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UNLABELLED: The differential diagnosis of acquired torticollis in infants and children comprises a large variety of disorders, including inflammation, neurologic disease or trauma. CONCLUSION: Spinal cord tumours in patients with ALL are a rarely reported entity and secondary spinal involvement in leukaemia presenting as head tilt is even more uncommon.
Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and T-cell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by 'error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.
Chromosomal translocations t(4;11)(q21;q23) are associated with a group of acute lymphoblastic leukemias with very poor prognosis. From the complete sequences of the breakpoint cluster regions of the human MLL and AF-4 translocation partner genes, a novel set of 66 oligonucleotides that facilitates the rapid identification of translocation breakpoints by PCR analysis of genomic DNA was designed. For each breakpoint, a pair of optimally snited primers can be assigned, which improves the monitoring of the disease during treatment. Comparison of the breakpoints with the corresponding parental sequences also contributes to our better understanding of the illegitimate recombination events leading to these translocations.
Peripheral mobilized parental CD34+ progenitors were isolated and used for the hematopoietic reconstitution after a myeloablative therapy in 23 pediatric patients with various diseases. Fourteen donors were human leukocyte antigen (HLA) three-loci mismatches, 6 donors were two-loci and 3 donors were one-locus mismatches. For depletion of T-lymphocytes, a positive selection of the mobilized peripheral CD34+ progenitors using the method of magnetic-activated cell sorting (MACS) was used. The purity of the CD34+ cells after MACS-sorting was 98-99%, the average number of transplanted CD34+ cells was 14.2 x 10(6)/kg (range 5.4-3.9 x 10(6)/kg) and the average number of infused T-lymphocytes was 1.4 x 10(4)/kg. Due to this low T cell number, only a short-term or no prophylaxis of graft-versus-host disease (GVHD) was necessary and no GVHD was seen. A significant GVHD was only seen in patients after add-back of donor T-lymphocytes, which was performed in some patients for prevention of relapse or in patients who showed a transient mixed chimerism. Since the B lymphocyte contamination of the isolated CD34+ cells was low in the range of 0.2%, no Epstein-Barr virus (EBV)-associated lymphoproliferative syndrome was observed. A primary engraftment was seen in 18 patients. Nonengraftment and rejection occurred in three and two patients, respectively. In four of these 5 patients, a second transplant using purified CD34+ cells from the same donor after an immunological reconditioning regimen resulted in a complete and sustained hematopoietic reconstitution. The speed of the immunological recovery was dependent on the number of transplanted CD34+ cells and was more rapid if this number was > 20 x 10(6)/kg. Eleven of the 23 patients are alive and disease free with a median follow-up of 12 months (range 2-30). The main cause of death was relapse (7 patients), and only one fatal infection was seen. Our data suggest that the transplantation of megadoses of haploidentical CD34+ cells is a realistic therapeutic option for patients who otherwise have no suitable donor, and an alternative to the use of unrelated cord blood.
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The antimicrobial activity of a silver-impregnated polymer catheter (the Erlanger silver catheter) was demonstrated by determining the microbial adhesion to the surface of the catheter and by measuring the rate of proliferation (viability) of microorganisms at this site. On the surface of a catheter impregnated with silver, according to previously described methods, the bacterial adhesion of Staphylococcus epidermidis is reduced by 28-40%. Bacterial proliferation on the surface of the catheter and biofilm production are also substantially reduced by the elution of free silver ions from the catheter matrix. Bacteriostatic and bactericidal activities can be determined. The antimicrobial efficacy of the silver catheter is not reduced by blood components. There is no loss in antimicrobial activity for weeks after preincubation in water or phosphate buffered saline. The antimicrobial activity depends on the extent of the active silver surface.
To date there have been no standard methods for assessing the thrombogenicity of central venous catheters. A procedure for testing the thrombogenicity of intravenous lines such as the silver-impregnated catheter by continuous blood flow in vitro was therefore developed. For this test, fresh blood was drawn from healthy human donors and anti-coagulated with sodium citrate (1:9). All material tested (catheter tubes with and without silver manufactured in the same way, polyethylene tubes and tubes with potentially thrombogenic material) were perfused through their lumen with anticoagulated blood for up to 31 hours. Blood samples were collected at different times from the test system at sites before and after the perfusion of the test catheters. The hemoglobin concentration, erythrocyte, leukocyte and thrombocyte counts and markers for thrombin activation (thrombin-antithrombin III-complex, F1 + 2)-prothrombin fragments) and for hyperfibrinolysis (d-dimers) were determined. No thrombin activation or signs of hyperfibrinolysis were detected in any material tested. Polyethylene tubes were found to cause hemolysis, as shown by a decrease in hemoglobin content from 15 g% to 4.5 g%. Tecothane tubes with and without silver did not induce hemolysis.
The Erlanger silver catheter consists of a new form of polyurethane, which contains finely dispersed metallic silver. The aim of this study was to establish the biocompatibility of this intravenous catheter by investigating the acute cytotoxicity of extracts from the Erlanger silver catheter on human fibroblasts and lymphocytes. Extracts of the Erlanger silver catheter were not cytotoxic for MRC-5 human fibroblasts nor for sensitized phytohemagglutinin (PHA)-stimulated human lymphocytes. The addition of silver powder of up to 2% by weight to the basic catheter polyurethane Tecothane led to no increase in acute cytotoxicity in comparison with untreated Tecothane. The Erlanger silver catheter is a new intravenous catheter with good biocompatibility.
It is difficult to make the clinical diagnosis of catheter-related infections using the available and established definitions of the HICPAC (Hospital Infection Control Practices Advisory Committee) of the CDC (Centers for Disease Control, definitions of nosocomial infections). The scoring system shown here is a modification of these definitions and has enabled the causal relationship between the catheter and clinical episodes of systemic infections to be quantitatively graded. The scoring system included the following criteria: height and rate of rise of body temperature, attendant shivering, identification of pathogens in blood and/or catheter tip cultures, improvement in the clinical course after catheter removal, signs of catheter exit site inflammation and results of diagnostic tests for other possible sources of infection. These criteria were graded using points and weighted according to their specificity. The comparative evaluation of 65 episodes of systemic infections using the scoring system and the diagnostic criteria of HICPAC showed agreement in 85%. No case was graded "false-negative." In nine of ten false-positive cases additional findings supported the presence of a catheter-associated infection. This scoring system appears, therefore, to be more sensitive than existing diagnostic criteria, without loss of specificity.
A central venous catheter with a new form of silver impregnation of the internal and external surfaces was investigated for antimicrobial activity and tolerance in patients in a controlled comparative, prospective and randomized clinical study. Commercially available catheters with no antimicrobial activity were used as controls. One hundred sixty-five catheters were included in the final evaluation. All catheters were percutaneously inserted for the first time with a duration of > or = 5 days and a microbiological examination of the catheter tip. Catheter location (> 90% internal jugular vein), mean duration of catheterization (8-9 days), patients' age and diagnosis were comparable in both groups. Silver-impregnated catheter tips showed an incidence of colonization in 14.2/1000 catheter days and control catheters in 22.8/1000 catheter days. This represents a reduction of 37.7%. Catheter-associated infections were diagnosed in the silver group in 5.26/1000 catheter days and 18.34/1000 catheter days in the control group, indicating a reduction rate of 71.3% (P < 0.05, chi 2-test). No complications or side effects were documented in either group.
Central venous long-term catheters offer reliable, large-lumen vascular access with high flow rates for delivery of nutrition or for cell-containing infusions and perfusions. Catheter-associated infections (CAI) pose the greatest threat to such vascular access, despite existing preventive measures. In this article one prospective and one retrospective study of CAI in pediatric therapy are presented. Study I: A retrospective investigation from 1990 through 1995 of 60 conventional long-term catheters in 50 patients. The total number of days in which the catheters were in place was 11,818. The calculated CAI incidence was 1 per 1,000 days of catheter insertion. Bacteriologically demonstrated CAI (identical isolate on the catheter tip and in a blood culture) occurred in three instances (5%). Five cases (8.3%) were diagnosed with a therapy-resistant, septic clinical picture. Study II: A prospective, randomized comparison of long-term silver-impregnated (Erlanger silver catheters) and control catheters (Quinton Instrument Co.) was made with 41 patients (20 with a silver catheter, 21 with a Quinton catheter). To date, the silver catheters have been distinguished by sterile bacteriological findings, whereas three cases of CAI have been demonstrated with the comparative catheters. One patient recently underwent intensive care after becoming unstable with signs of septic shock and demonstrable Pseudomonas aeruginosa, and two other patients manifested coagulase-negative staphylococci on the catheter tips. In three of nine control catheters an incidence of 1.18 per 1,000 days of indwelling catheters was found, whereas no CAI has occurred with the eight microbiologically tested silver catheters.
The regulatory effects of IFNgamma on CD95 expression and CD95-mediated cell death were investigated in three high-risk pro-B acute lymphoblastic leukemia (ALL) lines that carry the chromosomal translocation t(4;11)(q21;q23). These leukemias are characteristically refractory to conventional chemotherapeutic treatments operating through the induction of apoptosis. However, the mechanisms leading to increased cell survival and resistance to cell death in these leukemias are largely unknown. Interferon-gamma (IFNgamma), a potent inhibitor of hematopoiesis, acts in part by upregulating CD95 and sensitizing cells to CD95-induced apoptosis. The t(4;11) lines SEM, RS4;11, and MV4;11 expressed low levels of CD95, but were completely resistant to CD95-mediated death. Addition of IFNgamma markedly upregulated CD95 expression in SEM (8-9-fold), RS4;11 (2-3-fold), and MV4;11 (2-3-fold) lines. However, after treatment with IFNgamma, only an 11% increase in sensitivity to CD95-mediated cell death was observed in SEM cells, whereas RS4;11 and MV4;11 cells remained resistant. Cycloheximide, but not actinomycin D or brefeldin A, increased CD95-specific cell death only in IFNgamma-treated RS4;11 cells by approximately 12%. Abundant levels of Bcl-2 and Bcl-XL, known to inhibit CD95-signaling in some cells, were present suggesting a possible role for both molecules in the resistance to CD95-mediated cell death. Resistance of the leukemic blasts to CD95-mediated cell death and the failure of IFNgamma to substantially sensitize the CD95-signaling pathway may contribute to the highly malignant phenotype of pro-B ALL with translocation t(4;11).
Chromosomal translocations t(4;11) are regularly associated with a specific type of acute leukemias and probably initiate the development of this disease. It has been proposed by others, that these translocations are mediated by recombinases of the immune system. The breakpoints on both derivative chromosomes for three t(4;11) leukemia-derived cell lines and primary blasts from two patients have been analysed here in detail. The results revealed that: (a) multiple double- or single-stranded DNA breaks must have occured near the translocation breakpoints on both participating chromosomes; and (b) DNA fragments flanked by these breaks must have either been deleted, inverted or duplicated during the translocation process. We found no evidence for the involvement of specific target sequences and recombinases of the immune system. Similar characteristic features were observed by re-interpretation of published t(6;11) and t(9;22) translocation data. Therefore we present a new model for the generation of these translocations which poses, that these translocations are reciprocal but not balanced at the fine structure level and that the DNA damage-repair machinery is likely involved in producing the final structure of the translocation breakpoint.
UNLABELLED: Acute renal failure is a rare but very severe side-effect of methotrexate (MTX) treatment requiring extracorporeal detoxification. A new detoxification regime, consisting of a haemodiafiltration (HDF) and continuous plasma resin perfusion (PRP) over anion exchange resin column with high affinity for MTX, was used for treatment of MTX-induced acute renal failure in a 2-year-old boy with medulloblastoma. More than 99% of MTX was removed from plasma by perfusion over the resin column resulting in a marked decrease of MTX plasma half-life. No haematological complications occurred. CONCLUSION: The use of PRP alone or in combination with HDF is a new and effective treatment strategy for extracorporeal detoxification of MTX.
The AF-4 gene on human chromosome 4q21 is involved in reciprocal translocations to the ALL-1 gene on chromosome 11q23, which are associated with acute lymphoblastic leukaemias. A set of recombinant phage carrying genomic fragments for the coding region and flanking sequences of the AF-4 gene were isolated. Phage inserts were assembled into four contigs with 21 exons, and an intron phase map was produced enabling the interpretation of translocation-generated fusion proteins. The gene contains two alternative first exons, 1a and 1b, both including a translation initiation codon. The translocation breakpoint cluster region is flanked by exons 3 and 6 and two different polyadenylation signals were identified. Polyclonal antisera directed against three different portions of the AF-4 protein were produced and used to detect a 116 kD protein in cellular extracts of human B-lymphoblastoid and proB cell lines. In mitogen-stimulated human peripheral blood mononuclear cells the AF-4 antigen was predominantly located in the nucleus. The AF-4 gene is a member of the AF-4, LAF-4 and FMR-2 gene family. The members of this family encode serine-proline-rich proteins with properties of nuclear transcription factors. Comparison of AF-4 protein coding sequences with the LAF-4 and FMR-2 sequences revealed five highly conserved domains of potential functional relevance.
Peripheral stem cells were mobilized and collected in 20 children with stage 4 neuroblastoma. A total of 37 leukaphereses were performed in the 20 patients. The mean number of collected cells was 5.6 +/- 2.4 x 10(8)/kg (range 1.9-10.5), and the number of collected CD34+ progenitors was 6.1 +/- 6.3 x 10(6)/kg (range 0.75-21.7). CD34-positive selection was performed using the CellPro method. Of the adsorbed cells, 42 +/- 20% (range 4.3-76.6) stained positively for CD34, and the number of positively selected CD34+ cells was 2.0 +/- 1.9 x 10(6)/kg (range 0.09-7.1). The mean recovery of CD34+ cells was 36 +/- 20% (range 6-67). For detection of contaminating neuroblastoma cells before and after CD34-positive selection, a murine antidisialoganglioside GD2 antibody (14.G2a) was used, followed by the alkaline phosphatase antialkaline phosphatase (APAAP) method. Before the positive selection, various numbers of contaminating neuroblastoma cells were found in the leukaphereses of 7 patients. After positive selection, neuroblastoma cells were still detectable in all 7 patients, with a mean log depletion of tumor cells of 1.41 +/- 0.45 (range 0.69-2.13). In 1 patient, contaminating neuroblastoma cells were found only after CD34-positive selection. In 15 of the 20 patients, high-dose chemotherapy was performed, and positively selected CD34+ cells were reinfused in 12 patients. In 10 of these, the mean time to reach > 0.5 x 10(9)/L granulocytes was 12.3 +/- 1.7 days (range 10-16). One patient died at day 7 due to sepsis, and in 1 patient the backup was given at day 15. Because of the low number of collected CD34+ cells, 3 patients were grafted with a combination of unmanipulated PBSC and CD34+ progenitors. In summary, we have shown that positive selection of peripheral CD34+ progenitors is feasible in pediatric patients. However, strategies to improve the recovery of the CD34+ cells and the purging efficacy of this method (i.e., higher enrichment of CD34+ cells, combination of positive and negative selection methods) should be evaluated further.
The human ALL-1/MLL/HRX gene on chromosome 11q23 is the site of many locally clustered chromosomal alterations associated with several types of acute leukemias, including deletions. partial duplications and reciprocal translocations. Structurally variant proteins derived from an altered ALL-1 gene presumably make essential contributions to the malignant transformation of hematopoietic progenitor cells. The ALL-1 gene is spread over approximately 92 kb and consists of at least 37 exons. An exon/intron map including the position of the 3'-end of the gene and a detailed restriction map were produced and an updated map is presented. Data from other laboratories were incorporated where compatible. Exon/intron boundaries were sequenced and an intron-phase analysis was performed. The results are expected to contribute to a better understanding of those structural alterations of the gene that conserve the open reading frame and produce presumably oncogenic variants of the ALL-1 protein. They will also facilitate the rapid molecular diagnosis of structural alterations of this gene and the choice of therapeutic options. Mechanisms that may potentially account for the striking clustering of the translocation breakpoints in the breakpoint cluster region of the gene are discussed.
A group of 28 children was investigated after hematopoietic stem cell transplantation for evidence of hepatitis G virus (HGV) infection. HGV RNA was detected in 14 of the 28 patients (50%) and persisted in 9 of 11 patients with follow-up samples for up to 32 months. Whereas thrombopoiesis was delayed in 2 of the 14 HGV-RNA-negative patients (14.3%), 6 out of 14 (42.9%) patients in the HGV-RNA-positive group had a delayed thrombopoiesis and 2 of the latter group had to be retransplanted because of complete graft failure. These were the only cases with hepatitis C virus (HCV) coinfection. Significant liver diseases were also found only in these 2 patients with HGV and HCV coinfection. These results suggest that HGV infection may significantly influence the engraftment in patients after hematopoietic stem cell transplantation, particularly if coinfection with HCV occurs.