A retrospective observational single-centre study on the burden of immune thrombocytopenia (ITP).

BACKGROUND: German data on economic consequences of immune thrombocytopenia (ITP) are limited. PATIENTS AND METHODS: A retrospective, observational study based on chart review of adult patients with a confirmed diagnosis of ITP was conducted at a German university hospital. Costs are presented from the hospital perspective. RESULTS: Of 50 eligible patients, 45 could be classified by disease duration: 19 patients < 3 months (38%, newly diagnosed ITP), 12 patients ≥ 3 to < 12 months (24%, persistent ITP), 19 patients ≥ 12 months (38%, chronic ITP). Complications included 85 bleeding events in 43 patients, including 3 intracranial haemorrhages. Documented were 955 outpatient visits in 43 patients (86%) and 92 inpatient hospital admissions in 45 patients (90%). Of the 46 patients (92%) treated, all received corticosteroids, 25 (50%) intravenous immunoglobulin, and 7 (14%) further therapies. 12 patients (24%) underwent splenectomy. Average total direct medical costs (mean (standard deviation)) were 17,091 (18,859) per patient, 12,749 (11,663) in 17 newly diagnosed ITP patients with a 0.88-month (0.65 months) average disease duration, and 29,868 (29,397) in 13 chronic ITP patients with a 33.5-month (16.8 months) average disease duration. Inpatient stays were the main cost drivers. CONCLUSION: These data concerning current healthcare provision for ITP patients in Germany indicate considerable resource consumption and the need for more effective treatment options in individual patients.

The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis.

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 DeltaglnR 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.

The CD70/CD27 pathway is critical for stimulation of an effective cytotoxic T cell response against B cell precursor acute lymphoblastic leukemia.

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 lymphoblastic 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 lymphoblastic 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 lymphoblastic leukemia.

High expression of CD40 on B-cell precursor acute lymphoblastic leukemia blasts is an independent risk factor associated with improved survival and enhanced capacity to up-regulate the death receptor CD95.

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 antigen-presenting capacity and sensitivity to proapoptotic signals. Yet, although CD40 ligation does result in significant up-regulation 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.

High nerve growth factor receptor (p75NTR) expression is a favourable prognostic factor in paediatric B cell precursor-acute lymphoblastic leukaemia.

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.

Leukemia vaccines.

Evidence that immunological effector mechanisms contribute to the elimination of leukemic blasts in allogeneic bone marrow transplantation supports the concept that the immune system plays a prominent role in the control of leukemic disease. For patients with high-risk acute leukemia, relapse prevention in the setting of minimal residual disease is paramount. This review discusses vaccine strategies aimed to stimulate a leukemia-specific immune response in vivo.

Transgenic IL-2 expression in Ewing tumor cell lines after transfection with Starburst dendrimers and cationic liposomes.

Immunotherapy with IL-2-transduced cells requires efficient methods of gene transfer. Nonviral methods have been studied intensively in recent years. This study examined whether tumor and fibroblast cell lines established from Ewing tumor patients could be efficiently transfected with the IL-2 gene. Starburst dendrimers (Superfect), a novel transfection reagent, were chosen for a transfection study and optimal conditions for gene transfer were evaluated. Three Ewing tumor cell lines and 3 fibroblast cell lines obtained from Ewing tumor patients were analyzed. The concentration of IL-2 in the supernatant of transfected cells was measured by ELISA. All three Ewing tumor cell lines transfected by Starburst dendrimers yielded higher IL-2 levels than after lipofection. In contrast to lipofection, expression of IL-2 increased with time and peaked later. In one of three tested fibroblast cell lines, transfection using Superfect yielded higher IL-2 levels. IL-2 production was generally lower in fibroblasts as compared to Ewing tumor cell lines. Given the low toxicity of Superfect reagent and the high efficiency of transfection, this method seems to be ideal for clinical studies on the immunotherapy of Ewing tumors.

Rapid-cycle PCR and fluorimetry for detection of mycobacteria.

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 culture-positive specimens and offers an excellent alternative to previously established nucleic acid amplification-based techniques for the diagnostic mycobacterial laboratory.