01) and 154% increase in IL-6 mRNA levels (p < 0.01). The latter finding is inconsistent with

murine studies, which reported that expression of these cytokines was dependent on the presence of IRF-8 [35-37]. This distinction cannot be attributed to any abnormality in the IRF-8 gene present in CAL-1 cells, as multiple sequencing experiments verified that the WT form of this gene was being expressed (sequences compared to the NCBI reference NM_002163.2, data not shown). As nuclear IRF-1 ALK inhibitor and IRF-5 protein levels continued to rise through 6 h after “K” ODN stimulation (Fig. 2A), their effect on the continued production of IFN-β and IL-6 was examined. siRNA-mediated knockdown of

IRF-5 led to a significant decrease of IFN-β and IL-6 mRNA levels through 9 h, whereas the knockdown of IRF-1 still had no effect on cytokine mRNA levels (p < 0.05; Fig. 4C). Overall, these data https://www.selleckchem.com/products/LY294002.html indicate that IRF-5 (but not IRF-1) contributes to “K” ODN upregulation of IFN-β and IL-6 in human pDC, and that IRF-8 negatively regulates the expression of these genes. Previous studies examining other cell types found that IRF-5 and/or IRF-7 could form complexes with MyD88 [15, 17, 38]. To examine this issue in human pDCs, CAL-1 cells were transfected with a plasmid encoding HA-tagged MyD88 [39]. Analysis of the lysate generated from unstimulated cells showed that HA-tagged MyD88 co-precipitated with both IRF-5 and Clomifene IRF-7 (Fig. 5). When stimulated with “K” ODN, the amount of IRF-5 associating with HA-MyD88 decreased while IRF-7 association remained unchanged (Fig. 5). These results demonstrate that human IRF-5 and IRF-7 both complex with MyD88 in resting CAL-1 cells. Upon CpG triggering, the association of IRF-5 with MyD88 decreases, presumably reflecting the activation/translocation of IRF-5 from the cytoplasm to the nucleus. IRF-5 and NF-κB p50 both translocated to the nucleus of CAL-1 cells within 1 h of CpG stimulation (Fig. 2) and both contributed to the upregulation of IFN-

β and IL-6 (Fig. 3 and 4). These findings raised the possibility that IRF-5 might directly interact with p50. An immunofluorescence-based assay was used to examine the nuclear localization of each transcription factor. Consistent with the results in Figure 2, exposure of CAL-1 cells to “K” ODN led to the accumulation of both IRF-5 and p50 in the nucleus (Fig. 6A). To determine whether these transcription factors were associating in the nucleus, a PLA was employed. PLA generates a signal only when the proteins of interest are in close physical proximity (<40 nm distant, [40]). Thirty minutes after treating CAL-1 cells with “K” ODN, significant nuclear co-localization of IRF-5 with NF-κB p50 was detected (p < 10−4 when compared to unstimulated cells, Fig. 6B and C).

It includes the previously mentioned pIgR, as well as a receptor which can re-internalize IgA–antigen complexes from the gut lumen [94]. This second receptor is also expressed by M cells. Antigens complexed with IgA are addressed to DCs from PP, inducing the production of TGF-β and IL-10 [95]. There is growing evidence

that the biological process of immune tolerance to food and microbial antigens is not confined solely to lymphocytes; conversely, LY294002 cost all the cells in the human intestine play a role in shaping the attitude of the organism towards molecules present in the gut content. Our review emphasizes the participation of enterocytes in this orchestra of mechanisms which preserve the equilibrium

between activation and tolerance in the gut mucosa. The ultimate goal of this equilibrium is to decide more clearly when and against which it is necessary to fight back in order to preserve our AZD4547 manufacturer integrity as an organism. In this context, enterocytes constitute more than a physical barrier against foreign substances from the gut; they are capable of reacting intelligently to the heavy antigenic load of the gastrointestinal tract. Through their diverse array of receptors, anti-microbial peptides and regulatory cytokines, enterocytes are true immune-competent cells. The fineness of the immune mechanisms displayed by enterocytes, in conjunction with the complex design of the local lymphoid tissue, is yet to be elucidated. A better understanding of ‘who and how’ is responsible for developing oral tolerance will ultimately offer us the tools for manoeuvering in a wide range of clinical situations. This work was funded by the Romanian National Council of Scientific Research – CNCS (PD_477). The authors have no conflicts of interest to declare. “
“Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition TCL by TLR2. FSL-1 was internalized into the murine

macrophage cell line, RAW264.7. Both chlorpromazine and methyl-β-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake.

However it was not fully investigated that how reserve capacity of single kidney for healthy kidney donor changes after unilateral nephrectomy. The aim of this study was to assess the change of remaining single kidney function after kidney donation and evaluate predictive pre-donation factor for reserved single

kidney capacity in donors. Methods: Total 74 kidney donors who underwent 99mTc-DTPA Scintillation-Camera renography before and after kidney donation were included in this study. The renography measured singl-kidney glomerular filtration rate (sk-GFR) of both kidney before donation and post-donation GFR of remaining kidney during 12 months in donor. We investigated the factors that are associated with reserved capacity of remaining single kidney after donation, such as age, BMI, BSA, serum creatinine for Cock-Croft Gault’s fomula and MDRD GFR, 24 hr urine collection for creatinine clearance MLN8237 manufacturer and kidney volume measured by abdomen CT. Results: After uninephrectomy the mean of serum creatinine increased significantly

(P = 0.000, Topoisomerase inhibitor from 0.77 to 1.07 mg/dL) and the mean measured GFR by the renography declined (P = 0.000, from 112.9 to 74.9 ml/min/1.73 m2). Nevertheless the mean of serum creatinine and mGFR was stabilized during 12 months follow-up period (mGFR at Post-donation, P = 0.165 [6 month 74.9 ± 18.2 vs 12 month 81.4 ± 14.8 ml/min/1.73 m2]). The sk-GFR of remaining kidney significantly increased by 33.6% after uninephrectomy (sk-GFR, P < 0.01 [Pre-nephrectomy 57.9 vs Post-nephrectomy 77.5 ml/min/1.73 m2]). By univariate linear regression BMI, total mGFR, sk-GFR of remaining kidney and total kidney volume at pre-donation was included as independent predictors of change of sk-GFR. Among these, BMI (P = 0.013) and sk-GFR of remaining kidney at pre-donation (P = 0.019) was statistically related to reserved single kidney capacity in multivariate regression analysis. Conclusion: After kidney donation, reserved single kidney capacity showed selleck compound significant increase due to adaptive hyperfiltration, especially more compensatory

response in donor with lower BMI and sk-GFR of remaining kidney at pre-donation. TSUCHIMOTO AKIHIRO1, NAKANO TOSHIAKI1, MASUTANI KOSUKE1, MATSUKUMA YUTA1, KITADA HIDEHISA2, NOGUCHI HIDEKO1, TSURUYA KAZUHIKO3, TANAKA MASAO2, KITAZONO TAKANARI1 1Departments of Medicine and Clinical Science, Graduated School of Medical Sciences, Kyushu University; 2Departments of Surgery and Oncology, Graduated School of Medical Sciences, Kyushu University; 3Departments of Integrated Therapy for Chronic Kidney Disease, Graduated School of Medical Sciences, Kyushu University Introduction: Lymphangiogenesis is often observed in both diseased native kidney and kidney allograft, and correlates with interstitial inflammation. However, there is little information about the clinical significance of lymphatic vessels in kidney allograft.

The function of circulating IgD has been debated for some time, but it was recently shown to bind to an unknown receptor on basophils, and cross-linking of IgD on the basophil surface leads to the production of inflammatory anti-microbial products and IL-4 (17). IL-4 from basophils was also recently shown to be crucial in the initiation and maintenance of TH2 responses

(18–20). Therefore, it is tempting SCH727965 concentration to speculate that hookworm suppresses the IgD response in infected individuals to suppress the development of a potentially host-protective TH2 response. All data on humoral responses to hookworms in humans have come from blood serum studies. However,

in the context of a parasite that resides in the gut lumen, such as hookworm, the mucosal and faecal antibody titres may be important in immunity. A recent study in the hamster model of Ancylostoma ceylanicum infection showed detectable levels of DAPT manufacturer parasite-specific IgA in the faeces of multiply infected hamsters, associated with resistance to re-challenge (21). Further studies in human hookworm-endemic populations are needed to see whether the mucosal IgA response is important in resistance, as this may have implications for vaccine design. Studies on the cytokines produced in hookworm infections show variable results: experimental and endemic (chronic) infections result in different cytokine profiles, indicating that repeated infection in endemic areas may induce a qualitatively and quantitatively different response (5,22). However, differences in techniques used may also have a role here: many studies use whole blood culture rather than PBMC purified cultures, which can result in lower concentrations of some cytokines (23), possibly leading to levels falling below the limits of detection. In addition, some groups have stimulated cell cultures with antigens derived from the dog hookworm,

A. caninum, rather than antigens from human hookworms because of the difficulty in obtaining the latter (24–26). Gastrointestinal parasitic infections Histamine H2 receptor have been long regarded to induce polarized TH2 responses, with production of IL-4, IL-5, IL-13 and IgE, which are necessary for their expulsion (27). TH2 responses have been shown to be somewhat effective against controlling hookworm infections, with elevated IL-5 positively correlating with resistance to reinfection after drug cure in humans (28). In recent years, evidence has mounted that the immune response to hookworms may not be as simple as a polarized TH2 response. As mentioned previously, immune responses differ between experimental primary infection and responses in presumably multiply exposed endemic populations.

A complete understanding of their function and regulation will therefore be critical to disrupt one of the most pathological effects of Plasmodium infections. In an effort

to improve functional annotation and increase our understanding of the parasite’s biology, a number of research groups have been leveraging biochemical metabolic profiling and metabolomics strategies (40). Metabolomics is the study of the entire repertoire of metabolites, i.e. small molecules such as amino acids, sugars and fatty acids that are known to perform critical functions in various biological processes. Correlation analyses of transcriptomics, proteomics and metabolomics data are a powerful way to identify new metabolic pathways as well as genes that encode for specific enzymatic functions (41,42). While the study of metabolomics in Plasmodium is still in its infancy, it has already uncovered important biological insights with possible implications in terms of adaptation, evolution and host–pathogen check details interactions (43–45). Functional genomics suffers from the lack of tools to analyse the malaria parasite’s genome. For example, gene silencing using RNAi cannot be used in Plasmodium because the machinery does not exist in the parasite; gene knockout experiments are time-consuming processes not Selleck Nutlin-3 compatible with large-scale high-throughput analyses. However, in the past few years, a transposon-based mutagenesis approach in Plasmodium has been developed (46). A Plasmodium-specific

selection cassette was added to the lepidopteran transposon piggyBac and transfected in parasites together with a transposase-containing helper plasmid (47). Random insertional mutants are obtained by multiple integrations of the transposon at TTAA recognition sites. Recent studies used piggyBac-based approaches to validate candidate parasite-specific

secreted proteins (48) or identify genes that are essential for the parasite’s proliferation (49). Used in combination with other genomics and proteomics analyses, piggyBac-based strategies could provide a better understanding of the parasite’s biology and its interactions Suplatast tosilate with its hosts. The data of large-scale and functional genomic analyses must be accessible and intelligible for practical and efficient usage. The task belongs to the informatics and bioinformatics fields that can provide the necessary tools. Up to now, data depositary banks and the Web-based databases such as PlasmoDB (http://plasmodb.org/plasmo/) have greatly facilitated the access, the comprehensive visualization and the analysis of large data sets. Gene predictions and annotations, new drug target identifications and discoveries of vaccine candidates all resulted from various genome-wide analyses. However, it is critical that such resources remain well maintained and free for maximized accessibility. Indeed, a systemic view of the malaria parasite’s biology can only be achieved with the successful integration and accessibility of the data from various origins.

[12] This review deals with the cellular pathology of ALS, with special reference to the relationship between BBs and skein-like inclusions. BBs are small round eosinophilic inclusions, 1–5 μm in diameter, observed in the brainstem motor neurons and spinal anterior horn cells in ALS. Ultrastructurally, the inclusions are composed of homogeneous, electron-dense granular matrix surrounded by vesicular

and tubular structures. They are considered to be originated from the endoplasmic reticulum[13, 14] and are immunolabeled with antibodies against cystatin C, transferrin and peripherin.[15-17] Skein-like inclusions are made of bundles of filaments, 15–20 nm in diameter. It is now known that TDP-43 is a major component of ubiquitinated BMS-907351 concentration inclusions in ALS and FTLD-TDP with or without motor neuron disease.[2, 3] Thus, these neurodegenerative disorders comprise a new disease concept, namely that of “TDP-43 proteinopathy”. Until now, phosphorylation, ubiquitination and abnormal cleavage are the known pathological modifications of TDP-43.[2, 3, 18] TDP-43 immunohistochemistry revealed overt inclusions of filamentous structures (skein-like

inclusions) or compact, round morphology (round inclusions) in motor and non-motor neurons in TDP-43 proteinopathy.[2, 3, 19-24] Proteinase K treatment following heat retrieval enhances the immunoreactivity for native TDP-43 in controls as well as for native and phosphorylated TDP-43 in ALS and FTLD-TDP.[25] A significant number of TDP-43-positive neuropil threads are found in selleckchem lesions, in which routine immunohistochemistry revealed that the predominant inclusions are cytoplasmic. Although recent studies have shown that BBs are immunonegative for TDP-43,[23] Resveratrol we hypothesized that the co-localization of BBs

and skein-like inclusions indicates a certain relationship between these two inclusions. To elucidate this hypothesis, we quantitatively examined the spinal cord and brainstem motor nuclei by sequential staining of the same sections with HE and an antibody against phosphorylation-independent TDP-43.[12] Twenty-two patients with sporadic ALS were utilized in the present study. Serial sections were cut from paraffin blocks of the fourth lumbar segment in 20 cases, the hypoglossal nucleus in six cases and the facial nucleus in five cases. The data of spinal cords (cases 1–4, 6–11 and 13–20 in Table 1) have been previously reported.[12] The results are shown in Tables 1 and 2. BBs were found in the spinal anterior horn in 16 of 20 cases (80%), in the hypoglossal nucleus in all six cases (100%) and in the facial nucleus in four out of five cases (80%). The average incidence of anterior horn cells with BBs and TDP-43 inclusions relative to the total number of neurons was 16.9% and 41.1%, respectively (Table 1). The incidence of co-localization of BBs and TDP-43 inclusions was 15.

B and T cells also showed altered secretion of cytokines and chemokines after LL-37 and LPS treatment compared with LPS alone 14. In B cells, LL-37 limited class switching and cell proliferation after LPS/IFN-γ treatment 15. Immunizing mice with OVA and mCRAMP led to an increase in specific anti-OVA

IgG as compared with immunization with OVA alone 13, while a fusion of LL-37 and M-CSFRJ6-1 improved the specific immune response to tumors in Tigecycline concentration mice 16. The extent to which these responses are influenced by APCs and innate immunity is still unclear and many aspects of the relationship between cathelicidins and the adaptive response are largely unknown. Additionally, most in vivo studies have focused on injecting cathelicidin into rodents instead of examining its endogenous effects on adaptive immunity. A study by Kin et al. 17 in this issue of the European Journal of Immunology brings new understanding to the role of cathelicidins in adaptive immunity by isolating populations of B and T cells

from peritoneal lavage and the spleen in WT and Camp−/− mice lacking the gene for mCRAMP. Intriguingly, it was found that the response to, and expression of, IL-4 was altered in the Camp−/− mice and this affected both T and B cells. IL-4 is a key regulator of adaptive immunity that leads to an increased humoral response by promoting Th2 cell development 18. Under IL-4-induced

Selleck JAK inhibitor Interleukin-2 receptor Th2 conditions, IL-4 was significantly increased in the Camp−/− T cells and the expression was reduced to WT levels when mCRAMP was added. In contrast, CD4+ T cells from Camp−/− mice showed a similar expression of IFN-γ as WT CD4+ T cells when both were cultured under IFN-γ-induced Th1 conditions 17. IL-4 also enhances class switching in B cells, increasing IgG1 and IgE expression in mice 19. In the Kin et al. study 17, B cells isolated from WT and Camp−/− mice showed no differences in IgM and IgG3 expression when cultured with LPS, or in IgG2c levels when CD40L/IFN-γ was used as a stimulus. Surprisingly, when the B cells were cultured with CD40L/IL-4, the Camp−/− cells showed decreased IgG1 and IgE expression. The antibody levels were restored to those of WT cells when mCRAMP was included in the culture conditions. The decreased IgG1 production was determined to be from reduced mRNA expression rather than changes in class switching. Kin et al. 17 further demonstrated a relationship between mCRAMP and B and T cells by injecting mice with type 1 and 2 antigens or T-cell-dependent antigens 17. T-cell-dependent antigens require Th2 cells to activate B cells and produce antibody, whereas type 1 and 2 antigens are T-cell independent and do not require a Th2 signal.

However, Nikora et al. note that decision making after death is often easier for whānau when the deceased has previously made their wishes known,[6] suggesting that in Māori society the wishes of the individual are used to inform whānau decision making, at least after death. To facilitate whānau involvement and support there needs to be enough warning that a discussion is planned for whānau to attend if possible. ACP may be seen by Māori Ibrutinib patients as a way to assist whānau with future decision making or it can be used as an opportunity to make health care professionals aware of the cultural

practises that will be important to them in their final days and after death (see case example in section 6 on Advance Care Planning). There is currently work underway by the Māori Tools Task Team of the New Zealand Advance Care Planning Co-operative on ACP tools with a Māori focus. The need for this has been endorsed by the ‘Kia Ngāwari: Investigating the end-of-life experiences and cultural needs of Māori and their whānau’ research project led by Dr Tess Moeke-Maxwell of Waikato University.

NVP-BKM120 This research is still being analysed but the patient cohort includes Māori with renal failure and in preliminary analysis it has been identified as a concern that Māori whanau do not always appreciate that renal failure, even for those who choose renal replacement therapy, is a life limiting condition (personal communication, Dr Tess Moeke-Maxwell). Engaging Māori patients and whānau in the open discussion of illness and prognosis that is part of ACP is one way to address this issue. The Māori concept of whānau is generally more inclusive than the New Zealand European concept of family. Family

meetings are often appreciated and well attended. Even small children may Org 27569 be included. Providing sufficient space for a dozen or more people can be helpful and at least one New Zealand renal unit has a collection of toys for children to play with during whānau meetings. Inviting whānau to open a meeting with a karakia or prayer can be an opportunity to respect the importance of taha wairua. As with any family meeting, it is likely to be helpful to ask all those present, including hospital staff, to introduce themselves and their role at the beginning of the meeting. There will often be a whānau spokesperson or people who will be identified by whānau (NG). When decisions are being made by whānau the goal is to reach consensus or kotahitanga. When this is not achieved the whānau usually defer to more senior family members. Silence or withdrawal from the discussion often represents protest or dissent rather than agreement.[6] It is usually appropriate to offer the opportunity for whānau to close a meeting with a karakia, particularly if they have chosen to open with one.

The Th1 cells secrete high levels of interferon-γ (IFN-γ) and IL-2, and

drive immunity against intracellular pathogens but also promote autoimmunity. Interleukin-12, in synergy with IL-18, drives Th1 differentiation, in large part via induction of T-bet (T-Box expressed in T cells), a master regulator transcription this website factor that controls the expression of IFN-γ.14 Interleukin-12 signals through JAK2 and Tyk2, and activates mainly STAT4, also a key transcription factor for Th1 commitment4 (Fig. 2). Indeed, STAT4-deficient CD4+ T cells do not produce IFN-γ following IL-12 or Listeria monocytogenes stimulation,15,16 and STAT4-deficient mice fail to secrete IFN-γ in response to Toxoplasma gondii and therefore die as the result of an uncontrolled parasite burden.17 It later emerged that STAT4 controls T-bet expression,18,19 with which it then collaborates for efficient binding to the Ifng promoter1 and to induce both IL-18Rα

and IL-12Rβ2.3 The STAT4 also induces tumour progression locus 2 (Tpl-2), a serine threonine kinase essential for T-bet and STAT4 up-regulation and so essential for optimal IFN-γ secretion.20 Therefore AZD8055 research buy STAT4 not only promotes the expression of IFN-γ and T-bet, but also of other genes that consolidate the Th1 phenotype (Fig. 2), as summarized in Table 1. Importantly, IFN-γ also facilitates the development of Th1 cells in a positive autocrine feedback loop,21 and STAT1-deficient T cells have reduced T-bet levels following infection,22 although IFN-γ secretion does not seem to be affected. Moreover, several studies Metalloexopeptidase have shown that JAK3 and STAT5 activation by IL-2 enables optimal IFN-γ secretion.23,24 Indeed, JAK3-deficient T cells fail to secrete IFN-γ,23 whereas

IL-2-mediated STAT5 activation is required for optimal IFN-γ secretion.23,24 STAT5 binds the first conserved non-coding sequence upstream of the Ifng promoter, which suggests that it might permit T-bet access.23,25 Therefore, STAT1 and STAT5 contribute to Th1 differentiation by enhancing T-bet and IFN-γ expression, respectively (Fig. 2). SOCS1 is a key inhibitor of IFN-γ signalling26,27 and blocks IFN-γ-mediated STAT1 activation by targeting JAK2 and IFN-γRα chain28 (Fig. 2). The SOCS1-deficient mice also have enhanced type 1 IFN responses, which render them more resistant to viral infection.27 Importantly, SOCS1 is up-regulated during Th1 commitment29 and not surprisingly, SOCS1-deficient T cells proliferate strongly in response to IL-12,30 which enhances their polarization towards the Th1 lineage.31 However, these cells also secrete elevated levels of IL-4, and exhibit heightened IL-4-mediated STAT6 phosphorylation, suggesting that SOCS1 could also be an important regulator of Th2 differentiation.

These five mutations are associated with 70–90% of all ethambutol-resistant

isolates (Johnson et al., 2006). The early and rapid detection of multidrug resistance is essential for efficient treatment and control of M. tuberculosis. The culture-based methods for detection of M. tuberculosis infection and Cell Cycle inhibitor drug susceptibility testing (DST) usually take more than 1 month due to the slow growth of this bacterium. The use of molecular methods for the identification of mutations in the resistance genes may offer the means for rapid screening of the drug resistance among the M. tuberculosis isolates and initiation of early treatment. In Jordan, although the incidence rate of tuberculosis declined in 1990–2010, the number of MDR-TB reported cases increased (WHO, 2010). In the present study, three find more different allele-specific PCRs (AS-PCR) that were previously optimized and validated were carried out directly with purified DNA to detect mutations in several codons in the rpoB, katG, and embB genes of M. tuberculosis isolates (Mokrousov et al., 2002a, b, 2003). The AS-PCR primers are used to amplify and discriminate between two alleles of a gene simultaneously. One benefit of AS-PCR is that it combines the amplification with detection events

without the necessity for additional probes or enzymes. A total of 100 M. tuberculosis-resistant strains were selected randomly from sputum cultures of tuberculosis patients obtained from the stock cultures of the Directorate of Chest Diseases and Foreigners Health (referred to as the TB Center for short), Ministry of Health in Amman, Jordan. Histamine H2 receptor These isolates were recovered from sputum specimens of adult patients diagnosed with pulmonary tuberculosis who were referred to the TB Center from eight cities in Jordan in 2007. Multiple isolates from the same patient were avoided. Species identification of the isolates was confirmed in the TB Center using a combination of standard microbiological tests: colony morphology, acid-fast staining, and conventional biochemical tests. The study was approved by the University Internal Review Board. The simplified version of the indirect proportion method was performed in the TB Center on Löwenstein–Jensen

medium against isoniazid, rifampicin, and ethambutol, at 0.2, 40, and 2 μg mL–1, respectively, according to standard procedures (Canetti et al., 1969). Each M. tuberculosis isolate was inactivated by a touch swab placed in an Eppendorf tube containing 500 μL of 1 × Tris-EDTA buffer (pH 8.0), and tubes were incubated at 80 °C in a water bath for 20 min. The M. tuberculosis H37Rv, and a collection of five to eight randomly selected clinical isolates that were susceptible to all the three test drugs were also included as reference strains in the study. DNA was extracted from the M. tuberculosis isolates using standard protocols as described previously (Van Soolingen et al., 1991). All PCR amplifications were carried out in GenAmp 9700 (Perkin Elmer). Each run of AS-PCR included M.