The library consists of approximately 2 × 109 independent transfo

The library consists of approximately 2 × 109 independent transformants and was screened using a modified ELISA as described previously22 using recombinant human IL-2 (Peprotech, Rocky Hill, NJ) adsorbed to plates as the target antigen. After several rounds of phage panning purification, a small panel of phage expressing scFv SAHA HDAC price (phscFv) was tested for the ability to bind human IL-2 in the presence of a neutralizing anti-human IL-2 monoclonal antibody (eBioscience, San Diego, CA). A recombinant form of a Plasmodium falciparum protein (accession number XM_001347271) and the phscFv from SGPP (structural

genomics of parasitic protozoa) that reacts with it,24 was used as a control to check for specificity of inhibition with the anti-human IL-2 neutralizing antibody. In brief, 0·5 μg/ml human IL-2 or SGPP in PBS was used to coat the ELISA plate, the wells were washed and 2 μg/ml anti-human IL-2 neutralizing antibody (MQ1-17H12; eBioscience), or blocking buffer was added. Supernatants containing individual phscFv clones were then added and phage binding was detected using an anti-M13 phage horseradish peroxidase (HRP) -conjugated BTK inhibitor mouse antibody (GE Healthcare,

Buckinghamshire, UK). The ELISA plate was developed by adding 50 μl o-phenylenediamine (Sigma-Aldrich, St Louis, MO) in 0·1 m citrate buffer pH 4·5 and 0·04% H2O2, stopped by adding 50 μl/well 2 m H2SO4 and the absorbance was read at Branched chain aminotransferase 490 nm. The DNA from phscFv-2 was isolated and used as the starting material for the construction of the scFv human IL-2 fusion construct. The human IL-2 cDNA in pBR322 (ATCC, Manassas, VA) was PCR amplified using primers (Table 1) which added an N-terminal SalI site, the PSAcs (HSSKLQ) and a C-terminal EcoRI restriction site. This insert was then directionally cloned into pBluescript (Stratagene, La Jolla, CA) using the SalI and EcoRI restriction sites. The (GGGGS)x linker of various repeat lengths was cloned into pBluescript using the EcoRI and KpnI restriction sites. The human IL-2 scFv was PCR amplified

(Table 1) from the M13 phage DNA from the phage clone scFv-2 and the 6 × His tag and the KpnI and BamHI restriction sites were added. This insert was then cloned into the pBluescript human IL-2/PSAcs/linker plasmid and shuttled into pcDNA 3.1 and subsequently cloned into the pVL1392 expression plasmid as described above. The generation of recombinant baculoviruses for the expression of proteins in insect cells has been described previously.25,26 Recombinant viruses were created using the pVL1392 transfer vector and the BD BaculoGold™ transfer vector system (BD Biosciences) as described by the manufacturer. Initial virus production was performed in Spodoptera frugiperda (Sf-9) cells cultured in Sf-900 II SFM media (Gibco®; Invitrogen) and after several passages a high-titre stock was obtained.

3, without lesion) had weak blood T cell responses against peptid

3, without lesion) had weak blood T cell responses against peptide E6/2, with mean 28 specific SFC/106 PBMCs. All three patients with a positive ELISPOT–IFN-γ assay exhibited proliferative responses directed against the same or other E6 or E7 peptides. T cells from six (nos 1, 2, 3, 4, 6, 9) of the 10 patients with initial Akt inhibitor proliferative responses still responded

12 months later, one (no. 8) lost detectable responses and three patients (nos 11, 13, 14) were lost of sight (Fig. 3). In the six responder patients, the recognized specificities were different from those observed initially, with a broadening of peptide recognition concomitant with a change on the recognition level of some specificity. E6/2 (14–34) and E6/4 (45–68) peptides were always the two that were recognized most strongly by four (nos 1, 3, 6, 9) and three (nos 3, 4, 6) patients, respectively. Four of these patients (nos 1, 3, 4, 9) received destructive treatment and remained free of vulvar lesions 1 year later, patient 2 had persistent lesions without improvement despite imiquimod therapy

and patient 6 relapsed 12 months after the inclusion in the study. Patient 1, who had cleared more than 50% of her lesions spontaneously, had no detectable ex-vivo blood T cell effector cells 12 months later (data not shown). The two patients with a low initial ex-vivo ELISPOT–IFN-γ response (nos 3 and 13) also had no detectable circulating effector cells 12 months later, despite the persistence of the lesions in patient 13 (data not shown). In contrast to HLA class I molecules, class II molecules accommodate

peptides of various sizes. We therefore Romidepsin in vivo submitted the whole E6/2 and E6/4 peptides directly to HLA-DR-specific binding assays, as these molecules are involved frequently in T cell epitope presentation. E6/2 (14–34) peptide bound to three of 10 HLA-DR molecules (Table 3). At least one of these three HLA-DR molecules, DR3, DR7, DR15, was shared by all except one responder studied. E6/4 (45–68) peptide bound to six of the 10 HLA class II molecules, DR1, DR4, DR7, DR11, DR15, DRB5, all shared by our patients. The HLA class I molecules binding of 12 short synthetic peptides (8–10-mers) included into E6/2 (14–34) and E6/4 (45–68) large peptides was tested against seven supertypes of Methamphetamine HLA class I molecules (Table 4). Every short peptide was able to bind to at least one HLA class I molecule. Binding affinities ranged between 10−4 M (low HLA binders) and 10−9 M (high binders). Specific blood T CD8+ and CD4+ cells play an essential role in the defence against HPV, as observed previously in immunodeficient patients who are more susceptible to HPV persistent infections [9]. The high frequency (62%) of proliferative responses observed in classic VIN patients in the present study is in accordance with previous reports of CIN3 [22]. In contrast, other groups found far fewer proliferative responses (approximately 20%) in CIN3 [31–33].

krusei as C inconspicua/norvegensis,Candida tropicalis, or Geotr

krusei as C. inconspicua/norvegensis,Candida tropicalis, or Geotrichum capitatum. In contrast, all C. krusei strains were correctly identified by MALDI TOF MS. In conclusion, species identification by MALDI-TOF MS was proven to be consistent with ITS sequence analysis; the technique has a resolving power comparatively ATM inhibitor as high as ITS sequence analysis. “
“Metergoline, a serotonin receptor antagonist, was evaluated for its antifungal activity against the opportunistic human fungal pathogen Candida krusei by a broth microdilution assay. The minimal inhibitory concentration and minimal fungicidal concentration of metergoline

against C. krusei were 4 and 8 μg ml−1 respectively. Significant synergism was found in combination of metergoline with amphotericin B (fractional inhibitory concentration index: 0.375–0.5) by a chequerboard assay. Metergoline also inhibited extracellular phospholipase secretion in a dose-dependent manner, which may be a possible action mechanism of metergoline on C. krusei. “
“The fungicidal properties of purified CAY-1, dissolved silver ion and ethylenediamine tetraacetic

acid (EDTA) separately were studied in vitro as were the abilities of silver and EDTA to enhance CAY-1 fungicidal Enzalutamide clinical trial properties. Non-germinated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium verticillioides (Fusarium moniliforme), Fusarium oxysporum and Fusarium solani were incubated separately with CAY-1 (0–24.8 μg ml−1), silver (0–111.1 μg ml−1), and EDTA (0–2400 μg ml−1). Controls consisted of non-germinated or germinated conidia in test medium. To assess combined activity, compounds, based on the sub-lethal doses of each as defined in the initial experiments, were combined and tested in bioassays. Controls for the mixed sets consisted of non-germinated or germinated selleck conidia only or with the sub-lethal CAY-1 test

concentrations. The minimum inhibitory concentrations (MICs) for CAY-1 and silver, both separate and combined, were determined. Viability assays showed CAY-1 activity only against the germinating conidia of A. flavus, A. niger and F. solani. Silver was active against the germinating conidia of all fungi and the non-germinated conidia of F. oxysporum and F. solani. Combined silver and CAY-1 produced significant viability loss at concentrations not effective separately. EDTA was not fungicidal separately and did not enhance CAY-1 fungicidal properties. MIC data showed that CAY-1 plus silver had an additive effect. Results indicate that dissolved silver was fungicidal in vitro and enhanced the fungicidal properties of CAY-1 at concentrations ineffective when tested separately. “
“Candida peritonitis is a potentially life-threatening infection after abdominal transplantation, although there is scant information regarding its incidence and outcome.

A further consideration relates to variations in antibody

A further consideration relates to variations in antibody

levels in a given individual’s serum samples, collected at different times. The most reactive serum is generally called the ‘peak serum’. This may have been collected selleck products several years earlier, with the ‘current serum’ showing quite different reactivity. As an example, the peak serum may show a clear positive CDC crossmatch result, but as the antibody levels have fallen in subsequent sera, so too may the degree of cell lysis in the assay. This may render the CDC crossmatch negative. Nevertheless, the antibodies found in the peak sera may still be of relevance, increasing the risk of early rejection as a result of this prior sensitization and the resulting immunological memory. For this reason, patients on transplant waiting lists have sera collected at frequent intervals; variations can be monitored

and newly appearing HLA antibodies can be detected. In interpreting crossmatches a basic understanding of HLA expression is required. The genes encoding check details HLA are found on chromosome 6 and are inherited en bloc; such that half of each individual’s HLA (an allele) will be from each parent.9 HLA is divided into class I and class II. Class I molecules are HLA A, B and C while class II molecules are HLA DR, DP and DQ. Class I molecules are expressed on all nucleated cells while class II molecule Progesterone expression is restricted to cells such as antigen presenting cells, for example, dendritic cells, macrophages and B cells. Importantly for transplant rejection pathophysiology, both class I and II HLA

can be expressed by vascular endothelial cells.9 Most rejection responses are thought to be due to differences in HLA between donor and recipient, with the HLA mismatched antigens serving as the targets in antibody-mediated rejection. Non-HLA antigens may generate rejection responses but in general this is thought to be less common.1 There are important differences in HLA expression between T and B cells, which influence the interpretation of the crossmatch. T cells do not constitutively express HLA class II so the result of a T-cell crossmatch generally reflects antibodies to HLA class I only. B cells on the other hand express both HLA class I and II so a positive B-cell crossmatch may be due to antibodies directed against HLA class I or II or both. Hence, if the T- and B-cell crossmatches are positive the interpretation is that there may be either single or multiple HLA class I DSAb/s or a mixture of HLA class I and II DSAbs.

Higher FGF23 concentrations have been consistently associated wit

Higher FGF23 concentrations have been consistently associated with increased risk of mortality at all stages of CKD, independent of traditional renal and cardiovascular risk factors.[91-94] In animal studies FGF23 excess as a result of direct intracardiac administration of a mutant FGF23 (and where klotho is absent) has been shown to lead to left ventricular hypertrophy and provides a plausible mechanism of direct cardiac injury at the high concentrations observed in advanced disease.[95] The significance that these experiments were carried out with mutant FGF23 resistant to furin PXD101 in vitro protease digestion is not known. However, supporting independent links between FGF23

and cardiovascular outcomes and mortality is the integrity of such associations after adjusting for phosphate, PTH and vitamin D levels.[91-94] It has yet to be established whether specifically lowering FGF23 or antagonizing its action would yield clinical benefit. Indeed, antagonizing FGF23 with a specific antibody increased vascular calcification and mortality in animals with renal impairment.[96] The downregulation of klotho expression in tissues where it is expressed has been linked to enhancement of the klotho-independent effects of FGF23 in other tissues. One explanation is that with less

binding to the klotho–FGFR Talazoparib complex, more FGF23 is left in the circulation to bind ‘off-target’ to other FGFR, where specificity to the receptor is low, yet ligand present in excess so causing activation of other low specificity FGFR at non-physiological sites. A consistent finding in CKD is the overall decrease in mKl expression in the kidney, parathyroid glands and vasculature.[97] Although human studies Selleck Neratinib of mKl have been

limited due to difficulty in obtaining tissue to determine expression, there appears to be good evidence of reduced kidney mKl expression in animal CKD models.[31, 98] A low level of sKl in plasma and urine of mice with CKD has also been reported.[31] Human studies reporting on associations between circulating sKl and renal function have been capricious even using the same assay (Table 1). Seiler et al. reported no correlation between sKl levels and renal function[43] while other investigators report an increase in sKl with declining GFR.[49, 50, 55] More than half of the human studies in patients with CKD however have documented a reduction in sKl levels with reduced GFR.[39-41, 52-54] The aforementioned issues with assay performance may underpin the apparent discordant results, but may also relate to differences in study setting or simply reflect intricacies of klotho metabolism, which as yet we do not understand. Nonetheless, reductions seen in mKl suggest a relative deficiency of klotho in CKD.

A (64%) and A/J (75%) mice In this study, we evaluated the in si

A (64%) and A/J (75%) mice. In this study, we evaluated the in situ localization of IFN-γ in the granulomatous response developed in the omentum of mice infected with P. brasiliensis. Herein,

the immunohistochemical evaluation allowed us to detect the presence of IFN-γ only in cells with lymphomononuclear morphology. Immunostained cells were located mainly at the periphery of the granulomas circumscribing macrophages, epithelioid cells, and giant cells around fungi in the center of the lesions. Other authors also demonstrated the presence of IFN-γ positive cells in cutaneous lesions of human PCM, and it was correlated to well-organized granulomas and maintenance of cellular immune response (Pagliari & Sotto, 2003). At 15 days after infection, the similar presence in both selleck products number of positive cells and intensity of IFN-γ staining detected in susceptible and resistant mice confirms earlier data that at this

time of infection the genetic background of susceptibility and resistance is not manifested yet (Fazioli et al., 1994). On the other hand, at later phase of infection, resistant mice showed a higher IFN-γ staining in lymphomononuclear cells compared with susceptible mice, suggesting the presence of protective immune mechanisms to the control of fungal dissemination through the high activation status of phagocytes, as previously described (Calich et al., 1994). Therefore, susceptible mice although able to produce IFN-γ since the early stage of the infection, could not control the fungal dissemination, as demonstrated by the several loose granulomas Protein Tyrosine Kinase inhibitor containing viable fungal cells, indicating their incapacity to control the infection, and confirmed by the higher fungal load in omentum lesions of B10.A than in A/J mice previously observed by the same authors (Nishikaku et al., 2008). Cellular distribution of IFN-γ was similar in mice infected with the slightly virulent isolate Pb265, showing positive immunostaining localized

at the periphery of the lesions in both mouse strains. Quantitative analysis demonstrated that IFN-γ positive cells were observed in both mouse strains at the early phase of Pb265 infection, but differently from the infection with the highly virulent Pb18 in which their positivity was increased; infection with Pb265 was associated with the presence of residual Pregnenolone lesions with lower number of IFN-γ positive cells, suggesting the inactivation of inflammatory/immune reaction and the resolution of the infection. The presence of IFN-γ has been observed in necrotic processes (Sugawara et al., 1998), whereas TGF-β has been associated with fibrosis in the granulomatous lesions (Wynn, 2004). In previous studies using the murine model of PCM, TNF-α (Nishikaku, 2003), and TGF-β (Nishikaku & Burger, 2003a) immunostaining was detected in macrophages and multinucleated giant cells, as well as in ECM components.

The TmLIG4-replacement cassette containing nptII was introduced i

The TmLIG4-replacement cassette containing nptII was introduced into the wild-type strain TIMM2789 by the ATMT method. Twenty-five G418 resistant-colonies were picked at random and tested for inactivation of the TmLIG4 locus by molecular biological methods. PCR with the primers Tmlig4/GW3F and nptII-RA suggested replacement of TmLIG4 in four clones. Southern blotting analysis confirmed the deletion without any additional

bands (Fig. 1). Two vigorously growing mutants, TmL28 and TmL36, were chosen for subsequent analysis. Microscopic and macroscopic buy GS-1101 observations of TmL28 and TmL36 strains did not reveal any unique morphology in comparison to the parental strain (data not shown). In addition, they showed the same growth ability on solid medium at various temperatures,

and on media containing chemical mutagens, as the wild-type TIMM2789 (Fig. 3). They displayed normal 3-deazaneplanocin A cost growth activity at 28°C and 37°C and growth inhibition at 42°C (data not shown). When the sensitivities of the TmLIG4Δ mutants and TIMM2789 to several mutagens (EMS, hydroxyurea and phleomycin) were compared, no remarkable differences in growth were observed (Fig. 3). These finding allowed the usage of TmLIG4-disruptant in further experiments. In many fungi, Lig4 plays an essential role in the nonhomologous integration pathway. Deletion of Lig4-encoding genes often leads to an increase in gene replacement frequency. The effects of TmLIG4 inactivation on gene targeting

frequency were estimated at different loci. The wild-type strain TIMM2789 and TmL28 were used as host recipients for these disruption experiments. With homologous fragments nearly 2 kb in length, gene replacement of TmKu80, tnr, TmFKBP12 and TmSSU1 was carried out using a hygromycin B resistance cassette as a dominant selectable marker. First, we attempted to disrupt tnr, which is an areA (31)/nit-2 (32) ortholog, encoding GATA-type transcription factors which activate genes involved in nitrogen catabolite repression. Replacement of tnr causes a decrease in growth activity of T. mentagrophytes on many nitrogen sources (14, 23). In a previous study, we Avelestat (AZD9668) used the wild-type TIMM2789 and TmKu80 disruptant as host cells for tnr inactivation (14). In TIMM2789, the homologous integration frequencies ranged from 3% to 13%, while the HI frequency was about 70% in the TmKu80-lacking strain. In this study, the disruption vector pAg1-tnr/T was introduced into both recipients by ATMT (Fig. 4). A total of 15 hygromycin resistant-colonies were randomly isolated for molecular biological analysis. The HI frequency was 40% in the wild-type and 80% in the TmLIG4Δ mutant (Table 2). Phenotypic analysis of tnrΔ mutants Tmt1 and TmLt8 showed altered growth ability which correlated with the nitrogen sources used (Table 3). Glutamine, glutamate and arginine supported vigorous growth of tnrΔ mutants.

Cells were incubated at 37°C in 5% CO2 for 72 h and pulsed with 1

Cells were incubated at 37°C in 5% CO2 for 72 h and pulsed with 1 µCi/well [3H]-thymidine (GE General Health, Mississauga, ON, Canada) during

the last 16 h. Disintegrations per minute (dpm) from triplicate wells were analysed. Data are presented as mean Venetoclax order dpm ± standard error of the mean (s.e.m.). The same experiment was performed three times using five to eight animals. Culture supernatant was collected from splenocytes 48 h after incubation with SEB and atorvastatin. IL-2 protein levels were quantified by the mouse IL-2 Duoset ELISA (R&D Systems, Minneapolis, MN, USA), as per the manufacturer’s protocol, and read using a SpectraMAX 250 plate reader (Molecular Devices, Sunnyvale, CA, USA). Similarly, TNF-α concentration was assayed in culture supernatant at 24 h and quantified by the mouse TNF-α Ready-SET-Go Kit (eBioscience, San Diego, CA, USA), as per the manufacturer’s protocol. In some experiments, MVA was also added to the SEB plus atorvastatin, and supernatants assayed for IL-2 and TNF-α as described. Results presented were representative of at least three independent experiments. Mouse vascular smooth muscle cells (SMC) (MOVAS) (generously provided by Dr M. Husain, Toronto General Hospital

Research Institute, Toronto, Ontario, Canada) were cultured [Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), sodium PD-1/PD-L1 activation pyruvate, non-essential amino acid, 2 mM l-glutamine and 10 mM HEPES] for 6 h with atorvastatin in addition to 25 ng/ml recombinant mouse TNF-α (eBioscience). In experiments to determine the effect of the mitogen-activated protein (MEK) 1/2 inhibitor U0126 (Cell Signaling, Beverly, MA, USA) on MMP-9 production, U0126 was used instead of atorvastatin. After the incubation period, the MOVAS cells were lysed with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and total RNA was isolated with a standard chloroform extraction method. Unoprostone Complementary DNA (cDNA) was synthesized using the GeneAmp

RNA PCR kit and murine leukaemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA). cDNA was then amplified by real-time RT–PCR following the manufacturer’s protocol with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and probe (Applied Biosystems) and the MMP-9 primers and probe set (Assays-on-Demand; Applied Biosystems) in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems). Data were collected and analysed using GraphPad Prism 4 software (GraphPad Software Inc, La Jolla, CA, USA). Relative quantities of PCR products were determined off the standard curve generated in each run from cDNA known to contain MMP-9 and expressed as a ratio against the housekeeping gene GAPDH. Real-time RT–PCR was performed in at least three independent experiments.

5a) In addition, IL-1β was capable of mediating its affect in th

5a). In addition, IL-1β was capable of mediating its affect in the absence of DCs and could amplify anti-CD3/CD28-mediated Treg proliferation at concentrations as low as 100 pg/ml, lower than the amount of IL-1β produced naturally by H. pylori-treated DCs (Fig. 5b).

We confirmed the role of IL-1β in HpDC-induced see more Treg proliferation by stimulating Tregs with HpDCs in the presence of a neutralizing IL-1RA. The addition of IL-1RA inhibited Treg proliferation, while anti-IL-6 and anti-TNFRII antibodies had no effect (Fig. 5c). These results suggest that IL-1β is the key inflammatory cytokine produced by DCs in response to H. pylori that is responsible for Treg expansion. Suppression of pathogen-responsive Teffs by Tregs at a site of infection is key to determining pathogen persistence/clearance and the degree of tissue injury caused by local inflammation. To determine, therefore, whether H. pylori affects the suppressive capacity of Tregs, ImmDcs and HpDCs were used to stimulate allogeneic Teff in the presence and absence of 1:1 Tregs for 5 days and suppression of proliferation calculated. HpDCs impaired suppression by Tregs when compared to co-cultures selleck chemical stimulated with ImmDCs (Fig. 6a). To rule out the possibility that proliferation of Teff impurities in the

Treg population caused an apparent loss of suppression, we repeated the experiments with CD25hi Tregs and CD4+CD25− Teff FACS-sorted to >98% purity. As before, suppression of Teffs was still impaired significantly by HpDCs (Fig. 6b). To determine whether the loss of suppression was mediated

by IL-1β, Tregs and Teffs were co-cultured at a 1:1 ratio and activated with HpDCs in the presence of IL-1RA. Antagonism of IL-1β resulted find more in partial restoration of suppression (Fig. 6c), suggesting that suppression of Teffs by Tregs is abrogated by IL-1β produced by HpDC. To determine the capacity of Tregs to inhibit the effector function of Teffs, we measured proinflammatory cytokine concentrations in supernatants of Teffs, Tregs and 1:1 Treg : Teff co-cultures stimulated by immDCs or HpDCs. IL-17 production was not detectable in this system, and IFN-γ production was not inhibited by Tregs in co-cultures stimulated with HpDCs, whereas ImmDC-stimulated Tregs could suppress IFN-γ production. (Fig. 6d). Taken together, these data demonstrate that the presence of H. pylori instructs DCs to inhibit Treg-mediated suppression of Teffs in an IL-1β-mediated manner. Persistence of H. pylori is the result of both resistance against the local gastric microenvironment and immunological evasion [32]. Despite making physical contact with immune cells in the lamina propria [33], H. pylori evades immune clearance through a variety of mechanisms including its unique site of colonization, modulation of adhesion and alteration of the host immune response [34]. H.

Data are the mean ± SEM of at least three independent experiments

Data are the mean ± SEM of at least three independent experiments, unless differently

specified. The Student’s t-test Dabrafenib cell line was used to determine result significance (p ≤ 0.05). This work was supported by grants from the: Associazione Italiana Ricerca sul Cancro (AIRC, “Code: IG – 10565 Funding source: 5 PER MILLE MIUR 2008 to L.V.; AIRC, Code: IG-9366” to M.G.); the European Network for Cancer Research in Children and Adolescents (ENCCA) to L.V.; Associazione Italiana Glicogenosi (AIG) to L.V.; Progetti di ricerca di Ateneo Università di Torino-Compagnia San Paolo, Special Project Microstructure and Nanostructure to M.G.; Regione Piemonte Progetti strategici Piattaforma innovativa Biotecnologie per le Scienze della Vita: Project IMMONC to F.N. F.R. was supported by a fellowships Ku-0059436 in vivo from AIRC. PBMCs and DCs were derived from the peripheral blood of healthy donors from the blood bank under an Institutional Review Board-approved protocol. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting

information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. “
“During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with Smoothened human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens.

A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets. Tuberculosis (TB) remains one of the leading infectious diseases throughout the world accounting for about 8.8 million incident cases in 2010 (Griffiths et al., 2010; WHO, 2011). India alone accounted for 2.0–2.5 million cases in 2010, thus contributing approximately 26% of all TB cases worldwide (WHO, 2011). According to National Tuberculosis Control Programmes (NTPs), 2.6 million new cases of sputum smear-positive pulmonary TB (PTB), 2.