin D status and LL-37 or hBD-2. In conclusion, our data suggest that LL-37 and hBD-2 may play an important role in the pathogenesis of TB in children. Further in vivo studies on this should be conducted. Acknowledgements The authors thank O Uysal, Department of Biostatistics and S Declorix, Department of Foreign Languages, Bezmialem Vakif University Medical Faculty, Istanbul, for their valuable contribution. Conflict of interest: none declared.

References 1 Bals R, Wang X, Zasloff M, Wilson J M. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA 1998; 95: 9541–9546.

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2 Bals R, Wilson J M. Cathelicidins-a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 2003; 60: 711–720. 3 McCray P Jr, Bentley L. Human airway epithelia express a betadefensin. Am J Respir Cell Mol Biol 1997; 16: 343–349. 4 Jia H P, Schutte B C, Schudy A, et al. Discovery of new human beta-defensins using a genomics-based approach. Gene 2001; 263: 211–218. 5 Bals R, Wang X, Wu Z, et al. Human beta-defensin 2 is a saltsensitive peptide antibiotic expressed in human lung. J Clin Invest 1998; 102: 874–880. 6 Durr U H, Sudheendra U S, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys Acta 2006; 1758: 1408–1425. 7 Rivas-Santiago B, Hernandez-Pando R, Carranza C, et al. Expression of cathelicidin LL-37 during Mycobacterium tuberculosis infection in human alveolar macrophages, monocytes, neutrophils, and epithelial cells. Infect Immun 2008; 76: 935–941. 8 Liu P T, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006; 311: 1770–1773. 9 Rivas-Santiago B, Schwander S K, Sarabia C, et al. Human beta-defensin 2 is expressed and associated with Mycobacterium tuberculosis during infection of human alveolar epithelial cells. Infect Immun 2005; 73: 4505–4511. 10 M´endez-Samperio P, Miranda E, Trejo A. Expression and secretion of cathelicidin LL-37 in human epithelial cells after infection by Mycobacterium bovis bacillus Calmette-Gu´erin. Clin Vaccine Immunol 2008; 15: 1450–1455. 11 Torun E, Cakir E, Aktas E C, Gedik A H, Deniz G. Intracellular cytokine and cathelicidin secretion from monocytes and neutrophils in childhood tuberculosis. Pediatr Infect Dis J 2013; 33: 224–226. 12 Yamshchikov A V, Kurbatova E V, Kumari M, et al. Vitamin D status and antimicrobial peptide cathelicidin (LL-37) concentrations in patients with active pulmonary tuberculosis. Am J Clin Nutr 2010; 92: 603–611.

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13 Cakir E, Kut A, Ozkaya E, Gedik A H, Midyat L, Nursoy M. Bronchoscopic evaluation in childhood pulmonary tuberculosis: risk factors of airway ınvolvement and contrubition to the bacteriologic diagnosis. Pediatr Infect Dis J 2013; 32: 921–923. 14 Stop TB Partnership Childhood TB Subgroup, World Health Organization. Guidance for national tuberculosis programmes on the management of tuberculosis in children. Chapter 1: ˙ Introduction and diagnosis of tuberculosis in children. Int J Tuberc Lung Dis 2006; 10: 1091–1097. 15 M´endez-Samperio P, Miranda E, Trejo A. Mycobacterium bovis bacillus Calmette-Gu´erin (BCG) stimulates human beta-defensin-2 gene transcription in human epithelial cells. Cell Immunol 2006; 239: 61–66. 16 M´endez-Samperio P, Alba L, Trejo A. Mycobacterium bovismediated induction of human beta-defensin-2 in epithelial cells is controlled by intracellular calcium and p38MAPK. J Infect 2007; 54: 469–474. 17 Rivas-Santiago B, Sada E, Tsutsumi V. Beta-defensin gene expression during the course of experimental tuberculosis infection. J Infect Dis 2006; 194: 697–701. 18 Lee H M, Shin D M, Choi D J, et al. Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes. Cell Microbiol 2009; 11: 678–692. 19 Castaneda-Delgado J, Herna´ndez-Pando R, Serrano C J, et al. ˜ Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis. Clin and Exp Immunol 2010; 161: 542–550. 20 Liu P T, Stenger S, Tang D H, Modlin R L. Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin. J Immunol 2007; 179: 2060–2063. 21 Selvaraj P, Anand S P, Harishankar M, Alagarasu K. Plasma 1,25 dihydroxy vitamin D3 level and expression of vitamin D receptor and cathelicidin in pulmonary tuberculosis. J Clin Immunol 2009; 29: 470–478.

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RESUME C A D R E : Les peptides antimicrobiens cath´elicidine LL37/hCAP-18 et la beta d´efensine humaine (hBD) sont des facteurs cl´es dans la r´eponse immunitaire inn´ee de l’arbre respiratoire. O B J E C T I F : D´eterminer les concentrations de LL-37 et de hBD-2 dans le liquide de lavage bronchoalv´eolaire (BAL) de patients p´ediatriques (age ˆ ,16 ans) atteints de tuberculose (TB) pulmonaire compar´es a` des enfants en bonne sant´e. M E´ T H O D E S : Nous avons mesur´e la concentration de ces peptides grace ˆ a` un test d’immuno-absorption ELISA. R E´ S U LT A T S : Un total de 40 enfants tuberculeux et 40 t´emoins en bonne sant´e ont e´ t´e enrol´ ˆ es dans l’´etude (age ˆ moyen 9,2 6 4,7 et 8,3 6 4,2 ans, respectivement ; P ¼ 0,97). Les deux groupes n’ont pas montr´e de diff´erence

statistiquement significative en termes de sexe, index de masse corporelle, poids ou taux de 25 hydroxy vitamine D. Le taux moyen de LL-37 dans le BAL e´ tait significativement plus e´ lev´e dans le groupe TB que dans le groupe t´emoin (0,95 6 1,33 ng/ml et 0,35 6 0,51 ng/ml ; P ¼ 0,01 ; t ¼ 2,54). Le taux de hBD-2 e´ tait e´ galement plus e´ lev´e dans le groupe TB, mais de fa¸con non significative (0,30 6 0,58 ng/ml et 0,14 6 0,30 ng/ ml ; P ¼ 0,11). Il n’y avait pas de corr´elation entre LL-37, hBD-2 et le taux de vitamine D. C O N C L U S I O N : Nos donn´ees sugg`erent que LL-37 et hBD-2 pourraient jouer un role ˆ important dans la pathog´enie de la TB de l’enfant. A notre connaissance, ceci est le premier rapport montrant les concentrations de LL-37 et de hBD-2 dans le BAL d’enfants atteints de TB pulmonaire. RESUMEN

M A R C O D E R E F E R E N C I A: La catelicidina LL-37 y las defensinas b (hCAP-18) son p´eptidos antimicrobianos que constituyen factores primordiales en la respuesta inmunitaria innata de las v´ıas respiratorias. O B J E T I V O: Determinar las concentraciones de LL-37 y hBD-2 en el l´ıquido del lavado broncoalveolar (BAL) de de edad) con pacientes pedia´ tricos (,16 a nos ˜ diagnostico ´ de tuberculosis (TB) pulmonar y compararlas con su equivalente en los ninos ˜ sanos. M E´ T O D O S: Se midieron las concentraciones de los pe´ ptidos mediante ana´lisis inmunoenzima´ticos por adsorcion ´ (ELISA). R E S U LT A D O S: Participaron en el estudio 40 ninos ˜ con TB y 40 testigos sanos (edad promedio 9,2 6 4,7 anos ˜ y respectivamente; P ¼ 0,97). No se 8,3 6 4,2 anos, ˜ observo´ una diferencia estad´ısticamente significativa entre los grupos con respecto al sexo, el ´ındice de masa

corporal, el peso relativo ni la concentracion ´ de 25hidroxivitamina D. La concentracion ´ promedio de LL37 en el BAL en el grupo con TB (0,95 ng/ml 6 1,33 ng/ ml) fue significativamente superior a la concentracion ´ en el grupo testigo (0,35 ng/ml 6 0,51 ng/ml; P ¼ 0,01; t ¼ 2,54). La concentracion ´ de hBD-2 tambi´en fue ma´s alta en el grupo con TB, pero la diferencia no alcanzo´ significacion ´ estad´ıstica (0,30 ng/ml 6 0,58 ng/ml y 0,14 ng/ml 6 0,30 ng/ml; P ¼ 0,11). No se observo´ una correlacion ´ entre la concentracion ´ de LL-37, hBD-2 y de 25-hidroxivitamina D. ´ N: Estos resultados indican que los CONCLUSIO p´eptidos antimicrobianos LL-37 y hBD-2 ejercen una importante funcion ´ en la patogenia de la TB en los ninos. Hasta el momento, este es tal vez el primer ˜ informe sobre la concentracion ´ de LL-37 y hBD-2 en el BAL de ninos ´ de TB pulmonar. ˜ con diagnostico