Ultrasound in abdominal and soft-tissue childhood PTLD (post-transplant lymphoproliferative disease).

AIM: Post-transplant lymphoproliferative disease (PTLD) is a rare condition, which should be well known to all paediatric medical facilities dealing with bone marrow and solid organ transplantation. The spectrum and the primary detecting modality of the initial imaging findings in paediatric transplant recipients with abdominal and soft-tissue PTLD should be studied retrospectively. METHOD: 7 children/adolescents (female: 4, male: 3; age: 3 - 19 yrs.; study period: 7 yrs.) after heart (5), kidney (1) or liver (1) transplantation were evaluated regarding their initial clinical and imaging findings of PTLD. RESULTS: 6 patients had a latent Epstein-Barr virus (EBV) infection. PTLD presented with clinical symptoms in only 5 patients (ileus: 2, soft-tissue swelling: 2, intussusception: 1) and was detected on routine abdominal ultrasound (US) controls in the remaining patients. US was the primary imaging modality in all children and led to suspecting PTLD in 6 patients. In the seventh case, US had been misinterpreted first. Compared to US, additional magnetic resonance imaging (MRI) and/or computed tomography (CT) better demonstrated the extent of the disease in 3 children, but were even inferior in another 3. There was no completely false-negative US examination during the study period. CONCLUSION: US is reliable for detecting as well as excluding abdominal and soft-tissue PTLD in paediatric patients after solid-organ transplantation and might even be superior to MRI/CT. Therefore, all patients with an increased risk of developing PTLD should be closely monitored by ultrasound. MRI/CT may be reserved for supplementary imaging in cases incomplete or equivocal on US, but are primarily essential in all patients with a localisation of PTLD not accessible by US.

Characterization of cytokine, growth factor receptor, costimulatory and adhesion molecule expression patterns of bone marrow blasts in relapsed childhood B cell precursor all.

Relapse of childhood acute lymphoblastic leukaemia (ALL) comprises a leading challenge of investigation. Characterization of leukaemic cells regarding their potency to express growth factors and surface molecules can provide insight into their aberrant biology. Thus, we analyzed bone marrow blasts from 10 children with relapsed B cell precursor ALL. The gene and protein expression of essential haematopoietic growth factors (IL-2, IL-4, IL-7, IL-10, IL-15, IFN-gamma, G-CSFR), their corresponding receptors as well as the expression pattern of adhesion molecules (ICAM-1, CD58) and costimulatory proteins (CD40, CD40L, B7.1, B7.2, CD28, MHC-I and II) was analyzed by RT-PCR and flow cytometry. Constitutive gene expression was found for IL-7, IL-10, IL-15 and IFN-gamma and their corresponding receptors. Flow-cytometric analysis showed that IL-10R, IL-7Ralpha, IL-4Ralpha and the gamma(c)chain are constitutively expressed, and that some cells bear the G-CSFR. IL-10 and IL-15 protein-producing leukaemic cells were easily detectable. The neoplastic cells mainly lack B7.1, and ICAM-1 is mostly decreased. Furthermore, high CD40, and, surprisingly, CD40L expression could be found. These studies show that ALL cells are likely to be sensitive to many growth factors and some factors are produced by the neoplastic cell itself. The secretion of IL-10 by leukaemic cells, and the absence or downregulation of conventional adhesion and costimulatory molecules might represent an effective mechanism of escape of immune surveillance in relapsed ALL.

Expression analysis and characterization of alternatively spliced transcripts of human IL-7Ralpha chain encoding two truncated receptor proteins in relapsed childhood all.

In the family of cytokines and cytokine receptors, alternative splicing of pre-mRNA is a frequently observed process that generates different protein isoforms from a single genetic locus. The splicing-derived cytokine receptor protein isoforms are mostly soluble receptors or show alterations in their cytoplasmic domain. It is possible that receptor abnormalities or a pathological ratio of different isoforms may contribute to leukaemia by circumventing normal growth factor control or altering the balance of proliferation and differentiation. IL-7 plays a critical role in early stages of both B and T cell maturation. Moreover, it stimulates the expansion of mature T cells including anti-tumour reactive cells as well as a number of T and B cell malignancies underlining its potential importance for deregulated lymphoid proliferation and leukaemogenesis. Here, we present detailed data on the expression of the interleukin 7 receptor alpha chain (IL-7Ralpha) in leukaemic cells from 210 children with acute lymphoblastic leukaemia (ALL) and describe two novel alternatively spliced transcripts of human IL-7Ralpha coding for truncated receptor proteins which are still capable of binding IL-7. IL-7Ralpha mRNA expression was more frequent in more mature pre-B ALL [91% (30/33)] than in common [81% (81/100)] or pro-B ALL [64% (18/28)], or even in T ALL [64% (29/45)]. These results are in concordance with flow cytometric analyses on the proportion of IL-7Ralpha bearing cells among total blast cell population. Our results lead us to assume that splicing derived IL-7Ralpha isoforms play a potential role in modulating IL-7 signal transduction and might be important for the pathogenesis of leukaemia.

Extensive alternative splicing of interleukin-7 in malignant hematopoietic cells: implication of distinct isoforms in modulating IL-7 activity.

Interleukin-7 (IL-7) plays a pivotal role in early stages of normal B and T cell development. In addition, IL-7 stimulates the proliferation of both antitumor reactive cells and a number of T and B cell malignancies, underlining its significance for leukemogenesis. However, its exact role in the process of pathologic maturation of lymphocytes and regulation of the immune response is not completely understood. As alternative splicing of pre-mRNA has been shown to be involved in the control of gene expression, and splicing-derived protein isoforms with antagonistic activity have been found, we assessed the mRNA-expression of IL-7 and its previously described alternative splice variant lacking exon 4, IL-7delta4, in leukemic cells from children with acute lymphoblastic leukemia (ALL). PCR of full-length IL-7 cDNA enabling the competitive amplification of both variants led to the amplification of diverse unexpected PCR products. The sequence data demonstrated the existence of three additional in-frame splice variants resulting from exon skipping of exon 3 or exon 5 or both in combination with exon 4. We named these IL-7delta3/4, IL-7delta4/5, and IL-7delta3/4/5. Furthermore, three out-of-frame splice variants were identified, IL-7(-56bpExon2), IL-7delta4(-56bpExon2), and IL-7delta3/4/5(-56bpExon2), in which, in addition to the aforementioned exon skipping, 56 bp of the 3' end of exon 2 are omitted. Our results led us to assume that splicing-derived IL-7 isoforms play a potential role in modulating IL-7-mediated biologic effects. Further studies are required to clarify the significance of the diverse IL-7 protein isoforms for the regulation of IL-7 function and the pathogenesis of leukemia.

Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.