Cellular regulation Protein Tyrosine Kinase inhibitor was determined using isolated vaginal and uterine epithelial/stromal

cells in vitro. Uterine and vaginal chemokine secretion is cyclically regulated with CCL20 at low levels but CXCL1 at high levels during high estradiol, generally mimicking estradiol effect in vivo. ERα but not ERβ regulated CCL20/CXCL1 secretion by uterine epithelial cells in vitro and vaginal CCL20 in vivo. Estradiol/SERMs failed to alter uterine CCL20 secretion in ovariectomized mice. Diminished uterine epithelial ERα staining following ovariectomy corresponded with estradiol unresponsiveness of uterine tissue. Estrogen receptors α regulates CCL20/CXCL1 secretion in the female reproductive tract, and ERα antagonists directly oppose the regulation by estradiol. Understanding ER-mediated antimicrobial chemokine expression is important to elucidate cyclic susceptibility to sexually transmitted pathogens. “
“Trichuris muris infection is an ideal model for

defining T-cell-driven immunity, and also provides essential insights that may impact on potential helminth therapies currently in development. Conflicting host variables determine the efficiency of such treatments and we have identified host-derived sex steroid hormones as key factors in the development of immunity. The female-associated hormone 17-β estradiol (E2) check details significantly enhanced the generation of a Th2 response in vitro; however, this stimulatory effect was found to be dispensable for the generation of immunity to Trichuris in the gender-biased IL-4KO mouse model. In contrast, the male-associated hormone dihydrotestosterone significantly inhibited the T-cell stimulatory capacity of DC and directly suppressed the immune response of male IL-4KO mice, with worm expulsion restored following castration. This finding was associated with dramatically reduced IL-18 mRNA expression suggesting androgens may act via this cytokine to suppress Th2 immunity to Trichuris. This study

has critical implications for the development and efficacy of potential helminth therapeutics and identifies host gender – Resveratrol specifically sex hormones – as important factors in the development of Th2 immunity in susceptible and immunocompromised mice. “
“This unit describes a method for in vivo delivery of oligonucleotides or plasmids using the hemagglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle, as a delivery system. Viral transfection methods generally show a higher transfection efficiency than nonviral methods for the delivery of genes to cells. However, in using these methods one must bear in mind that the introduction of a virus particle into a host carries a risk for leukemia induction and for creation of disturbances in immune function due to cytotoxicity. Curr. Protoc. Immunol. 91:10.17E.1-10.17E.9. © 2010 by John Wiley & Sons, Inc.

Results from an animal model support that the higher levels of Kyn in renal failure are attributed mainly to a combination of increased TDO activity and decreased kynureninase activity in the liver, and not to impaired renal excretion [16]. Conversely, the increased neopterin concentrations are attributed most probably to increased cellular immunity activation accompanying reduced renal function [18]. NVP-AUY922 cost Overall, the examined lifestyle factors associated with inflammation [3, 22, 24, 25, 35] were weaker

determinants of circulating markers of cellular immune activation and kynurenines compared to the biological determinants. Despite the fact that obesity is related to increased IFN-γ activity [4], BMI was not associated with neopterin in this or in a previous study [19]. In contrast, some studies indicate a positive association of BMI with neopterin [12, 22, 23], and inconsistencies might relate partly to the different study designs; one of the studies included mainly overweight and obese participants [22], whereas another presented only crude associations [23]. In contrast to the null findings for neopterin, we observed that overweight and obesity were associated positively with KTR and all kynurenines, except

AA, which is in line with previous studies on KTR [20, 21]. Thus, it is possible that kynurenines are involved in obesity and/or obesity-related conditions. Interestingly, HAA and HK can induce the formation of free radicals MLN0128 cost [36] and thereby may mediate oxidative stress associated with obesity [37]. Furthermore, XA can react with insulin and therefore may lead potentially to insulin resistance [4], a condition related strongly to obesity [37]. Finally, we observed recently that KA is a strong predictor of pre-eclampsia in obese women [38]. It has been shown that physical activity has an anti-inflammatory effect [24] and is associated with a reduction in visceral fat mass. In

the present study, physical activity was not associated with neopterin, KTR or kynurenines, except for a weak inverse Adenosine association between physical activity and KA. Previous studies on the short-term effect of intense exercise have reported an increase in both neopterin [39, 40] and Kyn [35]. Conceivably, short-term and habitual physical activity may have different effects on IFN-γ-mediated pathways, as demonstrated previously for several inflammatory markers [24]. In this community-based study we did not observe an association of current smoking with neopterin or KTR, as both Trp and Kyn were decreased slightly in moderate smokers and decreased further among heavy smokers; therefore, KTR was not changed in any of the groups. We also found a similar inverse association between smoking and all other kynurenines, except HK.

Furthermore, Selleckchem Imatinib both TREG cells and T effector (TEFF) cells from Lgals3−/− mice showed higher expression of Notch1 and the Notch target gene Hes-1. Interestingly, Notch signaling components were also altered in both TREG and TEFF cells from uninfected Lgals3−/− mice. Thus, endogenous galectin-3 regulates the frequency and function of CD4+CD25+Foxp3+ TREG cells and alters the course of

L. major infection. Galectins are a family of glycan-binding proteins composed of 15 members that are conserved throughout animal evolution and share sequence similarities in their carbohydrate-recognition domain [1-3]. Galectin-3, a widely distributed member of the family, plays pleiotropic roles in innate and adaptive immunity by regulating cytokine production, phagocytosis, chemotaxis, signaling, and

survival [4-7]. Through these mechanisms, galectin-3 has been proposed to control host immunity against several infectious agents [1, 6-8]. Yet, despite considerable evidence on the role of galectin-3 in the control of immune responses, its contribution to T regulatory (TREG) cell function during microbial attack has not yet been explored. TREG cells, either inducible or naturally occurring, suppress effector T (TEFF)-cell responses through different mechanisms including cell–cell contact and secretion of immunosuppressive cytokines such as IL-10, TGF-β, and/or IL-35 [9]. Interestingly, galectin-1 and -10 have been proposed to mediate the immunosuppressive activity of Foxp3+ TREG cells [10, 11] and galectin-3 has been postulated as a potential marker for human TREG cells [12]. In addition, Selleckchem Autophagy inhibitor galectin-3 Thiamet G increases the severity of autoimmune neuro-inflammation by decreasing the frequency of TREG cells [13], suggesting that this lectin might also influence the TREG cell

compartment during microbial infection. We took advantage of the availability of galectin-3-deficient (Lgals3−/−) mice on a BALB/c background in order to investigate the function of TREG cells during the course of Leishmania major infection. This experimental model has provided extensive information on the factors that regulate the development of CD4+ T helper (Th) cells in vivo [14] and has contributed to dissect the role of TREG cells during intracellular infections [15-18]. Here, we show that Lgals3−/− mice display higher frequency of TREG cells both in draining lymph nodes (LNs) and infection sites during L. major infection. Moreover, Lgals3−/− TREG cells produce higher amounts of IL-10, have enhanced suppressive capacity, and show altered Notch expression compared with wild-type (WT) mice. Thus, endogenous galectin-3 influences TREG cell number and function during parasitic protozoa infection. To investigate the role of galectin-3 within the TREG cell compartment, we first compared the outcome of L. major infection in Lgals3−/− and WT mice on BALB/c background.

In this study we demonstrated that the epithelial cells of the upper tract (Fallopian tube, uterus and cervix) and of the lower tract (ectocervix) constitutively produce Trappin-2/Elafin messenger RNA (mRNA) and protein. However, only the uterine cells consistently up-regulate Trappin-2/Elafin production upon stimulation with Poly(I:C), a synthetic viral double-stranded RNA (dsRNA) mimic. We also demonstrated that recombinant Trappin-2/Elafin can inhibit both

X4/T-tropic IIIB and R5/M-tropic BaL HIV-1 in a dose-dependent manner, possibly through a mechanism that involves direct interaction of virus and Trappin-2/Elafin. Finally, we demonstrated that CVL from both HIV-positive and HIV-negative women contain Trappin-2/Elafin, learn more with higher amounts present in HIV-negative women. In addition, women in the secretory phase of the menstrual cycle produced significantly more Trappin-2/Elafin Selleck GDC-0449 than women in the proliferative phase, suggesting hormonal regulation of this molecule in the FRT. Human uterine

and Fallopian tube tissue was obtained from women undergoing hysterectomy at Dartmouth-Hitchcock Medical Center (Lebanon, NH). Tissues used in this study were collected from patients with benign conditions, such as fibroids, distal from the site of pathology. The sections were examined by a pathologist and identified to be free of pathological lesions. A total of 11 different patients were used to obtain epithelial cells from the uterus, Fallopian tube, endocervix and ectocervix. All work on human subjects was carried out with the approval of the Dartmouth College and Miriam Rebamipide Hospital, Brown University Institutional Review Boards. Approval to use tissues was previously obtained from the Committee for the Protection of Human Subjects (CPHS). Epithelial cells were isolated as previously described elsewhere.46,47 Briefly, tissues were rinsed with 1 × phosphate-buffered saline (PBS) and minced into

1–2 mm fragments before subjecting them to enzymatic digestion for 2 hr at 37°. The enzyme mixture contained 3·4 mg/ml of pancreatin (Invitrogen Life Technologies, Carlsbad, CA), 0·1 mg/ml of hyaluronidase (Worthington Biochemical, Lakewood, NJ), 1·6 mg/ml of collagenase (Worthington Biochemical) and 2 mg/ml of d-glucose, in 1 × Hanks’ balanced salt solution (HBSS) (Invitrogen Life Technologies). After digestion, cells were dispersed through a 250-mm mesh screen, washed and resuspended in Dulbecco’s modified Eagle’s minimal essential medium (DMEM)/F12 complete medium without phenol red, supplemented with 20 mm HEPES, 2 mm l-glutamine (all from Invitrogen Life Technologies), 50 μg/ml of primocin (Invivogen, San Diego, CA) and 10% defined fetal bovine serum (FBS) (Hyclone, Logan, UT). Epithelial sheets were separated from stromal cells by filtration through a 20-mm nylon mesh filter (Small Parts, Miami Lakes, FL).

MEK5 induction of KLF4 is mediated by ERK5. MEK5/CA-transduced HDMECs are less responsive

to TNF, an effect partly mediated by KLF4. Conclusions:  MEK5 activation by LSS inhibits inflammatory responses in microvascular ECs, in part through ERK5-dependent induction of KLF4. “
“Please cite this paper as: Su S-W, Catherall M and Payne S. The Influence of Network Structure on the Transport of Blood in the Human Cerebral Microvasculature. Microcirculation 19: 175–187, 2012. In this article, we explore how the structural properties of miniature networks influence the transport of blood through the human cerebral microvasculature. We propose four methods for generating such networks, and investigate both how the resulting network properties match available experimental data from the human cortex and how these properties affect the flow of blood through

the networks. As the nature of such microvascular Selleck PXD101 flow patterns is inherently random, we run multiple simulations. We find that the modified spanning tree method produces artificial networks having characteristics closest check details to those of the microvasculature in human brain, and also allows for high network flow passage per unit material cost, being statistically significantly better than three other methods considered here. Such results are potentially extremely valuable in interpreting experimental data acquired from humans and in improving our understanding of cerebral blood flow at this very small length scale. This could have a significant impact on improving clinical outcomes for vascular brain diseases, particularly vascular dementia, where localized flow patterns are very important. “
“Please cite this paper as: Mahé G, Durand S, Humeau-Heurtier A, Leftheriotis G, Abraham P. Impact of experimental conditions on noncontact laser recordings in microvascular studies. Microcirculation 19:

669–675, 2012. Microcirculation, especially skin microcirculation, is a window toward systemic vascular function in magnitude and underlying mechanisms. Different techniques have been developed to assess the microcirculation. Among these techniques, laser technology is used to perform noninvasive microvascular Morin Hydrate assessments. In the 1970s, the laser Doppler flowmetry (LDF) technique was proposed to monitor microvascular blood flow. More recently, noncontact technologies including laser Doppler perfusion imaging (LDI) and laser speckle contrast imaging (LSCI) have improved the reproducibility of the microcirculation measurements and facilitated some clinical evaluations such as on wounds and ulcers. However, due to the absence of contact between tissue and sensors, it is likely that different technical and environmental conditions may interfere with microvascular recordings. This review presents major technical and environmental conditions, which may interfere with noncontact laser recordings in microvascular studies.

Multiple cellular communication molecules and pathways, including the NKG2D-MICA system, may be involved in iTreg cells-NK cell cross-talk 38. It was shown that the engagement of NKG2D on activated T cells and NK cells promoted antitumor NK and T-cell responses against epithelial MICA+ tumor cells 39. We also observed stronger killing of MICA+ tumor cells compared with MICA− cells and induction of NKG2D on NK exposed to iTreg cells. However, iTreg cells enhanced NK cell cytotoxicity against tumor cell targets independent of MICA expression on target cells (Fig. 3C). Also, AUY-922 we could not detect any NKG2D

ligands on iTreg cells, which suggests that a mechanism other than direct NKG2D-ligand interaction is involved in the iTreg cell–NK cell cross-talk. Further, it was reported that NK cells can spontaneously lyse certain

transformed cells. However, early in immune responses NK cells are further activated and recruited to tissue sites where they perform effector functions. It was recently reported that NK cells were capable of lysing pathogen-induced Treg cells, which expressed UL16-binding protein 40. In contrast, it was demonstrated that Treg cells in a tumor microenvironment kill NK cells in a granzyme-B-dependent fashion 41. It was even shown that NK cells are able to induce Treg cells, which resulted in immune suppression 42, and underscores the complex cross-talk between these two immune cell subsets. Although we have not yet identified the find more molecular mechanism of NK activation by iTreg cells, our data suggest that direct contact between both cell types is required. We have also observed that the parallel execution of the perforin and the FasL cytolytic pathway is utilized by iTreg cell-activated NK cells. To our knowledge, this is the first report about enhancement of anti-tumoral NK cell-function

which is mediated by induced regulatory T cells. Without any doubt, still much has to be learned about the interaction of NK cells and regulatory T cells in the tumor microenvironment. A ADAMTS5 better understanding of the cellular cross-talk between regulatory T cells and cells of the innate immune system will aid future rationale therapeutic manipulation of this T-cell subset in cancer therapy. This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board (Ethical Committee) of University of Duisburg-Essen. Blood donors provided written informed consent for the collection of samples. Fetal calf serum (Biochrom AG, Berlin, Germany) was heat inactivated for 30 min at 56°C (ΔFCS). RPMI 1640 culture medium, L-glutamine, streptomycin, and penicillin were purchased from Invitrogen (Karlsruhe, Germany).

Tetraspanins can potentially contribute to both adhesion-dependent and adhesion-independent DC migration. Tetraspanins are best characterized by their ability to molecularly interact with integrins — adhesion molecules important in regulating cell migration in many diverse cell types [2]. Tetraspanins regulate integrin function, as frequently observed in the impaired adhesion and migration of tetraspanin-deficient cells of various lineages [27, 29-31]. Similarly, we demonstrate that adhesion to fibronectin is impaired in CD37−/− DCs under low shear flow (Fig. 6A) implicating a role for CD37 in regulating

outside-in signaling of α4β1 and/or α5β1 integrins in DCs. Tetraspanins are also known to interact with the cytoskeleton Dabrafenib molecular weight via molecular interactions with ezrin/radixin/moesin proteins [37], and cross-linking tetraspanins at the cell surface can drive cytoskeletal rearrangement [38]. In DNA Damage inhibitor this study we observed impaired CD37−/− DC function in two processes known to require cytoskeletal rearrangement: integrin outside-in signaling, investigated by measuring adhesion under flow (Fig. 6A), as well as

cell spreading to form membrane protrusions (Fig. 6C–G). An effect of CD37 ablation on cytoskeletal rearrangement is also consistent with a recent report that the absence of another tetraspanin, CD81, results in inhibited integrin-dependent in vitro DC chemotaxis [28] and the formation of membrane protrusions, driven by

a dysregulation of Rac-1 activation. While the Guanylate cyclase 2C in vivo immunological effects of impaired migration of CD81−/− DCs were not studied [28], in the present paper it is clear that CD37 ablation profoundly affects in vivo DC migration which is the likely cellular mechanism that underlies the poor cellular immunity induced in CD37−/− mice. The next challenge is to unravel the molecular interactions of CD37 in DCs. C57BL/6 (WT), C57BL/6.CD37−/− (CD37−/−) [10], CD11cYFP, CD37−/−.CD11cYFP, and OT-I Ly5.1 mice were bred in house, or obtained from the Walter and Eliza Hall Institute (Melbourne, Australia). Mice were housed under SPF conditions within the Burnet Institute animal facility (Austin Campus), the AMREP Animal Services, or the Nijmegen Medical Centre and used between 8 and 12 weeks of age. In vivo multiphoton imaging was performed on 8–10-week-old female CD37−/−.CD11cYFP mice with CD11cYFP mice used as controls. The corresponding campus animal ethics committees at Austin Hospital, AMREP Animal Services, Monash Medical Centre, or Nijmegen Medical Centre approved all animal experiments. Mice were challenged subcutaneously with 1–5 × 106 cells from either RMA (C57BL/6 — T-cell lymphoma) or RMA-Muc1 as described previously [39].

Results: Higher baseline RRF was inversely associated with slope of

RRFD (β = −0.72; p < 0.001), phosphate levels (β = −0.18;p = 0.02), and Ca×P levels ((β = −0.18; p = 0.02) by simple linear regression tests. After adjusting for gender, Obeticholic Acid solubility dmso age, serum albumin level, baseline RRF and diabetes mellitus by multivariate lineal analyses, serum phosphate levels (β = −3.57; p < 0.001) rather than calcium levels (β = −0.09; p = 0.12) showed an inverse correlation to the slope of RRFD. Conclusion: After adjusting for baseline RRF, higher serum phosphate level was associated with rapid RRF decline in CAPD patients. CHAO MEI-CHEN, WU MEI-YING, PAI SHING-QUEI, KUO LI-CHUEH, LEE CHIEN-TE, CHEN JIN-BOR Division Caspases apoptosis of Nephrology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung Introduction: Infection is one of factors to influence the outcome in long-term peritoneal dialysis (PD) patients. A good quality of exit site care is a key component to avoid infectious events in PD patients. Present study was to investigate

the efficacy of film dressing on the exit site and its influence on quality of life (QoL) in PD patients. Methods: The study design was prospective, open-label, parallel-control. The observation was one year. Eighty patients were enrolled in one PD center, the mean age 48.3 ± 12.6 year-old. The number of patients was forty in each group. The subjects in study group used film dressing on the exit site and changed dressing every two weeks in outpatient clinic. The subjects in control group

cared exit site according to regular guideline by PD nurses. The analyzed variables included infectious events and questionnaires pertaining QoL. Results: The infectious rate in exit site was 0.25 most times / 100 patient month in study group vs 0.88 times/ 100 patient month in control group. Five patients had early withdrawn from the study group because of allergic reaction to dressing. In QoL analysis, there were higher score in satisfaction, stress reduction and psychological relaxation in study group than control group. Conclusion: An invention of film dressing on exit site had reached a favorable outcome in infectious control and QoL in PD patients. SEI YUMI1, MIZUNO MASASHI1, SUZUKI YASUHIRO1, IMAI MASASKI2, HIGASHIDE KEIKO1, SAKATA FUMIKO1, IGUCHI DAIKI1, OKADA NORIKO2, MATSUO SEIICHI1, ITO YASUHIKO1 1Nagoya Univeristy Graduate School of Medicine; 2Nagoya City Univeristy Graduate School of Medicine Introduction: Peritoneal dialysis (PD) therapy is one of the most important renal replacement therapies. Impairment of peritoneal function can limit the long-term efficacy of PD therapy. Peritoneal impairment is caused by several factors that occur during PD therapy, including exposure to peritoneal dialysate, catheter trauma and peritonitis.

Although the mechanism of LAG-3 function remains unclear, a conserved KIEELE motif in the cytoplasmic domain of LAG-3 is essential 2. In contrast to CD4, LAG-3 is only expressed on the cell surface of activated T cells 1, 7–10. LAG-3 surface expression is further regulated by two metalloproteases, ADAM10 and ADAM17, which cleave surface LAG-3, a proportion of which is both constitutive and TCR-ligation induced 11. Importantly, prevention of LAG-3 cleavage blocks T-cell proliferation

and cytokine secretion 11 suggesting that LAG-3 surface expression is under tight regulatory control. This observation raised the question of whether other mechanisms are used to control the expression and distribution of LAG-3. Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), which is another inhibitory molecule for T-cell activation, SCH727965 is mainly stored in DAPT intracellular compartments such as the trans-Golgi network, endosomes and lysosomes 12–17. Surface expression is tightly regulated by controlled internalization and trafficking to the plasma membrane. This raised the possibility that LAG-3 surface expression might also be regulated by modulating its intracellular storage and trafficking. In this study, we addressed the following questions.

First, what is the extent of intracellular storage and localization of LAG-3 versus its relative CD4? Second, what is the sub-cellular localization of LAG-3 and CD4 in activated T cells? Third, what is the fate of intracellular LAG-3? In order to determine cellular distribution of CD4 and

LAG-3, we performed intracellular staining for CD4 or LAG-3 using flow cytometry. Freshly isolated naïve CD4+ T cells do not express LAG-3 10; so naïve T cells were first stimulated with plate-bound anti-CD3 and anti-CD28 for 72 h and then treated with pronase to remove cell surface CD4 and LAG-3 from activated CD4+ T cells. Pronase treatment removed most of the surface CD4 and LAG-3 on activated T cells (Fig. 1A). While intracellular staining revealed that a relatively small amount (23%) of CD4 is present inside cells, in Histamine H2 receptor contrast a greater amount (49%) of LAG-3 appears to be retained intracellularly (Fig. 1A and B). One might speculate that the slightly lower LAG-3 surface expression compared with CD4 following T-cell activation and the increased percentage of intracellular LAG-3 versus CD4 is due to its continuous cleavage by the metalloproteases ADAM10 and ADAM17 that limits surface LAG-3 expression 11, 18. However, when T cells were treated with the metalloproteinase inhibitor TAPI (Calbiochem), cell surface LAG-3 expression was only slightly increased (data not shown). While prevention of LAG-3 cleavage by TAPI slightly changed the ratio of surface and intracellular LAG-3, the effect was small and not sufficient to account for the differences observed between LAG-3 and CD4. The extent of intracellular LAG-3 storage was also examined by Western blot analysis.

Recently, two serodiagnostic tests for TB have become available in Japan: the Determiner Tuberculous Glycolipid antibody test (Kyowa-Medex, Tokyo, Japan), which

detects mycobacterial cord factor by ELISA, and the MycoDot test (Wako Pure Chemical Industries, Osaka, Japan), which detects lipoarabinomannan by immunochromatography (5, 6). However, when there are only a small number of bacteria in the sample, both these tests have limitations, including low sensitivity and inability to exclude other mycobacteria. Mycobacterial protein fraction from BCG 64 is a M. tuberculosis complex-specific exocrine protein that shows reactivity with M. tuberculosis strain H37Rv and M. tuberculosis Aoyama B, because mpb64 is encoded in the RD2 region of the M. tuberculosis genome (7). Since only M. bovis and M. tuberculosis GDC-0068 manufacturer secrete MPB64, it is a protein with strong specificity for these two species. Mycobacterial protein fraction from BCG Selleck PI3K inhibitor 64 is found in the culture

fluid of M. tuberculosis and Mycobacterium bovis BCG and has been cloned using a single-probe method. The open reading frame of this gene is 618 bp long and the protein has an estimated molecular weight of 22.4 kDa (8). Nakamura et al. reported that the MPB64 skin patch test discriminates patients with TB from persons who have undergone BCG vaccination, and concluded that it should be useful for the diagnosis of active TB (9). Recently, Zhu et al. reported that sandwich ELISA based on an MPT64 antibody aptamer is useful for the serological diagnosis of pulmonary TB, both in sputum smear positive and negative patients (10). In this study, we assessed the usefulness of a dot-blot assay based

MPB64 antigen for detecting TB by testing of serum and urine samples. Our objective was to develop a simple diagnostic test for active TB that can be employed for fieldwork in developing countries. Serum and urine samples were obtained from 28 pulmonary TB patients with active TB who were attending special TB hospitals and had given informed consent. The diagnosis had been microbiologically confirmed by sputum smear microscopy and/or culture in all these patients. These patients were defined as having active TB, whereas culture-negative patients were Oxymatrine considered to have inactive TB. The mean age of the patient group was 62.4 years; the male:female ratio was 22:6. As a control, serum and urine samples were also obtained from 20 healthy donors who attended the same hospital but were not infected with M. tuberculosis. All these individuals were sputum smear- and/or culture-negative, had been vaccinated with BCG and gave informed consent for taking of the samples. The mean age of the control group was 50.9 years; the male:female ratio was 4:1. The study was approved by the Institutional Review Board of Kansai Medical University, and informed consent was obtained from each participant. The mpb64 gene (Gene bank accession No.E02088) was kindly donated by Dr. Mastuo, National Institute of Infectious Diseases.