Multiple viral infections after haploidentical hematopoietic stem cell transplantation in a child with acute lymphoblastic leukemia.

After allogeneic hematopoietic stem cell transplantation (HSCT), viral infections/reactivations are a frequent complication, sometimes with fatal outcome. Thus, early diagnosis is recommended by screening of whole blood or plasma preparations using highly sensitive molecular techniques that test for the most common viral pathogens, such as Epstein-Barr virus, cytomegalovirus, and adenoviruses (ADVs). Despite this approach, not every reactivation/infection can be adequately detected or excluded, even with highly sensitive polymerase chain reaction. Particularly after toxic treatment, uncommon infections or infections resistant to first-line treatment can occur, even in unusual locations. Herein, we present the case of a child with Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic HSCT who suffered from 5 different viral reactivations/infections, including acyclovir-resistant herpes simplex virus type 1 esophagitis, human herpesvirus 6 encephalitis, rotavirus gastroenteritis, respiratory syncytial virus pneumonia, and ADV esophagitis, despite routinely performed blood examinations for viral pathogens remaining unrevealing at all times.

Monoclonal antibodies against neuroblastoma. Production and preliminary characterization of their specificity.

Neuroblastoma (NB) is a common childhood tumor that originates from neuroblasts of the neural crest. In this paper we will describe the production of murine monoclonal antibodies (mAbs) to human NB cell lines. The hybridomas were selected by ELISA and immunofluorescence for antibody binding to multiple human neuroblastoma cultured cell lines, but not to hematopoietic cells and leukemic cells. The mAbs were characterized in terms of their ability to bind to human cell lines and tissues. The IgG2a and IgG2b mAbs may prove useful in the diagnosis of therapy of neuroblastoma.

Expression of CD34 and other haematopoietic antigens on neuroblastoma cells: consequences for autologous bone marrow and peripheral blood stem cell transplantation.

Autologous peripheral blood stem cells, obtained by CD34+ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34+ stem cell selection.

Neuroblastoma cells can express the hematopoietic progenitor cell antigen CD34 as detected at surface protein and mRNA level.

Recently, we have shown the expression of the hematopoietic precursor cell antigen CD34 on neuroblastoma cells. Here, we present the CD34 expression on 16 permanent neuroblastoma cell lines and primary cell lines at the mRNA level and the flow cytometric results on neuroblastoma cells grown in the same culture and split for flow cytometric analysis and total mRNA extraction. The flow cytometry was performed using a panel of anti-CD34 antibodies covering the epitope classes I to III. In eight neuroblastoma cell lines, CD34 mRNA expression could be detected and corresponded always with the protein surface expression. Alternatively, when CD34 mRNA expression was not seen, CD34 antigen expression ranged from negative to as high as 78%. Based on these results caution should be taken with transplants obtained by CD34+ stem cell selection from neuroblastoma patients.

Fatty acid composition of lymphocyte membrane phospholipids in children with acute leukemia.

The composition of phospholipid fatty acids (PLFA) of separated mononuclear blood cells (MNC) from patients with leukemia was established by high-resolution gas chromatography. Abnormal fatty acid concentrations are detected in the MNC membrane phospholipids in patients with acute lymphoblastic leukemia (ALL) without a deficiency of essential fatty acids (EFA). Significantly reduced relative levels of linoleic acid (4.35 vs. 7.82%; P<0.001) are found in the MNC-PL in patients with ALL as compared to a healthy control group. Moreover, the Delta6-desaturated fatty acids are increased: gamma-linoleic acid (3.56 vs. 0.17%; P<0.001), arachidonic acid (21.82 vs. 16.27%; P<0.05), docosatetraenoic acid (3.52 vs. 1.56%; P<0.001), docosapentaenoic acid (0.34 vs. 0.04%; P<0.001), octadecatetraenoic acid (0.53 vs. 0.23%; P<0.05), eicosatetraenoic acid (1.83 vs. 0.08%; P<0.001) and docosahexaenoic acid (2.77 vs. 1.54%; P<0.001). A increased Delta(6)-desaturase activity is postulated as the cause for the increased level of desaturate products or the increased Delta6-activity index (Ratio of gamma-linoleic acid+dihomogamma-linolenic acid to linoleic acid) (1.21 vs. 0.27; P<0.001). The Delta6-enzyme activities measured using linoleic acid and alpha-linoleic acid as substrate underscore these findings (Delta6(n-6); 2.49 vs. 0.65 and Delta6(n-3); 2.75 vs. 1.12 nmol x h(-1)/10(8) MNC). In contrast, patients with acute myeloid leukemia (AML) do not show any significant differences in the lymphocyte membrane PLFA and no Delta6-desaturase abnormalities.

Model experiments for immunomagnetic elimination of leukemic cells from human bone marrow. Presentation of a novel magnetic separation system.

Optimal conditions for removing leukemic cells from human bone marrow with monoclonal antibodies (mAb) and magnetic immunobeads were investigated. Monodisperse 3 microns polystyrene microspheres containing magnetite were coated with affinity-purified rabbit antimouse IgG at 4 degrees C, pH 9.6 for 18 h. SKW-3 cells (T-CLL cell line) were marked with the supravital DNA stain Hoechst 33342, seeded into normal human bone marrow, and then incubated with the mAb CD1, CD6, and CD8 at 4 degrees C for 30 min. In preliminary experiments REH cells (cALL cells) and mouse anti-REH cell antibodies were used to find the most favorable conditions for the binding of magnetic beads to tumor cells. Optimal formation of cell-bead rosettes was achieved by rotating beads and tumor cells together at room temperature at a concentration of 1 x 10(7) cells/ml, a bead: tumor cell ratio of 100:1 and an incubation time of one hour. The novel magnetic separation apparatus consists of three polystyrene chambers connected by silicone rubber tubing. The chambers contain four steel inserts each equipped with 32 nickel wires, which are magnetized by permanent magnets in such a way that the inhomogeneous high gradient magnetic field could be established within the cell suspension containing the cells to be depleted. The fluid flow was established by a peristaltic pump. At a flow rate of 1.5 ml/min and a field strength of 160 kA/m, no beads could be detected in the purged marrow. A cocktail of the three mAb was more effective than any single antibody in forming bead-cell rosettes. Two sequential purging cycles were superior to one. The marrow recovered was highly viable as assessed by trypan blue dye exclusion and by growth of CFU-GM.

Cyclosporin A-induced graft-versus-host disease following autologous bone marrow and stem cell transplantation in hematological malignancies of childhood.

Cyclosporin A (CsA) can induce graft-versus-host disease (GVHD) following autologous bone marrow transplantation (ABMT) and autologous peripheral blood stem cell transplantation (APBSCT) in adults. We investigated whether GVHD can be induced following ABMT and APBSCT in childhood, and which cells are involved in the pathogenesis of this syndrome. We conducted a prospective study of 20 children and adolescents with hematological malignancies receiving CsA after ABMT and APBSCT. Skin biopsies were obtained on day 21 after transplantation or in the event of a rash. Immunophenotypic analysis of peripheral blood lymphocytes was performed on days 14, 21, 28 and 60 after transplantation. Clinical GVHD of the skin, confirmed by histological criteria, occurred in five patients. Five patients had no clinical GVHD but had acute GVHD alterations on routine skin biopsy. In all 10 patients with a positive skin biopsy for GVHD, CD4+ lymphocytes were the predominant cells in the epidermis. Immunophenotypic analysis of peripheral blood lymphocytes revealed a significantly increased CD4/CD8 ratio in patients with a positive skin biopsy (P < 0.01). Our findings indicate that it is possible to induce acute GVHD following ABMT and APBSCT in childhood. In addition, CD4+ lymphocytes play an important role in the pathogenesis of CsA-induced GVHD.

Pre-emptive therapy with rituximab for prevention of Epstein-Barr virus-associated lymphoproliferative disease after hematopoietic stem cell transplantation.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Therefore, early diagnosis of EBV reactivation and pre-emptive therapy may be clinically useful. We report three patients who presented with an extremely high EBV load in peripheral blood mononuclear cells and plasma without evidence of EBV disease. Following pre-emptive therapy with a single dose of rituximab, a concordant decrease of EBV-genome copies and B lymphocytes was observed. In all three patients, no EBV-associated LPD occurred. We conclude that pre-emptive therapy with rituximab appears to be effective for prevention of EBV-associated LPD after HSCT.

Successful treatment of Epstein-Barr virus-induced transverse myelitis with ganciclovir and cytomegalovirus hyperimmune globulin following unrelated bone marrow transplantation.

We report a patient who developed Epstein-Barr virus (EBV)-induced transverse myelitis 19 months after unrelated bone marrow transplantation (BMT). The disease was diagnosed by physical examination, serologic determinations, EBV-specific polymerase chain reaction in peripheral blood lymphocytes and cerebrospinal fluid, and characteristic magnetic resonance imaging scan of the spine. The patient was treated with ganciclovir and cytomegalovirus (CMV) hyperimmune globulin. He gradually improved and recovered completely within 4 weeks. This case suggests that ganciclovir and CMV hyperimmune globulin appear to be effective for the treatment of EBV-induced transverse myelitis in immunocompromised patients following BMT.

Analysis of cyclin D1 in de novo and relapsed childhood acute lymphoblastic leukemia.

In a retrospective analysis we investigated 59 children with de novo acute lymphoblastic leukemia (ALL) and 28 patients with relapsed ALL for the expression of cyclin D1 using RT-PCR. In addition, the relationships of cyclin D1 to the retinoblastoma tumor suppressor gene and the Ki-67-expression were analyzed. Cyclin D1-mRNA was detectable in 42 out of 58 patients with de novo ALL (72%) and in 26 out of 28 relapsed patients (93%). The blast cells of the relapsed patients contained significantly higher levels of cyclin D1-mRNA compared with the de novo group (p = 0.0007). The cyclin D1 expression was independent of the proliferative activity of the cells and inversely correlated to the expression of the retinoblastoma tumor suppressor gene (r = -0.27, p = 0.03). Prognostic considerations using Kaplan Meier estimates for the relapse-free interval showed that patients with high cyclin D1- mRNA levels had a poorer prognosis (p = 0.046).

[Selenium in phenylketonuria patients. Effects of sodium selenite administration]. / Selen bei Phenylketonuriepatienten. Effekte einer Natriumselenitsubstitution.

PATIENTS AND METHOD: 17 patients (8 female, 9 male; age 8.2 +/- 3.7 years) with phenylketonuria under phenylalanin restricted diet were investigated prior to and after 3 months of selenium substitution (sodium selenite, 115 micrograms Se/m2 BSA/d). Different parameters in blood were determined: selenium, glutathione peroxidase (Gpx) activity, thyroid hormones, blood cell count, lymphocytic antigen expression, muscle function and -enzymes, cardiac ultrasound. RESULTS: The main significant results of selenium substitution are: increased plasma-selenium, blood cell selenium, plasma-Gpx activity and left ventricular cardiac index as well as decreased plasma thyroxin, free thyroxin, reverse triiodthyronin, total cholesterol, mean erythrocyte and thrombocyte volume and lymphocytic CD2 expression. CONCLUSION: The data indicate metabolic and functional signs of selenium deficiency in patients with phenylketonuria without selenium substitution. We conclude that, despite of lacking clinical symptoms, a selenium supply in phenylketonuria patients under diet is necessary and should be performed with usefull peroral sodium selenite (115 micrograms Se/m2 BSA/d) initially, followed by a dosage between 30 and 60 micrograms Se/m2 BSA/d).

Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy.

Recent studies with very small numbers of patients showed that in some cases of childhood acute lymphoblastic leukemia (ALL), preleukemic cells are detectable on Guthrie cards that were used for newborn screening. We present here the largest series of ALL patients (n=32) in whom Guthrie cards were analyzed for the presence of preleukemic cells. Rearranged immunoglobulin heavy-chain genes were used as a marker for leukemic clones. We combined our set of patients with 17 previously published cases. Preleukemic cells were detected in 31 of all 49 patients (63%). Positive screening cards were not associated with patient's age at diagnosis but were almost always found in patients with hyperdiploidy (10/11; 91%; P=0.04). High birth weight is an established risk factor for childhood ALL. Positive screening cards were strongly associated with low birth weight (P=0.01). In conclusion, the majority of childhood B-precursor ALL arise prior to birth. In the search for causes of childhood leukemia we should concentrate on prenatal factors as well as postnatal factors. Our results suggest that autologous cord bloods could be a poor choice as the source of stem cells for transplantation in leukemia, which may contain preleukemic cells. Pending the development of suitable methods, childhood leukemia is a potentially screenable disease.

T-cell independent production of salivary secretory IgA after hematopoietic stem cell transplantation in children.

This study examined the recovery of secretory IgA (S-IgA) in saliva after hematopoietic stem cell transplantation (HSCT) in 35 children and young people between the ages of 3 and 27 years (mean=13.6), and compared this recovery with that of serum immunologic constituents. Reference values for human salivary S-IgA in saliva were obtained from 77 healthy control subjects between the ages of 7 and 25 years (mean=11.4). In the 35 patients, a nadir of secretory IgA concentrations in saliva (S-IgA) was observed between the 3rd and the 4th month, and a return to normal values 1 year after HSCT. Serum IgA concentrations reached their nadir in the 6th month, and normalized in the 18 months after HSCT. The recovery of T-helper cells (CD4+/3+) was also delayed to beyond 18 months. We found a significant correlation between the reconstitution pattern of S-IgA and that of T-helper lymphocytes, but no correlation was found between the post-transplant evolutions of S-IgA and serum IgA, or between S-IgA and T-helper cells. The recovery of S-IgA was more rapid than that of serum IgA and appeared to be T-helper cell independent.

[Acute lymphatic leukemia with pre-B-cell characteristics]. / Akute lymphatische Leukämie mit Prä-B-Zell-Charakteristik.

54 patients affected with acute lymphatic leukaemia (ALL) in the period from 1978-1984 were examined concerning the frequency of pre-B-ALL in comparison with other immunological subtypes of ALL. For this purpose the following markers were tested: 1. Cytoplasmatic IgM 2. Rosette formation with sheep erythrocytes 3. T-cell specific antigen 4. C-ALL antigen 5. Surface immunoglobulin By means of these markers the patients were divided into 5 immunological subtypes of ALL, viz. 1. Pre-B-ALL (16.6%) 2. O-ALL (37.1%) 3. C-ALL (27.9%) 4. T-ALL (16.6%) 5. B-ALL (1.8%) The immunological subtypes of ALL were compared by means of clinical parameters (age, initial leukocyte number, mediastinal tumor, initial CNS involvement). No difference could be detected between O-ALL, pre-B-ALL and C-ALL, whereas T-ALL showed a distinctly worse prognosis.

[Detection of glucocorticoid receptors in leukemia cells by dexamethasone-induced cytolysis and [3H]-dexamethasone binding]. / Nachweis von Glukokortikoidrezeptoren auf Leukämiezellen durch Dexamethason-induzierte Zytolyse und [3H]-Dexamathasonbindung.

The presence of glucocorticoid receptors on the leukemic cells of 33 patients affected with acute lymphatic leukemia (ALL) and 6 patients affected with acute myeloic leukemia (AML) was investigated by dexamethasone-induced cytolysis and [3H] dexamethasone binding. The tests undertaken proved that after 20 hours of incubation 9 of 26 non-T-non-B-ALL (c-ALL and unclassified ALL) and 2 of AML were lysed with dexamethasone; blood lymphocytes and bone marrow leukocytes of healthy donors, however, were not affected. Non-T-non-B-ALL and AML were able to bind essentially more [3H] dexamethasone than T-ALL. There existed no correlation between dexamethasone binding and dexamethasone-induced cytolysis.

[Lysis of leukemia cells with a monoclonal antibody-cocktail (e.g. VIP-pool) and human complement]. / Untersuchungen zur Lyse von Leukämiezellen mit einem monoklonalen Antikörpercocktail (sog. VIB-pool) und Humankomplement.

During the lysis of leukemic cells with a monoclonal antibody cocktail (the so-called VIB pool) and complement the attempt was made to replace rabbit serum as a complement source by human serum. For identifying the lysis of leukemic cells the complement-dependent in vitro cytotoxicity test was used and for excluding stem cell toxicity the CFU-c test according to PIKE and ROBINSON. In combination with the applied monoclonal antibody pool against B and c-ALL the human complement could be shown to be suitable to produce a lysis in the same manner as rabbit complement. Similarly to the pretested rabbit serum the treatment with the human complement had no impact on stem cell recovery. An optimal cytotoxic activity (95% against ALL blasts of patients, 100% against NALM) could be identified up to an antibody dilution of 1:32 with a volume percentage of 50% of human complement, an incubation temperature of at least 37 degrees C and an incubation time of 30 mins. With proved high reactivity against leukemic cells and lacking impairment of the haemopoietic power of the bone-marrow, this method can be recommended for "purging" protocol with the possibility of using human serum as a source of complement having advantages as far as clinical application is concerned.

[Residual placental blood and bone marrow as sources of hematopoietic stem cells for allogenic stem cell transplantation. Comparative analysis of hematopoietic potential]. / Plazentarestblut (PRB) und Knochenmark (KM) als Quellen hämatopoetischer Stammzellen zur allogenen Stammzelltransplantation. Vergleichende Analysen der hämatopoetischen Potenz.

Umbilical cord blood (UCB) contains a high number of hematopoietic progenitor cells, which can be used for a HLA-identical transplantation. To proof the usefulness of this source of stem cells for transplantation we compared the cellular composition of 19 specimen of bone marrow of healthy donors (BM) and 23 of UCB. We determined the cell number, the frequency of CD34+ progenitor cells and the CFU-GM as well as the T- and B-cell-subsets by means of flow cytometry and in vitro culture methods. The T-cell content was higher in UCB than in BM while the B-cell number was lower. The CD34+ progenitor number per ml was 2.9 fold higher in BM than in UCB, but the frequency and the proliferative capacity of the CFU-GM progenitor cells in the CD34+ population was 8.6 fold higher in UCB than in BM. First clinical trials have demonstrated the usefulness of UCB for allogeneic stem cell transplantation in children. It is possible to harvest 80-100 ml UCB per placenta containing 1.2 x 10(9) nucleated cells with 9.7 x 10(5) (4-15 x 10(5)) CFU-GM.

[Cryopreservation of bone marrow for the autologous transplantation in children]. / Kryokonservierung von Knochenmark zur autologen Transplantation bei Kindern.

In preparing the autologous transplantation of children a method for cryoconservation of bone-marrow was developed by means of investigating the donor's bone-marrow. This method is adapted to our conditions, can easily be practised and is cell-preserving. Quantity and quality of the stored bone-marrow cells were evaluated concerning their proliferation capability by means of CFU-c assays. The highest recovery in CFU-c (78%) and cells (98%) was observed if isolated mononuclear cells with cryoprotective addition of 5% DMSO, 20% of human albumin, and 20% of serum were slowly frozen at a controllable rate, stored in liquid oxygen and thawed very quickly. According to the elaborated method the remission marrow was taken from 15 children affected with malignant diseases for autologous reinfusion. The data gained here confirm the experimental experiences.

Interferon alpha-2a and external beam radiotherapy in the initial management of patients with glioma: a pilot study of the National Biotherapy Study Group.

The National Biotherapy Study Group conducted a phase I/II trial of alpha-interferon (IFN) plus radiation therapy (RT) in glioma patients to confirm the feasibility of combining these two modalities. Patients newly diagnosed gliomasreceived external beam RT as 180 cGy in 33 fractions over six to seven weeks, five days a week, and IFN at a dose of 3 MIU SC Monday, Wednesday and Friday of each week. IFN was increased to 5 MIU after two weeks and was given for up to 16 weeks. Patients were monitored for toxicity and failure-free and overall survival. There were 12 men and seven women with an age range of 24-77, and a median age of 64 years. There were 12 glioblastomas and seven advanced astrocytomas. Complete surgical resection was carried out in two patients, nine had a partial resection, and eight had a biopsy only. Two patients in the latter group deteriorated rapidly and received < 2 weeks of RT/IFN. One patient stopped IFN because of a skin rash, another stopped because of concurrent pneumonia, and one patient was noncompliant. RT and IFN were well-tolerated; 14 of the 19 patients completed the eight weeks of IFN/RT. However, only three patients took IFN for the maximum of 16 weeks. The only grade 4 toxicities noted were increases SGOT in three, increases alk phos in two, and severe fatigue in four patients. The median failure-free survival was two months, median survival was 7.5 months, and four patients survived beyond one year. The longest survivor was 29.1 months, and one patient is still alive after 20.7 months. IFN/RT can be safely co-administered in patients with gliomas. A randomized trial would be needed to establish clinical benefit.