Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine.

Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.

Induction of two distinct natural killer-cell populations, activated T cells and antineoplastic cytokines, by interleukin-2 therapy in children with solid tumors.

Children with poor prognoses regarding solid tumors have benefited from autologous bone marrow transplantation as a treatment modality. Posttransplantation adjuvant interleukin-2 (IL-2) therapy has previously been shown to improve prognosis in adults with neoplastic disease. The improved survival probability has been attributed to IL-2-mediated stimulation of the immune system and its antineoplastic activity. In this study, 10 pediatric patients with solid tumors in complete remission after autologous stem cell transplantation were treated with recombinant IL-2, which was administered in three 5-day cycles of continuous intravenous infusions with a 2-week rest in between cycles. We demonstrated that IL-2 therapy enhanced transplantation-related stimulation of the immune system, on both the cellular and humoral levels. Peripheral blood T and natural killer (NK) cells increased by the factors four and 14, respectively. Activation of the immune system was demonstrated by significantly elevated levels of soluble IL-2 receptor (IL-2R) and increased CD25 surface expression on T lymphocytes. IL-2 also induced significant proliferation of the CD56bright NK-cell subpopulation that appears post-bone marrow transplant (BMT) and is found only in minimal amounts in normal controls. In vivo and in vitro production of tumor cytotoxic cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was significantly stimulated following IL-2 therapy, further indicating IL-2-mediated stimulation of the antineoplastic activity of the immune system.

Treatment of donor T cell-mediated hematopoietic suppression after haploidentical bone marrow transplantation by T cell modulation in a patient with severe combined immunodeficiency.

An 8-month-old male patient with severe combined immunodeficiency syndrome was transplanted with maternal, haploidentical T cell-depleted bone marrow without prior conditioning therapy. Acute graft-versus-host disease developed 2 weeks post bone marrow transplantation (BMT) and was successfully treated with cortisone. After cortisone withdrawal the patient developed myeloid and B cell depression concomitant with T cell activation. For specific T cell modulation, treatment with the T cell receptor (TCR) alpha beta chain-binding MoAb BMA031 was initiated in combination with cyclosporin A. GM-CSF was given to enhance myeloid reconstitution. About 1 year post BMT, B cell and granulocyte counts were within the normal range with stable chimerism in both lineages. B cell proliferation tests were normal and first signs of in vitro immunoglobulin synthesis occurred.

Treatment of progressive or recurrent pediatric malignant supratentorial brain tumors with herpes simplex virus thymidine kinase gene vector-producer cells followed by intravenous ganciclovir administration.

OBJECT: The outcome for children with recurrent malignant brain tumors is poor. The majority of patients die of progressive disease within months of relapse, and other therapeutic options are needed. The goal of this Phase I study was to evaluate the safety of in vivo suicide gene therapy in 12 children with recurrent, malignant, supratentorial brain tumors. METHODS: After optimal repeated tumor resection, multiple injections of murine vector-producing cells shedding murine replication-defective retroviral vectors coding the herpes simplex virus thymidine kinase type 1 (HSV-Tk1) gene were made into the rim of the resection cavity. Fourteen days after the vector-producing cells were injected, ganciclovir was administered for 14 days. The retroviral vector that was used only integrated and expressed HSV-Tk1 in proliferating cells, which are killed after a series of metabolic events lead to cell death. The median age of the patients was 11 years (range 2-15 years). Treated brain tumors included seven malignant gliomas, two ependyminomas, and three primitive neuroectodermal tumors. The patients were treated with one of three escalating dose concentrations of vector-producer cells. Four transient central nervous system adverse effects were considered possibly related to the vector-producing cells. In no child did permanent neurological worsening or ventricular irritation develop, and tests for replication-competent retroviruses yielded negative findings. CONCLUSIONS: This Phase I study demonstrates that in vivo gene therapy in which a replication-defective retroviral vector in murine vector-producing cells is delivered by brain injections can be performed with satisfactory safety in a select group of children with localized supratentorial brain tumors.

Long-term haematopoietic reconstitution and survival without interleukin-7 in a murine syngeneic bone marrow transplantation model.

We created a syngeneic mouse bone marrow transplantation (BMT) model to examine the effect of endogenous interleukin-7 (IL-7) on long-term (>or=140 days) haematopoietic reconstitution and survival after BMT. Wild-type (WT) IL-7(+/+) and knockout (KO) IL-7(-/-) mice were lethally irradiated and transplanted with bone marrow. Survival is best (85.7%) in the group WT grafts transplanted into WT recipients (WT-->WT) with a trend towards poorer survival in the other groups (WT-->KO: 60%, KO-->WT: 50%, KO-->KO: 45.5%, differences statistically not significant). If the recipient is deficient for IL-7-producing cells, T- and B-cell reconstitution remain incomplete. If the graft lacks IL-7-producing cells there is a significant delay in T- and NK-cell reconstitution. Interestingly, in the absence of IL-7, T-cell reconstitution is neither delayed nor incomplete because of an expansion of TCRalphabeta(+)/CD4(-)/CD8(-) double negative T cells. Long-term survival and lymphocyte reconstitution after syngeneic BMT can occur despite the absence of IL-7.

[Lithium in the therapy of hematologic diseases in childhood]. / Lithium in der Therapie hämatologischer Erkrankungen des Kindesalters.

The literature as well as the author's data of this topic are reviewed. Lithium stimulates myelopoesis in vitro, especially via CSF-production. This effect is associated with a modulation of cyclic nucleotides. Lithium stimulates leukemic cell lines too. However, according to epidemiological data lithium does not play an etiological role in leukemia. Furthermore, lithium does not stimulate several tumor cell lines in culture. The effect of cytostatic drugs as well as remission rates are not lessened by lithium. In spite of increased production the functions of the granulocytes are not impaired. Because of the wide range of serum level variation serum level determinations are mandatory. To treat hematological disorders a serum level between 0.7 and 1.2 mmol/l should be achieved. Flame emission photometry and atomic absorption photometry are equivalent methods for determination of the serum level. CNS, thyroid gland, kidney, electrolyte balance, gastrointestinal tract have to be monitored for side effects. Lithium therapy has not be given in pregnancy and to breast feeding mothers. In neutropenia with increased susceptibility to infections lithium therapy including serum level monitoring can be given. Lithium reduces leukopenia and infections following cytotoxic chemotherapy for solid tumors. Current pediatric studies are investigating whether patients with chemotherapy induced neutropenia benefit from this effect in terms of increased and prolonged remission rates.

Receptor-specific inhibition of bone marrow erythropoiesis by recombinant DNA-derived interleukin-2.

Interleukin-2 (IL-2) induces differential secretion of lymphokines by IL-2 receptor (IL-2R)-positive and IL-2R-negative T cells. We studied T cell IL-2R-specific modulation of adult bone marrow erythropoiesis by recombinant IL-2 (rIL-2). I3-2R were induced by CD3 T cell surface determinant-triggering and analyzed by cytofluorography. Bone marrow monocyte and T cell-depleted (NAB-T) target cells were assessed for early erythroid progenitor expression (BFU-E) in the presence of 0 to 10(3) U/mL of rIL-2, rIL-2 had no significant effect on BFU-E expression in the absence of T cells or in the presence of IL-2R-negative T cells. rIL-2 caused a dose-dependent inhibition (75% to 90%) of BFU-E in the presence of autologous IL-2R-positive T cells. The addition of anti-IL2-receptor antibody to cultures containing rIL-2 plus IL-2R-positive T cells entirely abrogated rIL-2-mediated inhibition of BFU-E. In the presence of rIL-2 (10(2) U/mL) production of interferon gamma (IF-gamma) by adult marrow CD3-triggered IL-2R-positive T cells was increased 37- to 125-fold compared to IL-2R-negative T cells. rIF-gamma caused a dose-dependent (88% +/- 17% at 10(3) U/mL) inhibition of adult BFU-E in the presence of CD3-triggered autologous T cells. rIL2-mediated inhibition of adult BFU-E in the presence of IL-2R-positive T cells was partially abrogated (52% +/- 16%) following addition of monospecific IF-gamma antibody. These results demonstrate (a) rIL-2 modulation of adult marrow erythropoiesis is selectively dependent upon both the presence or absence of autologous T cells and the IL-2R status of these T cells; and (b) rIL-2-induced inhibition of adult marrow erythropoiesis is mediated in part by release of IF-gamma from IL-2R-positive T cells.

[Membranes properties of granulocytes in Job's-Syndrome with E. coli-septicemia (author's transl)]. / Membraneigenschaften der granulozyten bei einem Hiob-Syndrom mit E. coli-Sepsis.

Since the age of nine weeks a red haired girl suffered from purulent dermatitis and recurrent, systemic E. coli infections. She had an excessive hyperimmunoglobulinemia E, as well as impaired granulocyte adherence and chemotaxis. Though a sepsis was evident, the granulocytes exhibited a random FITC-Concanavalin A fluorescence. In spite of intensive treatment with various antibiotics and several granulocyte transfusions the child died at the age of 2 years and 11 months. As shown by the FITC-Concanavalin A distribution, the hyperimmunoglobulinemia E may have caused a decreased membrane fluidity causing the impaired adherence and chemotaxis. This could explain the pathophysiology of the Job's Syndrome.

Treatment of acute idiopathic thrombocytopenic purpura of childhood with intravenous immunoglobulin G: comparative efficacy of 7S and 5S preparations.

A prospective randomized study comparing 7S immunoglobulin G to a 5S IgG preparation for therapy of acute idiopathic thrombocytopenic purpura was conducted. The 5S preparation differed from the 7S preparation in that it lacked part of the Fc portion of the IgG molecule. Both groups were given 400 mg IgG/kg body weight over 5 days. All patients had platelet counts less than or equal to 30 X 10(9)/L before IgG infusion. Nine of the 10 patients in the 7S treatment group, compared with three of 10 patients in the 5S treatment group, responded to therapy with an increment in platelet counts greater than 100 X 10(9)/L within 4 days of completing the infusion treatment. Furthermore, the rate of increase of the platelets was more rapid in the 7S group. The results emphasize the efficacy of partial Fc for management of acute idiopathic thrombocytopenic purpura.

[Dose-effect relation in therapy of cytostatic-induced leukopenia with lithium carbonate]. / Dosis-Wirkungs-Beziehung in der Therapie zytostatisch induzierter Leukopenien mit Lithiumcarbonat.

Lithium is used increasingly for attenuation of chemotherapy induced leukopenia in infants and adolescents. Thus, pharmacological data are needed. The purpose of this study was to give recommendations for oral dosage yielding efficient serum levels. Furthermore, two methods of serum level determination were compared. Flame emission photometry using microsamples gave results comparable to atomic absorption photometry. The correlation of oral dose and serum level was investigated in a phase I study. A wide range of serum levels related to an identical oral dose was found. Therefore, frequent serum level determinations are needed to realize efficacy and to avoid toxicity. The correlation between increasing serum levels and leucocyte count was investigated in the first part of a phase II study. No significance was found; i.e. a linear progressing dose-effect relation does not exist. The time course of leukocyte count was analysed for two different serum level groups in the second part of the phase II study. A significant correlation was found in the group with a serum level greater 0,7 mmol/l.

[Effect of lithium on the proliferation of fibroblasts and tumor cell lines in vitro]. / Der Einfluss von Lithium auf die Proliferation von Fibroblasten- und Tumorzellinien in vitro.

The specificity of a proliferation-inducing effect of lithium was investigated. Cell lines of embryonal and adult solid tumors as well as fibroblasts were cultured in lithium-concentrations ranging from 0,5 to 5,0 mmol/l. Neuroblastoma-cell-lines SK-N-SH, SK-N-LO, IMR 32, osteosarcoma-cell-line SAOS 2, melanoma-line IgR 3 and a fibroblast-line were used in this study. Cell proliferation was measured with a 3H-TdR-incorporation-assay and a tumor-stem cell-assay, the fibroblast-proliferation was measured following growth as monolayer respectively. No stimulation of proliferation was observed. The data of this in vitro study are basic for the clinical evaluation of the benefit of lithium in attenuation of chemotherapy induced leukopenia in patients with solid tumors.

[Chemotaxis defect and recurrent E. coli meningitis]. / Chemotaxis-Defekt und rezidivierende E. coli-Meningitiden.

The increased susceptibility to bacterial infections due to a decreased chemotaxis of neutrophil granulocytes was investigated in a premature small for gestational age baby, who such suffered from recurrent E. coli-meningitis. Causes as anatomic lesions, brain abscess, septic granuloumatosus disease and the Chédiak-Higashi-anomaly were ruled out. At the age of 9 and 13 weeks chemotaxis function compared to age-matched and adult controls was diminished. Addition of serum from healthy adults increased directed migration without reaching normal levels. The study of the complement status revealed a diminution of CH50, the alternate pathway components and C6-C9. Thus, decreased chemotaxis is believed to originate from a combined intrinsic-extrinsic defect. No relapses of infection were observed during prophylactic treatment with ascorbate and cotrimoxazole. Intrinsic as well as extrinsic chemotaxis became normal at the age of 7 months. Simultaneously, complement status was found to approach normal levels. The relevance of haematologic-immunologic evaluation in cases of relapsing severe bacterial infections in preterm newborns is discussed.

[Diagnostic-therapeutic trial in infantile genetic agranulocytosis with leucorecruitin]. / Infantile genetische Agranulozytose (IGA). Leukorecrutin im diagnostisch-therapeutischen Versuch.

Subject of this report is the usefulness of a biologically specific mediator of the leucocytosis reaction (leucorecruitin) in the diagnostic evaluation of granulopenic diseases. According to our trial the application of leucorecruitin in healthy humans resulted in a significant increase of granulocytes in circulating blood. The therapeutic effect depends on the neutrophil storage pool. In a diagnostic-therapeutic trial in infantile genetic agranulocytosis with depleted storage pools, leucorecruitin failed to lift the low number of circulating granulocytes to a normal level. No side effects could be observed so far.

[Helper T-cell deficiency in infantile genetic agranulocytosis (M. Kostmann)]. / Helfer-T-Zellmangel bei Infantiler Genetischer Agranulozytose (M. Kostmann).

A female turkish infant with congenital infantile genetic agranulocytosis was examined for its distribution of lymphocyte subclasses. Imbalances of B cells and T-cell subpopulations were found. Compared to the normal control group a relative as well as an absolute deficiency of T helper cells and an increase of the absolute number of T suppressor cells were the most prominent findings. The importance of immunoregulatory T-cell subsets in the pathogenesis of infantile genetic agranulocytosis, disorders of hematopoiesis and granulocyte production, as well as primary and secondary immunodeficiencies is discussed.

[Pre- and retrorenal neuroblastoma without kidney displacement]. / Prä- und retrorenale Neuroblastome ohne Nierenverlagerung.

Pre- and retrorenal growing neuroblastomas did not produce any displacement of the kidney or any obstruction in the intravenous pyelogram. The diagnosis was done by ultrasound-scanning as well as biochemically before surgery. The reported cases show that sonography and determination of the catecholamine metabolites are essential in the diagnostic evaluation of abdominal tumors in childhood.

[Leukocyte count and granulocyte function of children under lithium carbonate therapy treated with cytostatic drugs. A pilot study]. / Leukozytenzahl und Granulozytenfunktion zytostatisch behandelter Kinder unter Lithiumcarbonat. Eine Pilotstudie.

The usefullness of lithium carbonate for attenuation of leukopenia due to chemotherapy was studied in a prospective randomized trial. Chemotherapy for solid tumors was applied in a paired manner 24 times without and 24 times with lithiumcarbonate. Simultaneously, nucleotid-dependent functions of granulocytes were examined. An attenuation of leukopenia by lithium was observed. Toxic side effects within the therapeutic range of the serum level (0.3-1.4 mol/l) were not detected. Lithium did not impair adherence, chemotaxis and degranulation of neutrophile granulocytes. The influences of lithium on stem-cell maturation and granulocyte function are discussed.

Induction of a CD3+/CD56+ lymphocyte population following gene therapy with transgenic IL-2 secreting fibroblasts in a child with peripheral neuroectodermal malignancy.

BACKGROUND: Adjuvant interleukin-2 (IL-2) therapy after stem cell transplantation can improve the prognosis of patients with Ewing tumors. This has been attributed to stimulation of the immune system and its antineoplastic activity, thus eliminating minimal residual disease. As the side effects of systemic IL-2 limit the dosage, attempts have been made to locally augment the concentration of IL-2 in the proximity of the tumor. To achieve this, fibroblasts and/or tumor cells can be genetically modified to secrete IL-2 and then be injected to generate tumor immunogen. PROCEDURE: In a preliminary clinical trial we assessed whether the administration of transgenic IL-2-secreting fibroblasts was feasible without major toxicity and whether it had any effect regarding the activation of the immune system. We treated an 11-year-old boy with a peripheral neuroectodermal tumor of the left neck in fourth relapse, who was refractory to all available therapy. We transfected fibroblasts of the patient with an IL-2 gene expression vector using a cationic liposome reagent. In 51Cr cytotoxicity assays we obtained lysis of this patient's tumor cells by IL-2-stimulated mononuclear cells (MNCs). Under CT-guidance we intratumorally injected IL-2 transgenic autologous fibroblasts. RESULTS: We observed no local or systemic toxicity. In addition, we found a rise in the CD3+CD56+ lymphocyte population, previously described as cytokine-induced killer cells. No other hematological parameter changed significantly. CONCLUSIONS: Our data suggest that the intratumoral injection of transgenic IL-2-secreting fibroblasts is feasible without major toxicity and may lead to an increase in CD3+CD56+ cells.

Granulocyte-macrophage-colony stimulating factor for prevention of neutropenia and infections in children and adolescents with solid tumors. Results of a prospective randomized study.

BACKGROUND: Chemotherapy is an essential modality of curative strategies in pediatric oncology. Dose and dose intensity are, above all, restricted by the myelosuppressive effects of cytotoxic drugs. Neutropenia constitutes an important risk of morbidity and mortality. Granulocyte-macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor that increases the number of circulating neutrophils as demonstrated in adults. METHODS: A prospective randomized study of the effects of GM-CSF was performed with 11 patients who were treated for solid tumors and received GM-CSF for 2 weeks starting 48 hours after completion of chemotherapy. Forty-two intraindividual identical chemotherapy-courses with and 42 without GM-CSF were compared. The monitoring program included the surveillance of the hematological reconstitution and the number and duration of infectious episodes. RESULTS: The average nadir of the absolute neutrophil count (ANC) with GM-CSF was higher than without GM-CSF. The average number of days with an ANC below 500/microliters was significantly reduced by GM-CSF. Fewer infectious episodes were observed among those who received GM-CSF therapy. Erythropoiesis was not significantly influenced by GM-CSF, whereas patients with GM-CSF therapy showed a longer thrombocytopenia without requiring more platelet transfusions. Rashes developed in two patients. CONCLUSIONS: In children and adolescents undergoing intensive chemotherapy for solid tumors, GM-CSF reduces neutropenia and infectious episodes at the cost of mild thrombocytopenia.

Circulating CD34+ cell counts as predictive parameter for the efficacy of peripheral stem cell apheresis in Ewing tumor patients.

The parameters used by different centers for starting peripheral stem cell apheresis following mobilization with chemotherapy and G-CSF are diverse. We retrospectively analysed 62 leukaphereses performed on eleven patients with Ewing Tumors and one patient with Synovial Sarcoma, all after treatment with standardized chemotherapy (according to the EICESS protocol) and subsequent administration of G-CSF. Blood samples, drawn before the apheresis and samples of the apheresis products were analysed by flow cytometry for their content of CD34+ cells, furthermore, the peripheral leukocyte count was determined. Our aim was to define a parameter that reliably predicts the efficacy of leukapheresis in these patients. We found a significant correlation (r = 0.85; p < 0.0001) between the absolute CD34 positive cell count prior to leukapheresis and the yield of CD34+ cells/kg body weight (bw) in the apheresis product. No correlation was observed between the leukocyte count prior to apheresis and CD34+ cells/kg bw in the apheresis product (r = 0.14; p < 0.28). In addition, we found no correlation between the number of CFU-GM in the apheresis product and the CD34+ cells/kg bw (r = 0.22; p < 0.35). Our data indicate that the peripheral CD34 positive cell count prior to leukapheresis is the best parameter for predicting the efficacy of peripheral stem cell apheresis in Ewing Tumor patients after treatment with standardized chemotherapy.