Several studies in mice have suggested that not only did Tfh cells produce IL-21, but IL-21 could also drive IL-21 production and Tfh cell differentiation.8,42,56,57 Subsequent studies, however, showed that disruption of IL-21 signals had little or no effect on Tfh cell development.35,58–62 IL-6 has also been shown to induce IL-21 production and Tfh cell generation.42,57,63 However, once again, while some studies have shown a decrease in Tfh cell generation in the absence of IL-6,42 others have failed to see any defect in the absence of IL-6.35,62,63 These discrepancies probably reflect a level of redundancy in the signals required for

generating Selleck Navitoclax Tfh cells. Indeed, both IL-6 and IL-21 signal through signal transducer and activator of transcription 3 (STAT3) and a recent paper by Eto et al.62 demonstrated that, although the absence of only one of these

cytokines did not affect Tfh cell numbers, the combined absence of IL-6 and IL-21 did result in a significant decrease. This decrease, however, was not complete, and a substantial number of Tfh cells could still be found. In this light it is interesting to note that a recent study demonstrated that another STAT3-activating cytokine, IL-27, can also increase IL-21 production and Tfh cell generation.64 Thus, the ability of all three of these cytokines to activate STAT3 contributes to a high level of redundancy in their requirement for Tfh cell generation. However, CD4+ CXCR5+ cells can still be identified even in the absence aminophylline of STAT3 itself, suggesting that it may not be absolutely required for the generation of Tfh cells,42,63 RAD001 nmr indicating that an even greater level of redundancy

exists. However, in the absence of STAT3 these CD4+ cells were very poor at supporting B cell responses, suggesting that STAT3 may be more important for the functional ability of Tfh cells even if it is not required for the cell surface expression of Tfh-associated molecules. The role of cytokines in inducing B cell helper function from naive human CD4+ T cells differs from that of mice in that it has been shown to largely involve IL-12, and to a lesser extent IL-6, IL-21 and IL-23.8,65 IL-12 induced the differentiation of cells expressing IL-21, CXCR5, ICOS and Bcl-6 that facilitated antibody production by B cells in vitro.8,65 Interestingly, several studies have found little effect of IL-12 on the production of IL-21 by murine CD4+ T cells,42,57,66 although another paper observed a significant proportion of IL-21 secreting cells in response to IL-12.62 These results suggest that different cytokine pathways may be involved in the generation of human versus murine Tfh cells. The key function of Tfh cells is to provide help to B cells to support their activation, expansion and differentiation and the formation of the GC. Several features of Tfh cells enable them to carry out these functions.

The criterion of six or more mutations in the IRRDR (IRRDR ≥ 6) was identified as the most powerful viral genetic factor that independently predicted SVR (15).

In another study curried out on a patient cohort in Yamagata Prefecture, Japan, we proposed that polymorphism in the secondary structure of the N-terminal region of NS3 of HCV-1b influences virological responses to PEG-IFN/RBV therapy, and that virus grouping based on NS3 polymorphism can also be used to predict the outcome of the therapy (16). In the present study, we further analyzed the Yamagata cohort for a possible DMXAA nmr relationship between heterogeneity of NS5A and the core regions of the HCV genome and virological responses to PEG-IFN/RBV therapy.

Fifty-seven patients who were chronically infected with HCV-1b, their diagnoses being based on detection of anti-HCV antibody and HCV RNA, and who had been seen at Yamagata University Hospital in Yamagata, Japan, were enrolled in the study. Their HCV subtypes were determined according to the method of Okamoto et al. (17). Patients were treated with PEG-IFNα-2b (Pegintron; Schering-Plough, Kenilworth, selleck chemical NJ, USA) (1.5 μg per kilogram of body weight, once weekly, subcutaneously) and RBV (Rebetol; Schering-Plough) (600∼800 mg daily, orally), according to a standard treatment protocol for Japanese patients established by a Hepatitis Study Group of the Ministry of Health, Labor and Welfare, Japan. All patients received >80% of the scheduled doses of PEG-IFN and RBV. Serum samples were collected from the patients before treatment and at intervals of 4 weeks during the whole observation period (72 weeks), and tested for HCV RNA titers as reported previously (18). The study protocol was approved beforehand by Lck the Ethics Committee at Yamagata University Hospital, and written informed consent for study participation was obtained from

each patient prior to treatment. Also, the study protocol conforms to the provisions of the Declaration of Helsinki. Hepatitis C virus RNA was extracted from 140 μL of serum using a commercially available kit (QIAmp viral RNA kit; Qiagen, Tokyo, Japan). Amplification of full-length NS5A and the core regions of the HCV genome were performed as described elsewhere (11, 18, 19). The sequences of the amplified fragments of NS5A and core regions were determined by direct sequencing without subcloning. The aa sequences were deduced and aligned using GENETYX Win software version 7.0 (Genetyx, Tokyo, Japan). To evaluate the optimal threshold of the IRRDR and ISDR mutations for SVR prediction, we constructed an ROC curve and calculated the AUC, sensitivity and specificity (11). Statistical differences in treatment responses according to NS5A and core sequence heterogeneity were determined by the χ2 test.

Many chemokine genes are clustered in defined chromosomal locations [39]. Two main clusters encode the essential inflammatory chemokines: the CXC cluster located in chromosome 4q12–21 and the CC cluster located in chromosome 17q11.2–q12. A potential explanation for this chromosomal arrangement is found in the evolutionary forces that have shaped the genome into gene superfamilies [40]. Over the course of evolution, gene duplication has

been a common event, affecting most gene families [41]. Once a duplication occurs, the two copies can evolve independently and develop specialized functions. This phenomenon explains the origin of chemokine clusters. An important characteristic of a chemokine cluster is that their genes code for many ligands that interact with a few receptors. Therefore, chemokine clusters act as single entities based on their overall function. The cluster of proinflammatory CC chemokines contains PF-01367338 datasheet 16 genes localized to a 2·06 Mb interval at 17q11.2–q12 on genomic contig NT_010799 (Fig. 1a). Four of these genes comprise the two closely related, paralogous pairs CCL3–CCL3L and CCL4–CCL4L[42]. Members within each pair share 95% sequence identity at both the genomic and the amino acid levels. Among all human chemokine genes, a singular characteristic of CCL3L and CCL4L, is that they are present in variable copy

numbers in the human genome. The CNV affecting CCL3L–CCL4L has been studied extensively since 2002 (when Towson et al. reported the first data about the extent of CCL3L–CCL4L Liproxstatin-1 research buy CNV in the Caucasian population [43]), although two groups had identified the existence of CCL3L–CCL4L as non-allelic copies of CCL3–CCL4 and as copy number variable genes 20 years ago [44,45]. The CNVR that includes CCL3L and CCL4L genes (and other non-related loci) seems to have been generated through a segmental duplication of a genomically unstable stretch of about 120 kb located on this region CYTH4 of

chromosome 17 [43–48]. In fact, the q arm of chromosome 17 of humans has multiple regions of genomic instability where gene duplications, chromosomal rearrangements and copy number variation are common [49,50]. Furthermore, the human CCL3L–CCL4L region shows evidence of complex homologous recombination events. For example, high-resolution CNV data reveal extensive architectural complexity in the CCL3L–CCL4L region, which includes smaller CNVs embedded within larger ones and interindividual variation in breakpoints [5,49]. One of the consequences of this complexity is that individuals may vary not only in the total copy number of CCL3L and CCL4L genes, but also their individual components. Underscoring this, although the copy number of CCL3L correlates with CCL4L, individuals average more copies of CCL3L than CCL4L[43,51,52]. Currently, gene copy numbers in humans range from 0 to 14 for CCL3L and from 0 to 10 for CCL4L with a strong population structure.

In the validation cohort of nine

patients, six had PGD grade 1, and for the remaining three there was no evidence to suggest PGD. All patients were extubated in the first 24 hr and none qualified for a PGD grade 2 or higher. A nearest centroid classifier18 was constructed from the 17 differentially reactive proteins identified (Fig. 2a), and was used to predict the PGD grades of the nine patients in this validation cohort (Fig. 2b). Here, five out of six patients see more having had PGD were correctly identified (83% sensitivity), and all three patients without PGD were classified as such (100% specificity), giving an overall classification accuracy of 89% (P = 0·048 by Fisher’s exact test). This is comparable to the classification accuracy in the test set (85%). Two recent studies have investigated gene expression differences

in donor lungs developing PGD9,10 Differential gene expression in each study was evaluated using Student’s t-test. Out of the 17 differentially reactive proteins identified, 15 proteins could be paired with gene expression in the first study,9 and six with expressions from the second study10 (Table 2). Comparing differences in IgM reactivity with differences in gene expression levels in the first study (study GSE8021 in Table 2), 12 out of 15 change in the Crizotinib nmr same direction (80% concordance, P = 0·04 by Fisher’s Exact Test), i.e. increased expression is significantly associated with increased reactivity and vice versa. The same conclusion is reached when calculating Pearson’s product–moment correlation (r = 0·63, P = 0·011), see Fig. 3(a). For IgG reactivity, no significant correlation with gene expression changes was observed (r = − 0·01, P = 0·98). Inspection of the P-values for the

differential expressions (study GSE8021 in Table 2) showed that none of them had P < 0·05, which is usually a standard threshold of significance. Still, five out of six genes displayed the same direction as well as magnitude of change when compared with the Pregnenolone second gene expression study (GSE9102 in Table 2), which is a significant correlation (r = 0·91, P = 0·013), see Fig 3(b). This study demonstrates that lung transplant recipients manifest widespread IgG and IgM autoantibody reactivity, and that specific patterns of reactivity to self-antigens discriminate between patients with and without PGD. It has been speculated that PGD may induce or accelerate chronic rejection in the form BOS, although conflicting results have been published.2 We observed no significant correlation between BOS and PGD grades among the 39 patients included in this study (Table 1). However, six (35%) out of the 17 informative proteins were also observed to be informative with respect to BOS.8 A two-factor analysis of variance including both BOS and PGD as factors in general confirms the significant differential reactivity with respect to both factors (Table 3 and Fig. S2).

This finding was supported partially in man by showing that DCs in H. pylori infected human gastric biopsies have a semi-mature phenotype and expressed DC-specific intercellular adhesion molecule-3-grabbing non-integrin (SIGN) [53]. In addition to this, the virulence factor vacuolating cytotoxin has also been shown to regulate DC maturation negatively [54], suggesting that the modulation of DC maturation plays an important role in H. pylori’s subversion of the immune response. The study presented here has focused on the effect of H. pylori-infected DCs on Selleckchem PCI 32765 naturally occurring Tregs, and whether or not infected DCs are able to produce IL-18 and induce de-novo Tregs has not been investigated.

However, many reports published in the last few years have confirmed that H. pylori

infection induced DC maturation and the release of IL-23 [10, 13, 55-57]. In conclusion, we have found that H. pylori expands Tregs in vitro and in vivo and subverts their suppressive function through the production of IL-1β from DCs. These findings question the role of Tregs at H. pylori-infected sites and provide mechanistic and therapeutic insights into the mechanisms of H. pylori-associated chronic gastritis and potential targets for the local treatment of inflammation associated with H. pylori in patients who do not respond to standard eradication therapy. The authors acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership CHIR-99021 mouse with King’s College London and King’s College Hospital IMP dehydrogenase NHS Foundation Trust. The authors acknowledge the support of the MRC Centre for Transplantation. This work

was funded by grants from the Medical Research Council (to B.A., P.M. and R.I.L.), the British Heart Foundation and Guy’s and St Thomas’ Charity Trust (R.I.L. and G.L.). The authors of this manuscript have no conflicts of interest to disclose. “
“Calreticulin (CRT) is a multi-functional endoplasmic reticulum protein implicated in the pathogenesis of rheumatoid arthritis (RA). The present study was undertaken to determine whether CRT was involved in angiogenesis via the activating nitric oxide (NO) signalling pathway. We explored the profile of CRT expression in RA (including serum, synovial fluid and synovial tissue). In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells (HUVECs) were isolated and cultured in this study for in-vitro experiments. Our results showed a significantly higher concentration of CRT in serum (5·4 ± 2·2 ng/ml) of RA patients compared to that of osteoarthritis (OA, 3·6 ± 0·9 ng/ml, P < 0·05) and healthy controls (HC, 3·7 ± 0·6 ng/ml, P < 0·05); and significantly higher CRT in synovial fluid (5·8 ± 1·2 ng/ml) of RA versus OA (3·7 ± 0·3 ng/ml, P < 0·05).

cDNA was synthesized using a high-capacity RNA-to-cDNA kit according to the manufacturer’s instructions (Applied Biosystems). Real-time PCR for RORγt, T-bet, Gata3, and AHR expression was performed using power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Primers utilized were: RORγt – 5′-GGCTGTCAAAGTGATCTGGA-3′ forward; 5′-CCTAGGGATACCACCCTTCA-3′ reverse; T-bet – Temsirolimus nmr 5′-CTGCCTGCAGTGCTTCTAAC-3′ forward; 5′-GCTGAGTGATCTCTGCGTTC-3′ reverse; Gata3 – 5′-ACTCAGGTGATCGGAAGAGC-3′ forward; 5′-AGAGGAATCCGAGTGTGACC-3′

reverse; AHR – 5′-CACTGACGGATGAAGAAGGA-3′ forward; 5′-TCGTACAACACAGCCTCTCC-3′ reverse. Expression was normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH). BALB/c mice were divided into three

groups of 5. Mice were shaved DAPT on the dorsum with electric clippers, and injected intradermally with 100 μL of PBS containing 530 pmol VIP, 530 pmol PACAP, or PBS alone. Fifteen minutes after injection, the mice were painted with 10 μL of DNFB (1% in acetone and olive oil (4:1)) epicutanousely at the injection site. Three days after immunization, mice were sacrificed and draining lymph nodes (axillary and inguinal) removed. Lymph nodes were mechanically disrupted and passed through a 70 μm nylon mesh to yield a single cell suspension. CD4+ T cells were isolated as described above. Ninety-six well flat-bottom plates were treated with 10 μg/mL of anti-mouse CD3 mAb in PBS overnight and washed. T cells were cultured (3 × 105 cells/well) in 250 μL of CM containing 2 μg/mL of anti-mouse CD28 mAb. Supernatants were collected 72 h after stimulation and cytokine content determined. Differences in average cytokine levels under different treatments at varying cOVA concentrations were analyzed using ANOVA. Average cytokine levels under each cOVA concentration were then compared between PACAP or VIP treatment and control groups. p-values were adjusted by controlling for the false discovery rate (FDR). For assessment of mRNA levels, effects of intradermal administration

of neuropeptides and effects of anti-IL-6 mAb on Ag presenting cultures, a linear mixed effects model was used to estimate the average level of the biomarkers under different treatments. This model takes into account many variations for each treatment both within and between plate and samples. Differences in the average level of the biomarker under pairs of experimental conditions of interest were evaluated using simultaneous tests for general linear hypotheses [[84]]. p-values were again adjusted for multiple comparisons by controlling the FDR. This work was supported by NIH Grant 5R01 AR042429 (R.D.G. and J.A.W.), the Jacob L. and Lillian Holtzmann Foundation (R.D.G.), the Edith C. Blum Foundation (R.D.G.), the Carl and Fay Simons Family Trust (R.D.G.), the Seth Sprague Educational and Charitable Foundation (R.D.G.), the Lewis B. and Dorothy Cullman Foundation (R.D.G.

HDAC9 [12] and HDAC6 [28] were recently shown to be important negative regulators of FoxP3; neither are effectively targeted by n-butyrate in contrast to TSA. The lack of FoxP3 induction may present an alternate option for a direct deactivation of stimulated effector CD4+ T cells. Gilbert et al. have proposed a role for cyclin-dependent

kinase inhibitor p21cip1 as a direct mediator for HDAC inhibitor–induced anergy in CD4+ T cells [11, 29]. Antigen-activated CD4+ T cells deficient Cabozantinib in p21cip1 were shown to be far less susceptible to n-butyrate-induced anergy in contrast to CD4+ T cells non-deficient in p21cip1. Furthermore, p21cip1 was shown to be highly upregulated within anergized CD4+ T cells. Alterations in genome-wide hyperacetylation may be responsible for the upregulated gene expression profile of p21cip1 that may then aid in anergy induction. n-Butyrate may also induce CD4+ T cell anergy through direct alteration of lysine acetylation on non-histone proteins.

One study determined that over one thousand non-histone proteins may be directly targeted by HDACs and HDAC inhibitors [4]. Evidence suggests that acetylation and deacetylation of proteins involved in a wide range of cellular processes play an important regularity role in controlling protein function [30]. In addition to the induction of genome-wide hyperacetylation mediated through the use of HDAC inhibitors, direct changes upon lysine residue acetylation on transcription factors or other important regulatory proteins within the anergized CD4+ T cells may be responsible for the observed n-butyrate-induced ZD1839 in vivo functional unresponsiveness. As a result, p21cip1 expression may be induced through still unknown pathways in addition to an increase in transcription through open chromatin access. The authors from thank Dr. Amy Scurlock and Mr.

Isaac Foote for contributing FoxP3EGFP mice. Drs. Uma Nagarajan and Richard Morrison provided helpful critical analysis of this manuscript. Michelle Phillips, Charles Foote Fleet III, Ashley Nelson, Dr. Horacio Gómez-Acevedo, Dr. Sarah Blossom, Chase Lambert, Meagan Kreps, Cemeka Agugbuem, Jenny Rau, James D. Sikes, Shelby Smith, Oliver Irlam and Ronni Stern offered instrumental assistance. This work was supported by the Arkansas Biosciences Institute. “
“The association of autoimmunity with antitumor immunity challenges a paradigm of selective surveillance against tumors. Aided with well-characterized models of robust autoimmunity, we show that self-antigen-specific effector T (Teff) cell clones could eradicate tumor cells. However, a tumor microenvironment reinforced by Treg cells and myeloid-derived suppressor cells (MDSCs) presented a barrier to the autoimmune effectors, more so in tumors than in healthy tissues. This barrier required optimal CTLA4 expression in Teff cells.

In striking contrast, such an increase was not evident in the spleens. These results indicated that the inflammation in K5-PLCε-TG mice is local and has no systemic impact. The observed close association between the CD4+ T-cell infiltration and the skin symptoms prompted us to compare the expression levels of various Th cell-derived cytokines in the skin between WT and K5-PLCε-TG mice by quantitative real-time RT-PCR (qRT-PCR) (Fig. 5). The expression

of both the Th1 cytokine, IFN-γ, and the Th17 cytokines, IL-17 and IL-22, was elevated in K5-PLCε-TG mice compared to WT mice at P9 and P26 but not P6 and 15 wk (Fig. 5). Immunostaining of the symptomatic skin showed that these Th cytokines were produced by CD4+ T cells (Fig. 6A–C) and that most of the infiltrating CD4+ T cells produce IL-22 (Fig. 6F). IL-17 was also produced by Gr-1+ neutrophils (Fig. 6D and E). The Th2 cytokine IL-4 showed a small increase Selleck AG 14699 with no apparent relationship with the skin symptoms (Fig. 5). These results suggested that

CD4+ T cells producing the Th1 and/or Th17 cytokines rather than those producing the Th2 cytokines were accumulated in the symptomatic K5-PLCε-TG mouse skin. In addition, Foxp3 was expressed Decitabine in the K5-PLCε-TG mouse skin at P9 and P26 (Fig. 5), suggesting the infiltration of Foxp3+ Treg. Consistent with this, their signature cytokine IL-10 5 showed a small buy Palbociclib increase at P26. Gene expression profiling of the whole skin (Fig. 5) also demonstrated a substantial increase of the expression of IL-12/23 p40 and IL-23 p19, which constitute the IL-23 heterodimer implicated in Th17 cell activation 4, 26, in K5-PLCε-TG mice at P6, P9, and P26. Moreover, the K5-PLCε-TG mouse skin showed elevated expression of not only IL-1α and IL-1β having pleiotropic functions in induction of inflammation 27 but also CCL20, chemokine (C-X-C motif) ligand (CXCL)1/2, and CXCL10, having chemoattracting functions for DC precursors 11 and Th17 cells 28, neutrophils 29, and Th1 cells 28, respectively. In

addition, besides cytokines, the expression of polypeptides implicated in the pathogenesis of psoriasis 12, 13, such as the cathelicidin antimicrobial peptide Camp (a mouse ortholog of human LL-37) and the S100 family proteins, was elevated in the K5-PLCε-TG mouse skin at P6, P9, and P26. We next examined the effect of PLCε overproduction on expression of the factors relevant to inflammatory diseases by using keratinocyte primary cultures established from K5-PLCε-TG mice (Fig. 7A). PLCε overexpression had no significant effect on the proliferation potential of cultured keratinocytes as assessed by BrdU incorporation; the frequencies of BrdU-positive cells were 43 and 35% for WT and K5-PLCε-TG (Line G), respectively, which is consistent with our previous data showing no proliferation defect in PLCε−/− keratinocytes 17.

Interleukin-15 (IL-15) produced by hyperplasic stroma and epithelial cells [22] selleck could attract T cells and support their expression of P. Augmented IL-15 production by prostate cells could

be a contributory factor facilitating the transendothelial migration of CD56+ NK cells to the stroma of BPH tissue and support their P expression [16]. In our study, flow cytometry analysis revealed negligible expression of P in T lymphocytes and NKT and NK cells in the PCa tissue; this may be attributable to tumour activity leading to the development of a chemical barrier around the tumour that probably inhibits TIL infiltration and activation [23, 24]. The increased production of reactive nitrogen species by the tumour actively models the tumour environment, leading to an altered chemokine profile and changes in capacity to recruit T lymphocytes [25]. In contrast to proinflammatory dominance in patients with BPH, increased levels of IL-4 have been observed in patients with androgen-independent PCa [26]. Furthermore,

IL-4 was shown to enhance the expression of the PSA gene, whose protein product is a prostate-specific glycoprotein overexpressed in patients with PCa [27]. PSA, because of its glycoprotein structure, could Erismodegib chemical structure support the local anti-inflammatory response and induce alternative activation of antigen-presenting cells, leading to inefficient activation of cell-mediated immunity [28]. In such cases, tumour cells could deeply invade surrounding tissues and enter systemic circulation. Negligible CD3+ T cell infiltration as well as reduced NK cell infiltrate with low P content was found in Monoiodotyrosine PCa tissue and this could be responsible for the inefficient control of tumour invasion. Moreover, a negative correlation between PSA values and overall

percentage of P+ cells and P-expressing T and NK cells in the prostate tissue was observed only in PCa patients, suggesting that the low percentage of P+ cells in the prostate tissue could be responsible for an increased risk of tumour development and progression. In conclusion, our findings showed that the low frequency of P+ lymphocytes, including T, NKT and NK cells, in prostate tissue of patients with BPH and, particularly, PCa could be the consequence of local tissue microenvironment and one of the mechanisms involved in the pathogenesis of prostate hyperplasia following malignant alteration. This investigation was supported by the grants from the Croatian Ministry of Science, Education and Sports (projects no. 062-0620096-0094 and 062-0000000-0220). The authors thank Ksenija Tulic for assistance in laboratory work. The authors declare that they have no conflict of interest. “
“Interleukin-15 (IL-15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models.

Thus, culture

is required to assess both bacterial viability and the drug susceptibility profile. However, culture is complicated and it takes several weeks to make a diagnosis. A tuberculin skin test is not always valid because prior BCG vaccination and previous infection with M. tuberculosis can affect the result. PCR is an effective method for early diagnosis of TB; however, it cannot distinguish between viable and dead bacteria. In addition, because similar positive results may be obtained regardless of the actual bacterial count, assessing the severity of infection is difficult. Furthermore, PCR is too expensive for wide use in developing countries. On the other check details hand, Sada et al. developed an effective test for the diagnosis of TB in 1990. This test is called MycoDot and can detect anti-mycobacterium antibodies (anti-lipoarabinomannan) in only 20 mins (5). Because only a small MK-8669 nmr test sample is required, the quantity and quality of clinical samples does not influence the results. A high percentage of patients with

negative sputum tests are positive by the MycoDot test, but it may not detect infection at an early stage when antibody production is low. In recent years, researchers have developed a diagnostic kit for TB based on production of interferon-γ after stimulation of T lymphocytes with M. tuberculosis antigen. In 1995, Andersen and colleagues Casein kinase 1 identified EAST-6 in the culture fluid of M. tuberculosis (11). M. tuberculosis-sensitized T lymphocytes recognize EAST-6 but BCG-sensitized T lymphocytes do not, allowing discrimination of infection with M. tuberculosis from prior BCG vaccination (12). In 1996, using a subtractive genomic hybridization technique, Mahairas and colleagues found that EAST-6 is located in region of difference 1 (13). A second-generation Quanti FERON-TB kit was developed by using EAST-6 and CFP-10 antigens, which occur in M. tuberculosis but not in M. bovis BCG and most non-tuberculous acid-fast bacteria,

markedly improving the specificity of this assay for M. tuberculosis (14, 15). However, to improve the control of TB in developing countries, there is also a need for simple diagnostic methods that are applicable in field settings. Sometimes sputum samples are not collected correctly. In contrast, it is easy and safe to collect urine samples. Itoh and colleagues reported that ELISA of urine samples showed adequate sensitivity and specificity for the diagnosis of visceral leishmaniasis, supporting the usefulness of diagnostic tests based on urine specimens (16). Therefore, we employed MPB64 protein to develop a specific and sensitive method for screening clinical samples to detect patients with active TB. This protein is secreted by only two bacterial strains, M. bovis and M. tuberculosis. Its expression has been clearly observed in M.