Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc 2 155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

In this study we used LightCycler PCR amplification and product detection by fluorescence resonance energy transfer probes to identify mycobacteria and differentiate between Mycobacterium tuberculosis complex, Mycobacterium avium, and other nontuberculous mycobacteria. Targeting the 16S rRNA gene, three different probes specific for mycobacteria, M. tuberculosis complex, and M. avium were constructed. As few as five genome copies of target nucleic acid were detected by the probes, illustrating the high sensitivity of the system. All 33 mycobacterial species tested but none of the closely related actinomycetes and other bacteria produced a specific fluorescence signal. A specificity of 100% was also demonstrated for the M. tuberculosis complex-specific probe and the M. avium-specific probe. Within 45 min, the LightCycler method correctly detected mycobacteria and specifically identified M. tuberculosis complex and M. avium without any post-PCR sample manipulation. In view of future clinical studies, we also constructed and tested an internal control which could be used to assure successful amplification and detection of mycobacteria. Monitoring of PCR inhibition will be essential for evaluation of this system for direct detection of mycobacteria in clinical specimens. Finally, we tested our system on sputum seeded with mycobacteria and were able to detect as few as 10 organisms. At present, this system is the fastest available method for identification and differentiation of mycobacteria from culturepositive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.

We have pursued our analysis of potential tumor-rejection antigens recognized on human melanoma by autologous cytolytic T lymphocytes (CTL). We reported previously that 3 distinct antigens (A,B,C) were recognized on melanoma cell line SK29-MEL in association with HLA-A2. Selection for melanoma-cell variants resistant to anti-A CTL revealed that antigen A consists of at least 2 determinants (Aa, Ab) which can be lost separately. Genetic linkage between Aa and Ab was suggested by concomitant loss of Aa and Ab in an immunoselected tumor-cell variant. This variant was also resistant to an autologous CTL clone restricted by HLA-B45, indicating that this CTL may also recognize a determinant of antigen A. Of 11 allogeneic HLA-A2 melanoma cell lines that were tested, 5 expressed both Aa and Ab, 1 expressed only Aa, and 1 only Ab. None of them was lysed by anti-B or anti-C CTL clones. A CTL clone derived from another HLA-A2-melanoma patient was found to have exactly the same lytic pattern as the anti-Ab CTL of the first patient. This suggested that it may be possible to elicit an anti-Ab response in many HLA-A2 patients. We conclude that there are at least 2 distinct antigens presented in association with HLA-A2 that are common to many melanomas and therefore constitute promising targets for specific immunotherapy.

For effective immunotherapy, maintaining the frequency and cytotoxic potential of effector cells is critical. In this context costimulation via the CD70/CD27 pathway has been proven essential. CD70 has been reported to be expressed to varying degrees on malignant B cells. However, in B cell precursor acute lymphboblastic leukemia, the most common childhood malignancy, the role of CD70 in stimulation of antileukemic T cell responses has so far not been delineated. Herein we demonstrate that in B cell precursor acute lymphboblastic leukemia expression of CD70 is low but can be induced upon blast activation via CD40. Both CD70 and CD80/CD86 up-regulated on CD40-stimulated blasts contribute to primary stimulation of T cell proliferation and cytokine production in an additive manner. These two signals also cooperate in the prevention of T cell anergy. In contrast to blockade of CD70 during the effector phase, inhibition of CD70-mediated costimulation during generation of antileukemic T cells prevents effector cell proliferation and reduces their cytotoxic capacity. Modulation of the CD70/CD27 pathway may thus represent a novel therapeutic approach for augmenting magnitude and quality of the antileukemic response in B cell precursor acute lymphboblastic leukemia.

Identification of the T-cell receptors (TCR) used by synovial cytotoxic T lymphocytes (CTL) of patients with reactive arthritis (ReA) may be crucial to better understanding the pathogenetic mechanism underlying the HLA-B27 association of spondylarthropathies. The authors, therefore, sequenced 25 TCRB chains from HLA-B27-restricted CD8+ CTL clones and two clonal lines specific for self- or Yersinia enterocolitica antigen isolated from synovial fluids of 3 HLA-B27+ patients with ReA and PBL of one healthy HLA-B27+ individual. Fourteen non-HLA-B27-restricted CTL served as controls. Both autoreactive and Y. enterocolitica specific HLA-B27-restricted CTL used a highly limited set of VB genes with preferential rearrangement of three closely related VB families (VB 13, 14, 17), suggesting that these families contain a preferred site for contact with the HLA-B27 molecule. In addition, the presence of limited TCRBJ usage, limited heterogeneity in CDR3 sequences and dominant clones from individual donors among these CTL indicate that TCRB chain usage is further restricted by a limited set of peptides bound to the HLA-B27 molecule. Limited TCR usage by SF CTL of ReA patients may lay a basis for therapeutical manipulation of the T-cell response in the spondylarthropathies.

CD40 and CD27, members of the tumor necrosis factor receptor (TNFR) family, are critical regulators of lymphocyte growth and differentiation. In B-cell precursor acute lymphoblastic leukemia (BCP-ALL), we prospectively assessed the impact of CD40 and CD27 on outcome in 121 children treated according to the CoALL06-97 protocol. Expression of both CD40 and CD27 was found to be significantly higher in low-than in high-risk patients as defined by standard clinical risk parameters such as age and white blood cell count. In addition, in multivariable analysis, a very high percentage of CD40 ؉ blasts at diagnosis was identified as an independent favorable prognostic factor for relapse-free survival. Of note, high CD40 expression particularly protected against late relapse. In B cells, CD40 is known to enhance both antigenpresenting capacity and sensitivity to proapoptotic signals. Yet, although CD40 ligation does result in significant upregulation of CD80/CD86 in our cohort, it is up-regulation of the death receptor CD95 that significantly correlates with the percentage of CD40 ؉ blasts. Thus very high expression of CD40 on BCP-ALL blasts is an independent prognostic marker indicative of superior relapse-free survival that may in part be due to CD40-dependent death receptor up-regulation. IntroductionAlthough the overall prognosis in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is good, identification of new prognostic factors may result in improved risk classification and treatment outcome. Long-term survival is critically influenced by blast sensitivity toward chemotherapeutic agents. Immunologic control mechanisms are also thought to play a role. [1][2][3] In normal B cells, homeostasis is maintained by an intricate regulatory network with the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor/ ligand family of proteins playing a pivotal role in T cell/B cell interactions. [4][5][6][7][8] As adequate stimulation of effector cells as well as susceptibility of the target cells to apoptotic signals are required for optimal immunologic control, the capacity of B cells to respond to CD40 ligand (CD40L) stimulation is critical. In normal B cells, activation via the CD40 receptor promotes differentiation and results in increased expression of costimulatory molecules and the CD95 death receptor, another member of the TNF receptor (TNFR) family. 7,9-11 To various degrees, the CD40 receptor is also expressed on acute lymphoblastic leukemia (ALL) of the B-cell lineage. 12 In analogy to mature B cells, activation via CD40 turns leukemic blasts into efficient antigen-presenting cells mediated by up-regulation of MHC, adhesion, and costimulatory molecules. [13][14][15][16][17] In addition to promoting their antigenpresenting ability, in chronic lymphoblastic leukemia (CLL) it has been shown that CD40 ligation also induces CD95 expression, 18,19 although little is known about CD40-dependent CD95 modulation in BCP-ALL. CD27, another member of the TNF receptor family, also plays an important role in lymphoid di...

Analysis of formal amino acid sequence identity between different TCRB chain (TCRB) hypervariable regions (CDR3) is commonly used to localize relevant sites of TCR antigen interaction or to yield indirect information on unknown corresponding antigens. However, this analysis sometimes fails to demonstrate expected concordances, e.g. between CDR3 from T cell clones of identical reactivity. Since this may be due to ignorance of physico-chemical parameters, we have now use hydropathy profile analysis as an additional method to examine TCRB-CDR3 and putative peptide antigens. Superimposed hydropathy plots (SHOP) of 20 TCRB-CDR3 from HLA-B27-restricted autoreactive and Yersinia enterocolitica-specific synovial cytotoxic T lymphocytes (CTL) isolated from patients with reactive arthritis (ReA) revealed restricted distribution of polar amino acids resulting in characteristically different SHOP profiles between the two CTL groups. Similarly, Yersinia-derived and self nonapeptides known to bind HLA-B27 differed in SHOP profiles. To validate the method we have extended SHOP analysis to published TCRB sequence data from additional TCRB-CDR3 from peptide-specific CTL but not in TCRB from HLA-B27-alloreactive CTL or non-HLA-B27-restricted control CTL. We here demonstrate that SHOP may improve TCR-CDR3 sequence analysis by detection of structural constraints which remain cryptic by conventional sequence analysis. Our data suggest that electrostatic properties rather than rigid sequence motifs determine T cell specificities.

Summary Nerve growth factor (NGF) plays a pivotal role in cellular survival/death decisions with the low affinity receptor p75NTR predominately transmitting anti‐proliferative signals. In spite of its established role in B‐cell function and identification as a prognostically favourable marker in a number of malignancies, little is known about the expression pattern and prognostic significance of p75NTR in B cell precursor‐acute lymphoblastic leukaemia (BCP‐ALL). p75NTR expression was prospectively studied on primary ALL‐blasts in a cohort of paediatric patients with common ALL (n = 86) and preB‐ALL (n = 34) treated within the Co‐operative study group for childhood acute lymphoblastic leukaemia (CoALL) protocol, CoALL06‐97. Flow cytometric analysis showed that almost half of the patients expressed no or negligible amounts of p75NTR (<10%). The median expression in patients expressing p75NTR beyond that threshold was 49% (range 11–100%). In patients classified as low‐risk at diagnosis, p75NTR expression was significantly higher than in high‐risk patients (P = 0·001). Of note, p75NTR expression was lower in the 21 patients who subsequently developed relapse compared with those remaining in remission (P = 0·038). Accordingly, relapse‐free survival was significantly better in patients expressing high surface p75NTR (P = 0·041). Thus, in this prospective analysis, high p75NTR expression was a strong prognostic marker that identified a group of paediatric ALL patients with favourable outcome.