malQ mutants were able to transmit from ticks to mice (Table 2). Ear, ankle, and bladder tissues were cultured for B. burgdorferi at 5 weeks post-tick feeding, demonstrating that dissemination following infection by tick bite also did not require MalQ (Table 2). Although MalQ seems to have no apparent role in the experimental enzootic cycle of B. burgdorferi

or in the ability of the spirochete to utilize glucose disaccharides, the malQ gene is conserved Talazoparib purchase in all sequenced genomes of Borrelia species, albeit encoding an unusual yet functional amylomaltase (Godány et al., 2008). Therefore, MalQ likely has a function that was not discernible in our tick–mouse model system, perhaps related to survival in the tick in nature. There is precedent for our apparently enigmatic results: ospD, encoding an outer surface lipoprotein, and chbC, encoding the chitobiose transporter, are conserved genes that are not essential in an experimental enzootic cycle (Tilly et al., 2004; Li et al., 2007; Stewart et al., 2008). Interestingly, our data indicate that B. burgdorferi can utilize trehalose, which may be physiologically relevant in the tick because trehalose is present in hemolymph (Barker & Lehner, 1976). This may be an important carbon and this website energy source as B. burgdorferi moves from the tick midgut via the hemolymph to the salivary glands during feeding and transmission. We thank

Christian Eggers for thoughtful and critical reading of the manuscript;

Aaron Bestor, Mike Minnick, Utpal Pal, Kate Pflughoeft and Kit Tilly for valuable discussions; Lou Herritt and Scott Wetzel for assistance with microscopy; the LAR staff for assistance with mouse experiments; Mike Norgard, Patti Rosa and Frank Yang for providing strains; Tom Schwan for providing antiserum against Borrelia; Philip Stewart for providing pBSV2; Pamela Stanley for providing chitobiose; Patty McIntire (Murdock DNA Sequencing Facility) for DNA sequencing; and Laura Hall and Beth Todd for excellent Axenfeld syndrome technical assistance. L.L.H.-H. and E.A.M. were supported by Watkins Scholarships from The University of Montana and Undergraduate Research Internships through the National Science Foundation EPSCoR program under Grants EPS-0701906 and EPS-0346458; L.L.H.-H. was also supported by an Undergraduate Research Award from the Davidson Honors College and an Honors Fellowship through the Montana Integrative Learning Experience for Students (MILES) program under Grant 52005905 from the Howard Hughes Medical Institute-Undergraduate Science Education Program; and E.A.M. was also supported by a Goldwater Scholarship. This research was supported by R01 AI051486 to D.S.S. and R21 AI88131 to D.D. and D.S.S. from the National Institutes of Health. “
“Systemic lupus erythematosus (SLE) is an autoimmune disease that involves dysregulation of B and T cells. A tolerogenic peptide, designated hCDR1, ameliorates disease manifestations in SLE-afflicted mice.

Furthermore, CD38− chronic lymphatic

leukemia cells show impaired chemotactic responses to CXCL12 in vitro, and, consequently, are thought to home less efficiently to lymphoid tissues 33, 34. The in vivo analyses of CD38-deficient mice have confirmed the impaired chemotactic migration of DCs and granulocytes towards chemotactic signals. CD38 activity also controls lymphocyte proliferation and apoptosis 23, which indirectly have an impact on leukocyte trafficking. CD38 can also regulate leukocyte traffic by interactions that are not dependent on its enzymatic activity 3, 23. On the cell surface, CD38 is normally expressed as a dimer, and is concentrated in lipid rafts. It can laterally interact with integrin α4 and CXCR4, classical adhesion and chemokine receptors, respectively, and this supramolecular complex may fine-tune leukocyte migration. Moreover, CD31, another classical adhesion molecule that is particularly important for leukocyte transmigration, is Gefitinib mouse a non-substrate ligand for CD38; ligation of CD38 by CD31, triggers signaling cascades in lymphocytes, and may also directly bind leukocytes to endothelial cells. CD157 triggers the same catalytic reactions as CD38, therefore also generating ADPR, cADPR and NAADP 23, 26; however, CD157 is attached to the cell membrane via a GPI-linkage, whereas CD38 is a transmembrane Cell Cycle inhibitor protein. CD157

is expressed both on endothelial cells and myeloid leukocytes and it interacts with integrins on the cell surface of monocytes. Via this integrin interaction, the

ligated CD157 triggers next signals that enhance the polarization of monocytes, and enhance their chemotaxis towards fMLP and transmigration through the endothelial monolayer 35. NAD+ can also post-translationally modify surface proteins 23, 26, 36. In this reaction, which is catalyzed by ectoenzymes belonging to the ADP-ribosyltransferase (ART) family, one or more ADP-riboses are covalently attached to specific amino acid residues. In terms of leukocyte trafficking, L-selectin and the purinergic P2X7 receptor on the leukocyte surface are two important targets of ARTs. In mice, ART2-modified L-selectin is rapidly shed from the cell surface, with potential consequences for leukocyte extravasation, and ADP-ribosylated P2X7 triggers signals, which ultimately lead to T-cell apoptosis 37, 38. Thus, extracellular NAD+ also functions as a classical danger signal, as well as regulating leukocyte traffic. Enzymes regulating extracellular ATP metabolism are intimately connected to leukocyte trafficking. The balance between ATP and its dephosphorylated products ADP, AMP and adenosine determines whether the microenvironment is pro-inflammatory (ATP), pro-thrombotic (ADP) or anti-inflammatory (adenosine). ATP and ADP mediate their effects by binding to the purino-receptor of the P2X and P2Y families, whereas adenosine binds to the G-protein coupled A1, A2a, A2b or A3 receptors 26, 39.

Negative control sections were stained with isotype immunoglobulin (Ig)G that resulted in no positive staining (data not shown) (samples were observed from all the patients described in the text). Fig. S3. Staphylococcal enterotoxin B (SEB) increases the levels of acid-related orphan receptor (ROR)γt in forkhead box P3 (FoxP3)+

regulatory T cells (Treg). Peripheral blood mononuclear cells Adriamycin (PBMC) were isolated from 10 healthy subjects and cultured in the presence of SEB (10 μg/ml) for 4 days. Cells were collected at the end and analysed by flow cytometry. (a,c) Dot plots indicate CD4+ FoxP3+ Treg before (a) and after (c) culture. (b,d) Histograms indicate RORγt+ Treg (open histograms). The solid histograms learn more indicate isotype immunoglobulin (Ig)G staining. “
“In a previous study, our group verified that 100% of mice survived to a lethal dose of Candida albicans following pretreatment with concanavalin-A (Con-A) for 3 days. This work proposed

to investigate whether treatment could mediate an adaptative immune response involving TH17 cells. A significant increase in IL-17 levels at 6 h postinfection was observed and was maintained up to 18 h in the Con-A group, whereas in control mice, a reduction in this cytokine was verified. In addition, TH17 cells develop in the presence of TGF-β, IL-1 β, and IL-6 that were increased significantly 2 h postinfection in Con-A-treated mice. Macrophages were involved in the process, engulfing greater numbers of yeast cells, and were activated through TNF-α and interferon-γ produced at significant levels at 2 h postinfection. A significant increase in IL-12 levels was also observed at 2 h postinfection. Thus, activated macrophages were probably more capable of killing and processing Candida antigens, signalizing an adaptative immune response. Macrophages from controls did not prevent yeast-to-hyphae transition and were partially destroyed, as shown

in scanning microscopy. These results suggest that treatment with Con-A Ribonuclease T1 facilitated the triggering of TH17 and TH1 responses via IL-17 and IFN-γ production, leading to the resolution of C. albicans infection. Candida albicans is a commensal organism found in the gastrointestinal and reproductive mucosa; however, in immunocompromised settings, C. albicans leads to oral and oropharyngeal, vulvovaginal, mucocutaneous or disseminated candidiasis (Villar & Dongari-Bagtzoglou, 2008). C. albicans may cause peritonitis when it reaches the peritoneal cavity through iatrogenic inoculation involving contaminated plastic devices and fluids during continuous ambulatory peritoneal dialysis (Michel et al., 1994; Goldie et al., 1996; Fourtounas et al., 2006). The immune responses to these different forms of disease are quite distinct, revealing the complexity of the anatomical basis for host defenses against C. albicans infection.

45 These encouraging outcomes have created a foundation of successful experiences of the CKD Preventive Project in Taiwan. More evidences for improving patients’ outcome and reducing health-care burden is awaited from the ongoing large-scale population, multi-centres collaborative researches on CKD Prevention

and Care Plan in Taiwan supported by the Institute for Biotechnology and Medicine Industry and National Health Research Institute of Taiwan. Taiwan has been recognized as an epidemic area of kidney disease with the highest incidence and prevalence rates of ESRD. Although its prevalence of CKD approximates the reported prevalence Selleck MK0683 of CKD from other Asian and Western countries, it might be underestimated because of low awareness of CKD. The impact of CKD on public health burden is worsening with increasing comorbidities and mortality, and huge medical expenses. More emerging potential risk factors for CKD drive us to pay more attention to these new high-risk groups with an extended screening program. The nationwide CKD Preventive Project with multidisciplinary care program has proved its effectiveness in decreasing dialysis incidence, mortality and medical

Ku-0059436 cell line costs. Provision of a more comprehensive preventive strategy and better care plan for CKD should be achieved by future international collaborative efforts and research. The Authors state that there is no conflict of interest regarding the material discussed in the manuscript. “
“Aim:  There is accumulating evidence that advanced glycation end products (AGE) play a role in cardiovascular disease (CVD) in patients with haemodialysis (HD). Carnitine deficiency is frequently observed in HD patients, which may also contribute to CVD. In this study, we examined whether carnitine deficiency was independently associated with increased tissue accumulation levels of AGE in HD patients. Methods:  One hundred and twenty-nine HD patients underwent determinations of blood

chemistries including serum level of carnitine. Tissue AGE levels were evaluated by measuring skin autofluorescence with an AGE-reader. Results:  Serum carnitine levels were significantly lower, while skin AGE levels were significantly higher in HD patients compared with healthy controls (P < 0.001). In univariate Casein kinase 1 analysis, β2-microglobulin (β2-MG) and carnitine (inversely) were correlated with skin AGE levels. Multiple stepwise regression analysis revealed that carnitine levels were one of the independent determinants of skin AGE levels (P = 0.024). When β2-MG-adjusted skin AGE levels were stratified by serum carnitine levels, a statistical significance and dose-response relationship were observed (P = 0.043). Furthermore, skin AGE levels were one of the independent determinants of serum carnitine levels as well (P = 0.012).

The current studies are limited by the fact that the measure of performance, infant looking time, has had only modest success as a measure of individual differences (e.g., Frick & Richards, 2001). It has been used primarily as a group measure that yields a categorical outcome in which performance is either above chance or not different from chance. Our studies therefore suggest that a gender difference in mental rotation ability exists, but may not be especially sensitive to revealing the magnitude of this difference. However, the recent work of Krogh, Moore, and Johnson (2013) suggests that progress may be achieved by examining

individual differences in posthabituation 5-Fluoracil looking times as a measure of mental rotation performance and correlating them with other measures. Krogh et al. eye-tracked 5-month-olds while they performed a mental rotation task and observed selleck chemicals llc that males allotted more visual attention to the top of the stimulus and that higher levels of this top-bias were associated with successful performance; by contrast, female visual attention was distributed more evenly throughout the stimulus and with no relation to performance. Additionally, Krogh et al. reported

a positive relation in females, but not in males, between mental rotation performance and prior tactile manipulation with the three-dimensional object to be presented in the looking time task. Taken together, the findings of Krogh et al. suggest that there may be different determinants of performance for male and female infants in mental rotation tasks. An additional possibility worth exploring in future work is whether DCLK1 males and females might be only quantitatively different, rather than qualitatively different, in their mental rotation abilities. Such a possibility might be manifested if females were found capable of performing at an above-chance level in the mental rotation task, but just needed more time to complete it. It is additionally worth

noting that the procedure used in our second study suggests that the gender difference exists between 3 and 10 months, but is not well-suited to determining whether this difference increases during that time window as has been reported for the time period between childhood and adulthood (Geiser, Lehmann, & Eid, 2008). Additional studies could examine whether a sex difference in infant mental rotation changes in magnitude over time. This research was supported by NIH Grant HD-46526. The authors thank Scott P. Johnson and an anonymous reviewer for helpful comments on the initial submission, and Paige Valeski and Laurie A. Yarzab for their assistance. “
“An abundance of experience with own-race faces and limited to no experience with other-race faces has been associated with better recognition memory for own-race faces in infants, children, and adults.

In clinical Fulvestrant purchase situations when a fungicidal antifungal is desirable, AMB may be used. “
“Department of Internal Medicine, Geriatrics and Nephrologic Diseases, Clinic of Infectious Diseases, S’Orsola Malpighi Hospital, University of Bologna, Bologna, Italy Autopsy studies remain an essential tool for understanding the patterns of fungal disease not detected ante mortem with current diagnostic approaches. We collected data concerning the microbiological

trends, patient clinical characteristics and sites of involvement for invasive fungal infections (IFIs) identified at autopsy in a single large cancer treatment centre over a 20-year period (1989–2008). The autopsy rate and IFI prevalence both declined significantly during the study period. The prevalence of Aspergillus

spp. decreased significantly from the first 15 years of the study (from 0.12 to 0.14 cases per 100 autopsies to 0.07 in 2004–2008; P = 0.04), with only Mucorales accounting for a greater proportion of IFIs over the duration of the study period (0.06 to 0.2 cases per 100 autopsies, P = 0.04). After 2003, moulds accounted for the majority of infections identified at autopsy in the spleen, kidney, heart and see more gastrointestinal tract. Despite a trend of decreasing prevalence from 1989 to 2004, invasive candidiasis increased in prevalence during later periods 2004–2008 (0.02–0.05 per 100 autopsies) with decreasing kidney, heart and spleen involvement. Despite a declining autopsy rate, these data suggest a decreasing prevalence overall of IFIs with changing patterns of dissemination in patients with haematological malignancies. Invasive fungal infections (IFIs) remain an important cause of death in patients with leukaemia and recipients of haematopoietic stem cell transplantation (HSCT).[1-3] The epidemiology of IFIs has shifted over the past two decades, paralleling advances in treatment and transplantation for haematological

malignancies, earlier IFI diagnosis and the introduction of new antifungal agents into C-X-C chemokine receptor type 7 (CXCR-7) clinical practice.[4-6] Since the 1990s, invasive aspergillosis has been the predominant IFI in patients with haematological malignancies,[1, 7] coinciding with the introduction and widespread use of fluconazole prophylaxis to reduce mortality associated with invasive candidiasis.[8] More recently, several cancer treatment centres have observed an increase in the prevalence of uncommon, but difficult-to-treat moulds such as Mucorales, Fusarium spp. and Phaeohyphomycetes.[3, 4, 6, 9, 10] The increase in these previously uncommon moulds has coincided with increasing antifungal resistance among Candida species[2, 11] and possibly also Aspergillus species.

There was no prior history of hypertension, hyperlipidemia or diabetes. None of the subjects used additional oral vitamins prior to or during the study period. The investigation was an open cross-over study aimed at reducing the influence of oxidative stress by strengthening the antioxidant defense. The purpose was to gain an insight into whether these antioxidants improve microcirculatory

flow in individual microvessels and if they increase their functional reactivity as assessed by vital capillaroscopy after PRH with and without a potent provocator, in this case the inhalation of cigarette smoke. The doses were chosen to be below the supraphysiological levels commonly used in most studies in Temsirolimus clinical trial this field. The aim was to examine moderate

levels close to what could be achieved by diet or additive vitamins in daily life. The subjects were first treated with 1 g of the water soluble antioxidant ascorbic acid t.i.d. (Friggs C-vitamin brustabletter®; Semper Foods, Stockholm, Sweden) for a period of two weeks to assess the microvascular response before and after treatment. In the 14 subjects who completed the second part of the study, the effect of the lipid soluble chain breaking antioxidant vitamin E (E-vimin®, 100 mg, capsules; Astra Zeneca AB, Södertälje, DAPT in vivo Sweden) t.i.d. was assessed in an identical manner. There was a wash-out period of at least four weeks after the treatment with ascorbic acid. Two subjects were excluded from the study due to too poor visibility of microvessels in the recordings to allow adequate quality in off-line analysis. In another subject, only the ascorbate analysis was of sufficient quality and the subject chose not to participate in the vitamin E part of the study. All subjects were examined by capillaroscopy before and after the intervention with ascorbic acid and vitamin E, respectively. Blood samples were collected

at the same four occasions. Blood samples were collected in connection with microcirculatory measurements at each occasion. Hemoglobin, total leukocyte count, platelet count, and fibrinogen were assessed. Lipid levels—cholesterol, HDL cholesterol, and triglyceride many levels—were assessed initially by standard enzymatic assays (Boehringer Mannheim GmbH, Mannheim, Germany). Plasma α-tocopherol and retinol were analyzed at each point of examination by high-performance liquid chromatography. Ascorbic acid levels in plasma were determined after precipitation with metaphosphoric acid as described by Kallner et al. [24]. Reactivity of microvessels was studied by intravital capillaroscopy. All sessions were video recorded and further evaluated using the Capiflow system (Capiflow®, Stockholm, Sweden). With this technique, CBV can be continuously assessed by a computerized dual-window cross correlation technique that allows a continuous analysis of the velocity in a specific capillary during the registration [4].

Intralymphatic injection into subcutaneous lymph nodes (ILIT) is a novel and potentially attractive alternative. Randomized controlled trials in more than 200 patients have shown efficacy in reducing symptoms, and immunomodulatory effects have been seen with doses a tiny fraction of those used in conventional SCIT. In a randomized study in hay fever sufferers, a short protocol of three intralymphatic injections of grass pollen extract over 8 weeks resulted in improvements in symptomatic and laboratory parameters comparable to that achieved NVP-BGJ398 nmr with conventional SCIT, even after 3 years [142]. No systemic reactions to ILIT occurred during these studies. Another

area of interest is the combination of SCIT with anti-IgE humanized monoclonal antibody. There is some evidence that this approach may induce a synergistic effect with respect to clinical Ku0059436 efficacy and enhance safety of accelerated protocols [143,144], but cost of treatment would be the important deterrent. Allergen-specific immunotherapy is a safe and effective method of treatment for allergic rhinitis and hymenoptera venom allergy, provided this is delivered in a safe and controlled environment with robust patient selection criteria and by a specialist with knowledge

and experience in this field. There is emerging evidence that allergen-specific immunotherapy may be indicated early in the course of allergic rhinitis in order to prevent progress of ‘allergic march’ and development of newer sensitizations. It is

likely that the future will see better vaccines with reduced allergenicity and greater immunogenicity in order to make them even more safe and efficacious. There may be a role for anti-IgE humanized monoclonal antibody alongside allergen immunotherapy, and studies are under way. Drug desensitization is gaining popularity, as recent reports have highlighted its success across Idoxuridine a range of drugs inducing immediate hypersensitivity responses. Understanding of the precise mechanisms underlying desensitization will pave the way to development of novel immunomodulatory therapies. Dr M. T. Krishna is a member of Standards of Care Committee of British Society for Allergy and Clinical Immunology and is the lead author of the guideline ‘Diagnosis and Management of Hymenoptera Venom Allergy’ (submitted for publication). “
“Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively.

Vasopressors were administered at treatment levels for shock. Neither developed flap compromise, suggesting that pharyngeal reconstruction with an ALT flap may be safely performed in the setting of continuous high-dose vasopressors. buy Pirfenidone © 2013 Wiley Periodicals, Inc. Microsurgery 34:237–239, 2014. “
“Intestinal malrotation results from failure of intestinal rotation and fixation during fetal life. We report two cases of esophageal reconstruction with free jejunal flaps

following total laryngopharyngectomy of hypopharyngeal and cervical esophageal carcinoma in which intestinal malrotation was detected during the jejunal flap harvesting. In both cases, the ligament of Treitz was absent, and the laparotomy incision was

thus extended to identify the jejunum. In case 1, harvesting an adequate length of the vascular pedicle of the flap was impossible because of the abnormal position of the pancreas; thus, Selleck Panobinostat a jejunal flap of maximal length was harvested for optimal pedicle positioning in the recipient site. In case 2, Ladd’s ligament prohibited the release of the jejunum from the ascending colon and required its dissection. Both patients underwent successful reconstruction. When the ligament of Treitz is absent during jejunal flap harvesting, investing the whole bowel by extended laparotomy incision is recommended. When anatomical abnormality caused by intestinal malrotation is detected, releasing an adhesion of the jejunum from circumferential

organs and identifying the adequate vascular pedicle of a jejunal flap are necessary. If harvesting the long vascular pedicle is impossible, a jejunal flap of maximal length should be harvested for optimal positioning for vascular anastomosis at the shortest distance Nintedanib (BIBF 1120) in the recipient site. © 2014 Wiley Periodicals, Inc. Microsurgery 34:582–585, 2014. “
“The conventional method of microvascular anastomosis with interrupted sutures is well proven method, with high successful rate. However, this method is time consuming, especially when multiple anastomosis are required. Even though several techniques have been described to minimize the time of anastomosis, none of these have been widely accepted.[1, 2] Vessel anastomosis with a continuous suture has the advantage of being faster than the conventional method but due to the high risk of stricture at the anastomotic site is not recommended for microvascular anastomosis.[3] Herein, we present a novel method of performing microvascular anastomosis, which combines the advantages of the continuous and interrupted sutures. After proper setup of the vessels, the anastomosis begins with the application of two 10-0 sutures at 0° and 180° angle (Fig. 1A). Then a loose running suture is applied at the anterior wall of the vessel. Depending on the size of the vessel, usually 3 to 4 passes of the suture are required, creating 2 or 3 loops, respectively. (Figs.

Briefly, E7 was removed from pSh-CRT-E7 using the restriction sites Mlu I and Not I and replaced with the ESAT-6–CFP10 fusion gene using the same sites to generate the plasmid pSh-CRT–ESAT-6–CFP10. This plasmid was then characterized through the use of unique sites in pShuttle such as Bgl II and EcoR V. pSh-CRT–ESAT-6 was created by deleting CFP10 from Raf inhibitor the pSh-CRT–ESAT-6–CFP10 plasmid by Spe I digestion. The pSh-ESAT-6–CFP10 plasmid was created by cloning the ESAT-6–CFP10 gene directly into the pShuttle using the sites Mlu I and Not I. CFP10 was removed from pSh-ESAT-6–CFP10 by Spe I digestion to create pSh-ESAT-6. All the pShuttle plasmids were recombined with the AdEasy plasmid (AdEasy system;

Agilent Technologies, Santa Clara, CA, USA) that contained the entire type 5 human adenovirus

genome except the E1, E3 and packaging regions. The recombinant adenoviral vectors were characterized with Spe I, Pme I and Mlu I restriction enzymes. These recombinant vectors were transfected into HEK293 cells for the generation of the adenovirus particles AdESAT-6, AdCRT–ESAT-6 and AdCRT–ESAT-6–CFP10. The recombinant adenoviruses were purified by caesium chloride. Analysis of protein expression by recombinant adenovirus.  To verify the expression of the proteins by the recombinant adenoviruses, Western blotting and immunofluorescence studies were performed. HEK293 cells were infected with the recombinant adenoviruses and were monitored daily for viral cytopathic effect, at which time the proteins were extracted with lysis buffer (0.14 m NaCl, 1.5 mm MgCl2, 10 mm selleck inhibitor Tris–HCl pH 8.0, 0.5% Nonidet P-40 and 1 mm dithiothreitol). The protein concentration of the cell lysates was Parvulin determined using a Micro BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA), and equal concentrations of protein (200 ng) were added to each well of a 12%

Bis–Tris gel (Fermentas, Glen Burnie, MD, USA). After electrophoresis, the proteins were transferred onto nitrocellulose membrane using a transblot semidry system (BioRad, Hercules, CA, USA). Protein bands were visualized using a primary polyclonal antibody against CFP10 (Abcam, Cambridge, MA, USA) diluted 1:500 and an alkaline phosphatase-conjugated goat anti-mouse IgG diluted 1:100 as the secondary antibody. Immunofluorescence was also used to detect antigen expression (Thermo Fisher Scientific Inc.). HEK293 cells were transduced with recombinant adenovirus and monitored daily for viral cytopathic effect, after which the cells were fixed with acetone/ethanol (1:1) for 10 min at −20 °C. The cells were then incubated with a primary antibody to ESAT-6 (Abcam ab13960 rabbit polyclonal) at a dilution 1:1000 for 1 h at room temperature, followed by incubation with an anti-rabbit antibody (Invitrogen mouse polyclonal) conjugated to Alexa 488.