Presence of tumor-associated macrophages (TAMs) in malignant find more tissue correlates frequently with worse disease

prognosis and higher propensity of metastasis [1-3]. Schematically, macrophages can be divided into two categories, representing two extreme phenotypes: inflammatory M1 and anti-inflammatory M2 macrophages. Other than the classical M1 macrophages endowed with antimicrobial and immune-stimulatory properties, the M2-skewed TAMs [1] dampen tumor-directed T-cell responses [4], stimulate angiogenesis [5-7], support tumor growth by cytokine supply [5, 8], and promote dissemination of malignant cells [1]. Despite our increasing knowledge of functional aspects of the tumor–TAM interplay, the ontogeny of tumor-resident macrophages is less well-understood. Macrophages in nonmalignant tissues can be of a dual, monocyte-dependent and/or monocyte-independent origin [9]. In the former case, blood monocytes extravasate to steady-state or inflamed tissues, where they terminally differentiate and replace aged or exploited macrophages.

This model proves its merit in case of acute inflammatory processes, in which a high demand for tissue macrophages exists due to their extensive turnover, but it fails to explain many phenomena observed under homeostasis or during chronic inflammation [10]. For instance, a plethora of highly FK228 chemical structure specialized tissue-resident macrophages proliferate in situ under steady-state [11-15] and inflammatory conditions [16-19] and are able to self-maintain without significant input of marrow-derived precursors. TAMs settle inflammatory and dynamically expanding tumor environments with an elevated demand for macrophages supporting growth of the neoplasm. Circulating conventional monocytes (Gr-1+/ Ly6C+), either of BM or splenic origin, were shown to contribute markedly to the TAM pool [7,

20, 21]. On the other hand, recent reports on proliferating TAMs in human breast malignancies [3] indicate that TAMs may possess the capability to self-maintain independently of blood-borne precursors. An important aspect of TAM biology is how the malignant milieu influences differentiation of macrophages for tumor’s own sake. Anacetrapib In this respect, the potent hematopoietic cytokine CSF1 was proposed to be one of the main players [6, 8, 22]. The ubiquitously expressed CSF1 was proven to foster the development of various populations of tissue-resident macrophages and the complete maturation of blood monocytes [12]. In mammary cancer, CSF1 produced by tumor cells was shown to drive accumulation of TAMs that supply the neoplasm with the crucial growth factor EGF [8]. Studies on human breast carcinoma patients revealed a link between elevated expression of STAT1 and markers of macrophage infiltration with an impact on disease outcome [23].

Low birthweight as an index of IUGR reflects

the congenital defects of organs, which are associated with CKD through their direct influence on nephron number and function, also through related metabolic disease-induced kidney damage (Fig. 2). However, the role of LBW in the pathogenesis of CKD is not completely explicit and results of former studies are often inconsistent. Although a recent meta-analysis confirmed that LBW increases the risk of CKD, the authors still suggested additional well-designed population-based studies.51 In addition, it is worth looking for an alternative index to birthweight to better reflect the influence of IUGR on human health. The Authors state that there is no conflict of interest regarding the material discussed in the manuscript. “
“Aim:  Catheter-related infection is a major cause of catheter loss in peritoneal dialysis (PD). We selleck chemical evaluated the effect of catheter revision on the treatment

of intractable exit site infection (ESI)/tunnel infection (TI) in PD patients who required catheter removal. Methods:  We reviewed learn more the medical records of 764 continuous ambulatory peritoneal dialysis (CAPD) patients from May 1995 to April 2011 at our hospital. One hundred and twenty six patients had more than one occurrence of ESI. Catheter revision was performed to treat intractable ESI/TI. Incidence of ESI, causative organisms and the outcomes of catheter revision were analyzed. Results:  The total PD duration of all patients was 32 581 months. Three hundred and twelve ESI episodes occurred in 126 patients and the incidence of ESI was 1/104 patient-months (0.12/patient-year). The most common causative organism was methicillin-sensitive Staphylococcus aureus (MSSA) (98 episodes), followed by Pseudomonas aeruginosa (63 episodes) and methicillin-resistant S. aureus (MRSA) (28 episodes). Among these, catheter revision was required due to intractable ESI/TI in 36 patients. The most common causative organism was MSSA (14 episodes) followed by P. aeruginosa (10 episodes) and MRSA (six episodes) in catheter revision cases. The outcomes of catheter

revision were as follows: ESI relapsed in 11 patients (30.6%) after catheter revision. Among them, five patients were treated with antibiotic treatment, two patients required secondary catheter revision, oxyclozanide four patients required catheter removal due to ESI/TI accompanying peritonitis. The catheter survival rate after catheter revision was 89.7% in one year. There were no statistical differences in the rates of ESI relapse after catheter revision between ESI caused by P. aeruginosa (5/10, 50%) and ESI caused by S. aureus (6/21, 28.6%). Conclusion:  Catheter revision may be an alternative treatment option to treat intractable ESI/TI before catheter removal is considered in PD patients. “
“Aim:  Glomerular infiltration of macrophages is a characteristic alteration of renal pathology in hyperlipidaemic renal injury.

Indeed, treatment with rhIL-10 significantly reduced both CD8+ and CD4+ T-cell proliferation (Fig. 7C), thus proving a central role of IL-10 in the regulation of the T-cell response to allogenic monocytes. In this study, we demonstrated the role of the IRAK4 kinase as a differential switch between TLR-induced pro-inflammatory and anti-inflammatory cytokine production. This observation is of interest as to date IRAK4 is mainly being viewed as a central executor of the MyD88 pathway that unselectively transduces all signals downstream of MyD88. As previously described in IRAK4-deficient mice [26], stimulation of IRAK4 knockdown monocytes with TLR4 and TLR2 ligands resulted in markedly reduced pro-inflammatory cytokine

secretion such as TNF and IL-12 (Fig. 2A–C) and find more concomitant reduction of the NF-κB subunits p50 and p65 responsible for the transcription of these cytokines (Fig. 2D). The results obtained are further in accordance check details with observations made in cells of IRAK4- and MyD88-deficient patients. There, TLR stimulation fails to activate the NF-κB pathway and cells display an impaired cytokine response after stimulation with agonists for TLR-1, TLR-2, TLR-4, TLR-5, TLR-7, and TLR-8 [17-20]. Similarly, siRNA-mediated silencing of MyD88 caused a significant reduction in TNF and IL-12 cytokine production in human monocytes (Fig. 4C,D), highlighting the cooperative interaction

of MyD88 and IRAK4 in TLR-induced synthesis of pro-inflammatory cytokines such as TNF and IL-12. IRAK4-deficient patients are more susceptible to infections with pyogenic bacteria, especially Gram-positive species such as S. pneumoniae and/or S. aureus [18]. Consistently, the predisposition to S. aureus infections was also reported in IRAK4-deficient mice [26]. Until now, little is known about the role of monocytes in response to these pathogens albeit these cells belong to those first to encounter bacterial pathogens in blood stream infections.

In this study, we tested the role of IRAK4 in the TLR-mediated response of human monocytes to bacterial infections. In particular, our results showed diminished IL-12 PtdIns(3,4)P2 secretion and elevated TNF and IL-10 levels after treatment with live S. aureus and S. pneumoniae (Fig. 1C). To further assess these findings we conducted MyD88 knockdown experiments, obtaining concomitantly reduced IL-12 and IL-10 secretion. However, TNF levels were only slightly diminished (Fig. 4E). This strongly suggests that IL-12 and IL-10 secretion evoked by bacterial infections is MyD88-dependent, whereas TNF production is also regulated in a MyD88-independent fashion, possibly triggered via TRIF or cytosolic PRR-induced IFN-I [27]. In general, bacteria represent complexes of multiple ligands that can be sensed by membrane-bound as well as cytosolic PRR. The most prominent difference between stimulation with bacteria and single TLR ligands was an increase in TNF release under IRAK4-silencing conditions (Fig. 1C) and under rapamycin treatment (Fig. 5B).

Samples were analyzed using a BD FACS Calibur flow cytometer and data were analyzed using FlowJo software. Isotype-matched selleck products PE- and FITC-conjugated mAbs of irrelevant specificity were used

as negative controls. Lymphocytes from either EAMG or CFA control rats (2 × 106/mL) were cultured in the presence of AChR R97-116 (10 μg/mL) with or without CGS21680 (30 nM). After a 72 h incubation, supernatants were collected and IFN-γ, IL-4, IL-17, and TGF-β levels were determined using respective ELISA kits (Shanghai Senxiong Biotech Industry Co. Ltd., China) according to the manufacturer’s instructions. The analyses were performed in triplicate and the results are expressed as the mean cytokine concentration (pg/mL) ± SD. For preventive treatment experiments, rats were given CGS21680 (0.5mg/kg intraperitoneally (i.p.)) in PBS starting 1 day before EAMG induction and every 3

days throughout the course of the experiment. Therapeutic treatment of EAMG consisted of 1.0 mg/kg CGS21680 administered OSI-906 datasheet i.p. every 3 days starting 29 days post immunization. Hind limb muscles were harvested, snap frozen in liquid nitrogen, and a cryostat used to generate 10-μm thick sections. We incubated the sections with biotin-conjugated goat antirat IgG (Sigma-Aldrich) for 1 h. Sections were then stained with tetramethylrhodamine-labeled α-BTX (Molecular Probes), FITC-labeled goat antirat C3 (Nordic Immunological Laboratories), and Alexa Fluor 350-labeled streptavidin. Sections were then analyzed using a fluorescence microscope (LSM 700, Zeiss). Data were expressed as mean ± SD. Differences between groups were analyzed using Graphpad software (Graphpad software, CA) and the two-tailed Student’s t-test for paired and unpaired data. p < 0.05 were considered Etofibrate statistically significant. This work was supported by Heilongjiang Provincial Innovation Found for Postgraduates (YJSCX2011-324HLJ, Na Li is the recipient), National Nature Science Foundation of China (81000511, Lili Mu is the recipient), China Postdoctoral

Science Foundation (20100480062, Lili Mu is the recipient), National Nature Science Foundation of China (81000536, Qingfei Kong is the recipient), China Postdoctoral Science Foundation (20100471094, Qingfei Kong is the recipient), National Nature Science Foundation of China (30901330, Bo Sun is the recipient), National Nature Science Foundation of China (81100883, Yumei Liu is the recipient), and the Harbin Medical University Cell Biological Engineering Center (1151gzx05). The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Figure S1. The A2ARagonist CGS21680 reduced the number of AChR antibody-secreting cells. Figure S2.

apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 °C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 μmol l−1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis

(β-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l−1. EGTA did not Metformin mw promote any significant reduction in the conidial growth, while PHEN and learn more EDTA, both at 10 mmol l−1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum. “
“Rhino-orbital zygomycosis is a life-threatening fungal infection generally occurring in patients with an

underlying disorder, such as diabetes mellitus with ketoacidosis or with immunocompromising factors, although it may rarely appear in healthy individuals. The study has been undertaken to discuss the clinical presentation, pathogenesis, diagnostic work up and management of this rapidly progressive disease. Four male patients having uncontrolled diabetes and presenting with signs and symptoms of rhino-orbital zygomycosis were studied D-malate dehydrogenase to illustrate the serious nature of the disease. All the four patients had rapidly deteriorating vision loss either unilateral or bilateral along with other nasal and orbital signs and symptoms. All the patients were put on liposomal amphotericin B and underwent orbital exenteration and pansinusectomy. One patient died, while the other three were successfully treated. Early

diagnosis is critical in the prevention of morbidity and mortality associated with the disease. There is need for a high index of clinical suspicion in immunocompromised patients. Timely medical-surgical treatment proves extremely important for prognosis. “
“Detection of Trichophyton rubrum in superficial skin infections by conventional methods is time consuming and not always successful. However, with modern molecular methods, an alternative is in sight. The aim of this study was to compare the detection of T. rubrum by conventional methods and by a direct specific PCR assay under routine conditions. Skin scrapings (n = 464) and nail samples (n = 230) collected from suspected tinea lesions were equally divided for KOH-mounts, cultures and PCR-analysis.

He was born at 25/40 with a bicuspid aortic valve and developed short stature and developmental delay. At the age of 17 months he underwent a left groin exploration for an impalpable

left testis. Removal of the left testis revealed click here a preserved vas deferens with an absence of normal testicular parenchyma. Genetic investigation revealed regions of long contiguous stretches of homozygosity in chromosomes 1, 2, 3 and 4 with microdeletions in exons 13 and 14 of the SMARCAL1 gene. The proteinuria did not relate to podocin, WT1 or LMX1B mutations. Conclusion: This is consistent with Schimke immunoosseous dysplasia, an autosomal recessive multisystem disease characterised by focal segmental glomerulosclerosis, immunodeficiency, azoospermia and spondyloepiphyseal dysplasia. 290 ADENINE PHOSPHORIBOSYLTRANSFERASE DEFICIENCY AS A CAUSE OF RENAL FAILURE A SHARMA1, M JAYABALLA1, T NG2, M TCHAN3, M VUCAK-DZUMHUR1 1Department of Renal Medicine, Westmead Hospital, Westmead, NSW; 2Institute for Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW; 3Department of Genetic Medicine, Westmead Hospital, Westmead, NSW, Australia Background: We describe a case of adenine phosphoribosyltransferase

(APRT) deficiency. This is a rare, autosomal recessive cause of chronic kidney disease (CKD) that is potentially preventable and treatable. Case Report: A 42 year old man was referred to our nephrology clinic with progressive, unrecognised, advanced CKD. On presentation he had stage 4–5 CKD with microscopic haematuria and proteinuria. He had a history of nocturia

and lethargy Pirfenidone datasheet but no history of nephrolithiasis, there was no family history of renal failure. A percutaneous renal biopsy was performed which demonstrated widespread deposition of multiple crystals within the tubules, along with extensive chronic interstitial fibrosis and sclerosis of nearly all glomeruli. The unusual brownish appearance of the crystals raised the possibility of 2,8 dihydroxyadenine crystals associated with APRT deficiency. Although no urinary stones could be analysed as the patient was not producing any in his urine, the absence of APRT activity in the serum and presence of adenine and 2,8 dihydroxyadenine in the urine confirmed the diagnosis. Allopurinol was commenced in an attempt to prevent Nitroxoline formation of new crystals and potentially break down the crystals already present. However, as shown by the biopsy, the patient unfortunately had extensive fibrotic changes and his renal function deteriorated further within the next two months. He therefore commenced renal replacement therapy and is currently on the transplant waiting list. Conclusions: This extremely rare cause of CKD is potentially avoidable if early diagnosis and management can be facilitated. Furthermore, an important post-transplantation consideration is to prevent allograft dysfunction with the use of a xanthine oxydase inhibitor.

A p-value of <0.05 was considered significant. This work was supported by grants R01AI063331 and R01DK091191 from the National Institutes of Health. L. F. was supported by a Research Career Development Award from the Crohn's and Colitis Foundation of America. We would like to thank Randal Kaufman and Yingjie Chen for Pkr−/− mouse femurs. We would also like to thank Peter Kuffa for help in generating anti-Nlrp3 antibody and Sharon Koonse for animal husbandry. Luigi Franchi is an employee of Lycera, a biotechnology

company specializing in the field of inflammation. Selleckchem LY294002 “
“Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral

glycosphingolipids, L-3 (GlcNAcβ1-3Manβ1-4Glcβ1-1’Cer) in AP-61 cells, and nLc4Cer (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1’Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the β-GlcNAc residue may play an important role in dengue virus binding to the host cell surface. Dengue viruses are the causative agents of dengue fever selleck chemicals and its associated complications, dengue hemorrhagic fever and dengue shock syndrome (1).

These lethal conditions may be caused by any of the four virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) (2). There is neither effective treatment, nor vaccines currently available for prevention of dengue diseases. A prerequisite for development of antiviral strategies against dengue virus is a better understanding of the infection and replication processes (3). In regard to invasion of the host cells, the virus must attach to the cell very surface via cellular receptor(s), but the viral receptor is still unclear. Several studies have demonstrated putative receptor(s) for dengue viruses. By using multiple approaches and different cell lines with different strains of dengue viruses, numerous candidates for dengue virus receptor(s) have been provided. Possible receptors for dengue virus on mammalian cells that have been identified include HS-type GAGs (1, 4–7), C-type lectins dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin and liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (8, 9), glucose-regulated protein 78 (10) and the non-integrin receptor, laminin receptor 1 (11, 12). In the case of receptors of mosquito cells, two glycoproteins with molecular masses of 40 and 45 kDa have been identified (13, 14).

Both types of monocytes are F4/80+ BGB324 nmr and CD86− 6. Data are accumulating on the presence of local tissue precursors for DCs and macrophages and the contribution of these precursors to DC and macrophage accumulation under pathological conditions. In organs, such as the skin and brain, local precursors for macrophages and Langerhans cells have been detected 9–11. We earlier described the presence of local precursors for macrophages in the fetal pancreas

of C57BL/6 mice 12. However, little is known about the origin of the DCs that accumulate in the pre-diabetic NOD pancreas and the factors driving this accumulation. It is generally assumed that these cells are inflammatory in nature and infiltrate from the circulation. However, previous studies from our group suggest that the early accumulation of DCs in the pre-diabetic NOD pancreas cannot only be explained by a massive influx of DCs and DC precursors from the blood. First, pro-inflammatory chemokines that normally attract monocytic cells (CCL2 and Luminespib chemical structure CCL3) could not be detected in the pancreas at the time of DC accumulation 13. Second, DCs and monocytes of NOD mice have an impaired migration towards pro-inflammatory chemokines in vivo and in vitro 13, although the contribution of other chemokines cannot be excluded. Finally, the depletion of phagocytic

cells with clodronate resulted in a late re-appearance of DCs in the NOD pancreas (28 days after depletion), while monocytes and DCs had already re-appeared in the blood and spleen 4 days after depletion. This late re-appearance suggests that pancreatic

DCs are not only replenished from the circulation 14. We therefore hypothesized that local precursors for DCs are present in the pancreas and that an enhanced proliferation and differentiation of these cells is responsible for the enhanced accumulation of pancreatic DCs initiating the islet autoimmune reaction. In this study, the presence of local pancreatic precursors for DCs, their proliferative capacity and the actual generation of DCs from these pancreatic precursors was investigated in the fetal pancreas and the pre-diabetic pancreas of NOD and control mice. The presence of precursors for DCs in the fetal pancreas was studied using the myeloid progenitor marker DOCK10 ER-MP58. ER-MP58 has previously been described by our laboratory as a marker for all myeloid progenitor cells in BM 15. A double staining with ER-MP58 and insulin was performed on the E15.5 pancreas of C57BL/6 and NOD/LTj mice using immunofluorescence (Fig. 1). The results showed that ER-MP58+ cells were present in and around the insulin positive islets of Langerhans in the E15.5 pancreas. To investigate the phenotype of this myeloid precursor in the pancreas a FACS staining was performed on fetal pancreas cells and compared with blood monocytes (4 weeks) from C57BL/6 and NOD/LTj mice.

This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific NVP-AUY922 nmr for RA (NSAIDs), in use

of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented

reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly find more decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with Oxymatrine consequent improvements in the clinical manifestations of

RA, remains to be determined. Rheumatoid arthritis (RA) is a common systemic autoimmune disease, characterized mainly by synovial hyperplasia and symmetric polyarticular joint disorders [1]. Although the pathophysiology of this disease is not fully understood, it is known that the chronic inflammatory nature of the disease causes the migration of leucocytes from the peripheral circulation into the synovial tissue and synovial fluid (SF) via their interaction with endothelial cells, cellular adhesion molecules, cytokines, chemokines and receptors. The synovial tissue of RA individuals becomes replete with mononuclear cells [2, 3] while the neutrophils constitute over 90% of cells in the SF [4]; these phenomena lead to the proliferation of fibroblast-like synoviocytes with consequent destruction of cartilage and bone [5]. The migration of neutrophils to inflammatory foci is thought to be initiated by the capture, rolling and subsequent firm adhesion of the cells on the endothelium in response to chemotactic molecules such as the CXC chemokines interleukin (IL)-8 and epithelial neutrophil activating peptide (ENA)-78 [6].

Hence, the efficacy of DNA vaccines against TB needs more improvement. Ag85A, a member of Ag85 complex, can induce strong T cell proliferation and gamma interferon (IFN-γ) production in most healthy individuals infected with M. tuberculosis or M. leprae and in BCG-vaccinated mice and humans, making it a promising candidate as TAM Receptor inhibitor a protective antigen. In experimental mouse models, DNA vaccines encoding Ag85A induce partial protection against experimental tuberculosis [7, 16] So it is needed to improve the efficacy of Ag85A DNA vaccine

by some measures. As it is known, ub–proteasome system plays a key role in antigen presentation through MHC class I pathway [17]. When a protein is fused to ub, the degradation of the protein in proteasome and presentation can be enhanced, resulting in an improvement of immune response. In this study, we demonstrated that UbGR-Ag85A fusion DNA vaccine was capable of improving the cellular immune response against Ag85A. Mice.  BALB/c female mice, 6-to 8-week old, were bred in the animal facilities of Luminespib research buy the Second Military Medical University (SMMU). All procedures performed on animals were conducted according to the guidelines for the care and use of laboratory animals of SMMU under protocols approved by the institutional Animal Care and Use committee

at the SMMU. Cell transfection.  The recombinant plasmid pcDNA3-Ag85A was transfected into P815 (H-2d a lymphoma cell line, from Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) cells by liposome (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacture’s instruction. After selection in medium supplemented with G418 (Sigma, St. Louis, MO, USA) (800 μg/ml), stable transfectants were subcloned by limiting DOCK10 dilution and then determined by RT-PCR and immunochemistry methods. Immunocytochemistry.  The expression of Ag85A protein was detected by immunocytochemistry. P815 stable transfectants were fixed in 4% paraformaldehyde for 10 min, placed on a poly-l-lysine-treated microslides, and then air-dried for 30 min. Slides were redehydrated

and blocked using 1% BSA in PBS plus 0.1% Triton X-100 (pH 7.2) for 1 h. Then slides were incubated overnight at 4 °C in a humid chamber with appropriate sera diluted at 1:20 in PBS from the patients infected with M. tuberculosis (provided by Dr. Xiao An with the permission of patients). After washing in PBS (three times for 10 min), the bound human immunoglobulin was detected by incubation for 24 h at 4 °C with goat anti-human-HRP-conjugated secondary antibody (Southern Biotechnology Associates, SBA, Birmingham, AL, USA) diluted 1:100 in PBS plus 1% goat serum. After washed in PBS (three times for 10 min), the interest antigen was coloured by DAB substrate, and the slides were counterstained with haematoxylin. Plasmid construction and preparation.  The cDNA of Ag85A is cloned from the genome of cultured Mycobacterium tuberculosis by PCR method.