
 subjects - this study was approved by the ethical and scientific 
 committees of the mexican institute of social security ( r-2008 - 3301 - 15 ) .
twenty - six tb 
 household contact individuals between 26 - 58 years old were recruited between march - june 
 2012 .
tb household contacts included individuals with no tb diagnosis confirmed who 
 lived in the same household as the index case or visited the house at least once per 
 week , adding a total cumulative indoor exposure with shared air of at least 40 h within 
 the three months prior to the index case diagnosis to maximise the identification of 
 possibly infected contacts .
contacts were first visited and interviewed between 
 march - december 2010 ( 1 - 37 months after the index case was diagnosed ) using a 
 standardised questionnaire .
thus tst and qft positive individuals on the present study 
 had at least two years of presumable latent tb infection . to discard active tb 
 infection ,
none of the patients had any systematic or autoimmune disease , neither steroids nor 
 other hormone therapy .
all patients were human immunodeficiency virus negative . written 
 informed consent was obtained from each volunteer prior to blood sample drawing . 

tst and qft - it - all blood samples for qft - it assays and flow cytometry 
 analysis were drawn before the application of the skin test .
each tst was performed by 
 intradermal injection of five tuberculin units of ppd tubersol ( aventis pasteur ltd , 
 canada ) into the forearm using the mantoux technique .
reactions were evaluated 48 h 
 after injection , with a positive result defined as an induration of  10 mm in the 
 transverse diameter read by medical trained personnel .
the elisa - based qft - it assay 
 ( cellestis inc , valencia , ca , usa ) was performed in two stages , according to the 
 manufacturer s instructions .
briefly , 1-ml aliquots of blood were drawn directly into 
 three blood collection tubes , one containing ( negative control ) , one containing t cell 
 mitogen ( positive control ) and one containing mtb - specific antigens , including esat-6 , 
 cfp-10 and tb7.7 ( tb - antigen tube ) . following overnight incubation ,
200 l of plasma 
 were removed from each tube and the concentration of ifn- was determined by elisa 
 ( cellestis inc ) , with a positive response defined as a tb antigen - nil ifn- 
 concentration  0.35 iu / ml .
the individuals were categorised into two groups according 
 to their reactivity to qft , meaning as positive or negative to the test . 

cell purification - blood samples were processed in a time frame no 
 longer than 2 h after blood drawing .
polymorphonuclear cells were isolated from 
 peripheral blood by using ficoll - paque plus ( ge healthcare , uppsala , sweden ) and lysis 
 buffer with ammonium chloride .
once isolated , cells were washed twice with sterile 
 phosphate buffer saline and their number and viability was determined by direct counting 
 and by trypan blue exclusion assay .
purity of polymorphonuclear cells was determined by 
 flow cytometry ( > 90% ) . 

analysis of amps by flow cytometry - amps production by 
 polymorphonuclear cells was determined as follows : 2  10cells were treated 
 with fixation and permeabilisation solution ( bd pharmigen , san jose , ca , usa ) .
the cells 
 were then washed with perm / wash buffer ( bd pharmigen ) and incubated with a goat 
 anti - ll-37 polyclonal antibody ( santa cruz biotechnology , santa cruz , ca , usa ) or goat 
 polyclonal anti - hnp-1 antibody ( serotec , oxford , uk ) .
the detection of these antibodies 
 was made with a rabbit polyclonal anti - goat labelled with phycoerithrin ( santa cruz 
 biotechnology ) .
after washing , the cells were analysed in a facs canto ii flow cytometer 
 ( becton & dickinson , nj , usa ) with the facs diva software ( becton dickinson ) . 

statistical analysis - data from the clinical features of the 
 participants , the proportion of positive cells as well as the mean fluorescence 
 intensities were analysed through a kolmogorov - smirnov normality test . after this
, one 
 of the following tests was performed to determine if significant differences existed 
 among groups : mann - whitney u test , student s t test 
 with and without welch correction , which was indicated in each figure accordingly .
also , 
 a non - parametric spearman correlation was performed in order to determine the possible 
 association between qft - it and tst continuous values and the production of hnp-1 and 
 ll-37 .

 clinical analysis - we obtained samples from 26 individuals , which 
 according to the tst and qft results , were divided into two groups , those who were 
 positive to tst and qft ( n = 12 , further referred as qft ) and those who were 
 negative for both tests ( n = 14 , further referred as qft ) .
none of the clinical features show statistical differences , except for 
 their reactivity to tst and qft ( p < 0.001 ) .
tableclinical features of participants  qft and tst negativeqft and tst positivep(n = 14)(n = 12)(mean  sd)(mean  sd)tst ( mm)5.93  7.4620.25  3.96 < 0.001 
 qft ( ui / ml)0.03  0.066.81  3.42 < 0.001 
 age ( years)49.00  23.4941.92  15.250.3801 
 weight ( kg)73.31  18.4768.18  10.880.4076 
 height ( m)1.62  0.121.60  .060.5749 
 hip ( cm)107.36  10.92102.17  7.720.1816 
 waist ( cm)92.14  13.5688.25  11.960.4488 
 body mass index ( kg / m)27.50  4.8226.49  3.890.5642 
 waist to hip ratio0.86  0.080.86  .070.8948 
 sex ( male / female)4/104/8not availablehaemoglobin ( g / dl)14.27  1.3614.49  1.710.7179 
 leucocytes ( 1 x 10cell/l)6.39  1.806.82  1.820.6996 
 lymphocytes
( 1 x 10cell/l)2.42  0.952.29  .450.8201 
 glucose ( mg / dl)78.64  27.6596.48  75.620.9180 
 creatinine ( mg / dl)0.81  0.270.75  0.170.8360 
 cholesterol ( mg / dl)196.29  35.05196.49  31.880.9877 
 triglycerides ( mg / dl)164.31  79.36235.88  119.140.1308 

 a : welch corrected unpaired t test ; b : 
 mann - whitney u test ; c : unpaired 
 t test ; qft : quantiferon - tb gold ; sd : 
 standard deviation ; tst : tuberculin skin test . 
 

a : welch corrected unpaired t test ; b : 
 mann - whitney u test ; c : unpaired 
 t test ; qft : quantiferon - tb gold ; sd : 
 standard deviation ; tst : tuberculin skin test
. 
 amps are increased in qft positive individuals - polymorphonuclear 
 cells were purified and analysed by flow cytometry to determine the proportion and 
 production of the amps hnp-1 and ll-37 in the two different groups .
results showed that 
 there is an increased proportion of ll-37 positive cells in the qft 
 individuals ( p = 0.0184 ) when compared with qft individuals ( fig .
1a ) , which correlates with the mean fluorescence 
 intensity ( mfi ) , suggesting an increased production of peptide on a per - cell basis 
 ( fig .
positive cells 
 analysis showed no differences between groups ( p = 0.3413 ) ( fig .
however , significant differences ( p = 0.0043 ) were 
 observed when analysed for the mfi between groups as shown in fig .
fig . 1 : cathelicidin ( ll-37 ) and human neutrophil peptide-1 ( hnp-1 ) production in 
 polymorphonuclear cells
+ and ll-37 + cells was determined by 
 analysis in a flow cytometer in polymorphonuclear leukocytes ( pmn ) gated cells 
 ( a , c ) .
also , ll37 + ( b ) and hnp-1 + ( d ) cells mean fluorescence intensity ( mfi ) 
 was analysed .
group 
 comparisons were made by means of a non - parametric mann whitney 
 u test . in quantiferon - tb gold ( qft)- ,
amps production correlates with qft and tst reactivity values - to 
 determine whether positive values of qft and tst correlated with increased production of 
 hnp-1 and ll-37 both variables were analysed for each individual . as shown in fig . 2a
, ll-37 production is correlated with tst 
 reactivity , as well there is an association with hnp-1 production ( fig .
similarly , when correlated with qft positivity a positive 
 correlation was found for both ll-37 and hnp-1 ( fig . 

2 : cathelicidin ( ll-37 ) and human neutrophil peptide-1 ( hnp-1 ) correlated 
 with quantiferon - tb gold ( qft ) and tuberculin skin test ( tst ) 
 continuous values .
correlation between tst positivity with ll-37 ( a ) and hnp-1 
 ( b ) mean fluorescence intensity ( mfi ) was analysed .
also , qft positivity was 
 correlated with ll-37 ( c ) and hnp-1 ( d ) mfi .
the spearman correlation 
 coefficient and the level of significance are shown for each graph .
p values of 
 more than 0.05 were considered not statistically significant , n = 26 . 

previous studies by our group have demonstrated the importance of amps to contain tb 
 infection either during primary or latent infection ( rivas - santiago et al .
it has been reported that -defensins have direct 
 antimicrobial activity against mtb , however other amps such as ll-37 and hnp-1 , mainly 
 produced by neutrophils are included in the mtb containing process through a wide 
 variety of immunomodulatory functions ( martineau et al . 
 2007 ,
2010 ) . during the 
 infection , neutrophils accumulate at sites of mycobacterial challenge and rapidly 
 phagocytose bacilli and release several neutrophil - derived chemokines , cytokines , 
 granule products , hnp-1 and ll-37 that can drive monocyte recruitment from blood .
after 
 monocyte recruitment and maturation , cytokine secretion by both neutrophils and 
 macrophages might drive further accumulation and activation of both cell types leading 
 to inflammation ( martineau et al .
a previous study reported that ll-37 and hnp-1 
 gene expression was highly increased above 20-fold in patients with active tb compared 
 with healthy volunteers and latent tb infected individuals , these data indicate that 
 these peptides might be increasing during progression of disease ( gonzalez - curiel et al .
other studies have linked the presence 
 of high amounts of neutrophils in granuloma with increasing pneumonia and low 
 mycobacterial control which lead to an active disease ( eum et al .
2010 ) , thus we hypothesise that since amps producing cells are 
 increased while tb become active , should be a positive correlation with hnp-1 and ll-37 . 

recent studies suggest that the use of qft conversion is indicative of an approximately 
 eight - fold higher risk of progression to tb disease ( compared with non - converters ) 
 within two years of conversion in a cohort of adolescents in a high - tb burden population 
 ( machingaidze et al .
2012 ) , we sought to 
 determine if there was a correlation between the expression of hnp-1 and ll-37 with tst 
 and qft continuous values .
our results indicated that individuals who have positive 
 values to tst and qft had statistical significances regarding production of ll-37 and 
 hnp-1 , in the same line we found a positive correlation between expression of amps and 
 the continuous values for qft and tst .
altogether these data suggest that during gradual 
 reactivation process there is an influx of pmns into the lung to contain bacilli growth 
 and to recruit monocytes .
one of the main activators markers in neutrophils might be 
 ll-37 , since has been reported that increment of ll-37 in pmn promotes the generation of 
 reactive oxygen species dose and time - dependently , via nicotinamide adenine dinucleotide 
 phosphate oxidase activation and intracellular ca(2 ) mobilisation . besides 
 ll-37 induces both mrna expression and protein release of alpha - defensins neutrophil 
 peptide 1 - 3 ( zheng et al .
2007 ) and promotes 
 monocyte adhesion ( wantha et al . 2013 ) during 
 infection .
furthermore , ll-37 is a keystone for effective antimicrobial activity since 
 it controls the release of inflammatory mediators while increasing antimicrobial 
 activity of neutrophils ( alalwani et al . 

the main limitation of this study is that it is a cross - sectional study and the 
 individuals with latent tb have not been followed longitudinally to determine whether 
 they develop disease .
the reduced sample size is another handicap of the study ; however 
 it is good enough to observe tendencies and to plan further studies with a bigger 
 population .
finally , the possibility that other infections or autoimmune diseases could 
 interfere with the levels of these peptides needs to be elucidated .
all together our results indicate that amps levels positive correlates with tst and qft 
 values .
due there is not an accurate biomarker of progression for tb in latent infected 
 individuals , this preliminary study suggests the use of hnps and ll-37 as promising 
 biomarkers for progression , however further studies need to be done to strength this 
 results .
 it has been reported that patients with progressive tuberculosis ( tb ) express 
 abundant amounts of the antimicrobial peptides ( amps ) cathelicidin ( ll-37 ) and human 
 neutrophil peptide-1 ( hnp-1 ) in circulating cells , whereas latent tb infected donors 
 showed no differences when compared with purified protein derivative ( ppd ) and 
 quantiferon-tb gold ( qft)-healthy individuals . 
 the aim of this study 
 was to determine whether ll-37 and hnp-1 production correlates with higher tuberculin 
 skin test ( tst ) and qft values in tb household contacts . 
 twenty - six tb household 
 contact individuals between 26 - 58 years old tst and qft positive with at last two 
 years of latent tb infection were recruited . 
 amps production by polymorphonuclear 
 cells was determined by flow cytometry and correlation between tst and qft values was 
 analysed . 
 our results showed that there is a positive correlation between levels of 
 hnp-1 and ll-37 production with reactivity to tst and/or qft levels . 
 this preliminary 
 study suggests the potential use of the expression levels of these peptides as 
 biomarkers for progression in latent infected individuals . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION