Acute encephalopathy as a primary manifestation of haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) is characterized anatomically by an infiltration of multiple tissues with lymphocytes and haemophagocytic histiocytes. First symptoms are usually hepatosplenomegaly, pancytopenia, and intractable fever. Up to 73% of those with HLH develop CNS involvement during the disease course. The peculiarity of the two patients presented here, a 20-month-old Italian female and a 4-year-old Moroccan female, is that the initial presenting neurological symptoms mimicked an encephalitis, anticipating the typical systemic symptoms by 1 and 4 months. They developed progressive encephalopathy accompanied by status epilepticus, one child developed a secondary hydrocephalus. In both children it was not possible to detect an underlying infection or malignant disease and there were no other cases in the family that suggested a familial form of HLH. Diagnosis and initiation of treatment was delayed because of the initial encephalopathic clinical picture and the late onset of the typical systemic features. As early diagnosis allows better therapeutical approaches, haemophagocytic lymphohistiocytosis should be considered in children with persistent or progressive findings of encephalopathy, especially in the absence of identification of a plausible pathogen.

Treatment of drug-resistant human neuroblastoma cells with cyclodextrin inclusion complexes of aphidicolin.

Treatment failure in most neuroblastoma (NB) patients is related to primary and/or acquired resistance to conventional chemotherapeutic agents. Aphidicolin (APH), a tetracyclic diterpene, exhibits specific cytotoxic action against NB cells. The purpose of this study was to compare antitumoral efficacy of APH in parental NB cell lines and cell subclones that exhibit drug resistance to vincristine (VCR), doxorubicin (DOX) and cisplatin. Due to poor solubility of APH in water, gamma-cyclodextrin (gamma-CD) inclusion complexes of APH were used for systemic treatment of xenotransplanted parental and VCR-resistant UKF-NB-3 tumours. APH and its gamma-CD inclusion complexes inhibited growth of parental and drug-resistant NB cells at equimolar doses in vitro. Growth of VCR-sensitive and -resistant NB tumors was inhibited at equal doses in a dose-dependent fashion in vivo. These results indicate that the specific cytotoxic activity of APH against NB cells in vitro and in vivo is independent of cellular mechanisms facilitating drug resistance to conventional chemotherapeutic drugs. Hence, taking into account our previous findings that APH acts synergistically with VCR and DOX, APH might be an additive tool for the therapy of NB and is suitable for evaluation in clinical studies of NB treatment protocols.

Therapeutic efficacy of G207 in a novel peripheral nerve sheath tumor model.

Nerve involvement poses a significant obstacle for the management of peripheral nervous system tumors, and nerve injury provides a frequent source of postoperative morbidity. The lack of suitable animal models for peripheral nerve tumors has impeded the development of alternative nerve-sparing therapies. To evaluate the effect of a multimutated replication-competent herpes simplex virus (G207) on the growth of peripheral nerve tumors and on nerve function, we developed a novel peripheral nerve sheath tumor model. Human neuroblastoma-derived cells injected into murine sciatic nerve consistently caused tumor development within the nerve sheath after 2 weeks followed by increasingly severe impairment of nerve function. Tumor treatment by a single intratumoral injection of G207 resulted in significant reduction of functional impairment, inhibition of tumor growth and prolonged survival. Direct injection of G207 viral particles into the healthy nerve sheath caused no obvious neurologic sequelae, whereas injections of wild-type virus resulted in uniform lethality. The results indicate that viral therapy might be considered as a safe alternative to surgical removal of tumors with peripheral nerve involvement.

Cytotoxicity of aphidicolin and its derivatives against neuroblastoma cells in vitro: synergism with doxorubicin and vincristine.

Disseminated neuroblastoma diseases are still indicated by a poor outcome despite treatment regimens including radiation therapy and high-dose chemotherapy with stem cell rescue. Therefore, new substances and treatment regimens are of interest. Aphidicolin (APH), a tetracyclic diterpene antibiotic produced by Cephalosporium aphidicola, has a specific toxicity for neuroblastoma cells. Furthermore, it was shown to enhance the effects of X-ray radiation and chemotherapy on malignant cells. To find new substances, 20 APH derivatives were tested for their anti-neuroblastoma efficacy in vitro in UKF-NB-2 cells. Five derivatives had antitumoral activity in neuroblastoma cells. A relationship between the structure and the antitumoral efficacy showed that the hydroxyl groups at C-3 and C-18 are essential for the antitumoral effects. Furthermore, antitumoral effects of APH in combination with doxorubicin and vincristine, both part of commonly used treatment regimens for disseminated neuroblastoma diseases, were tested in the neuroblastoma cell line UKF-NB-2. APH was found to act synergistically with vincristine and synergistically to additive with doxorubicin depending on the molecular ratio of the substances in combination. This may offer the chance to use APH and its derivatives as additional tools in the treatment of neuroblastomas.

Lyme borreliosis mimicking central nervous system malignancy: the diagnostic pitfall of cerebrospinal fluid cytology.

We report two children with acute loss of neurological functions and signs of an increased intracranial pressure. Imaging techniques ruled out space occupying lesions, whereas CSF cytology indicated CNS involvement of a non-Hodgkin lymphoma in the form of abnormal lymphocytic pleocytosis with malignancy criteria fulfilling lymphoid cells. CSF protein electrophoresis and Borrelia burgdorferi serology revealed neuroborreliosis which was successfully treated with antibiotic therapy. The malignancy mimicking cytology is based on a blastoid transformation of B- and T-lymphocytes due to the antigenic stimulus of B. burgdorferi infection. Lymphoid cells in the CSF of a patient with acute or chronic neurological symptoms raise the differential diagnosis of inflammatory etiology versus CNS lymphoma. Monomorphism and higher quantity of the lymphoid cells point to CNS lymphoma. A lower quantity and polyclonal pattern of lymphoid cells associated with an elevated protein fraction caused by intrathecal immunoglobulin synthesis suggest an inflammatory etiology.

Valproic acid for the treatment of pediatric malignant glioma.

Despite surgery and adjuvant cytotoxic therapy anaplastic astrocytoma, glioblastoma and diffuse intrinsic brain stem glioma continue to have dismal prognosis. Differentiation induction is a new approach taking into account that malignant glioma cells share many features with immature glial progenitor cells that are capable of terminal differentiation. The concept of differentiation therapy is currently evaluated for several pediatric malignancies with or without multimodal standard therapy. Valproic acid (VPA) is a branched chain fatty acid that is able to inhibit proliferation of neuroectodermal cells and to induce these cells along neuronal or glial lineage. Preclinical studies have shown that VPA inhibits growth of human and rodent glial tumor cells in vitro and induces a distinct mature glial phenotype. In addition, growth of human neuroblastoma cells is inhibited in vitro and in vivo and exhibits marked evidence of differentiation. Treatment of neuroblastoma and glioma cells with VPA was accompanied by changes of surface molecule expression that enhance immunogenicity and reduce their capability to metastasize. The antitumoral effects observed in preclinical studies were reached at concentrations that are readily achieved in patients treated with VPA for epilepsy. Epilepsy patients receiving VPA have significantly enhanced hemoglobin F levels, supporting the hypothesis that nontoxic levels of VPA can induce cellular differentiation. Broad clinical experience with VPA and its low toxicity encourage the evaluation of VPA in patients that have been submitted to postoperative combined chemo- and radiotherapy for pediatric malignant glioma.

Human cytomegalovirus infection decreases expression of thrombospondin-1 independent of the tumor suppressor protein p53.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis. It has been shown that promoter sequences of the TSP-1 gene can be transactivated by the wild-type tumor suppressor protein p53. As human cytomegalovirus (HCMV) infection inactivates wild-type p53 of various cell types, we investigated whether HCMV infection is associated with reduced TSP-1 production. We found, in conjunction with accumulated p53, that TSP-1 mRNA and protein expression was significantly reduced in HCMV-infected cultured human fibroblasts. To determine whether the observed TSP-1 suppression depends on p53 inactivation, the p53-defective astrocytoma cell line U373MG was infected with HCMV. In these cells TSP-1 expression was also significantly reduced by HCMV infection whereas expression of the p53 mutant variant remained unaltered. In both cell lines the decreased expression of TSP-1 mRNA occurred early after infection (4 hours), indicating that HCMV inhibits TSP-1 transcription during the immediate-early phase of infection before HCMV DNA replication. Inhibition of HCMV DNA synthesis by ganciclovir did not influence TSP-1 reduction whereas the antisense oligonucleotide ISIS 2922, complementary to HCMV immediate-early mRNA, completely prevented the HCMV-mediated TSP-1 suppression. These findings strongly suggest a novel role for HCMV in the modulation of angiogenesis due to p53-independent down-regulation of TSP-1 expression.

Aphidicolin glycinate inhibits human neuroblastoma cell growth in vivo.

Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the viability of normal human cells and a variety of other tumor entities. We evaluated the antitumoral effects of the water soluble ester aphidicolin glycinate (AphiG) on established human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. Furthermore, we explored the efficacy of direct intraneoplastic and systemic delivery of AphiG. Systemic administration of AphiG (60 mg/kg intraperitoneally, twice per day on 10 consecutive days) significantly suppressed tumor growth but was not able to induce any cures. In contrast, intratumoral AphiG injections (60 or 40 mg/kg/twice a day for 4 days) induced complete tumor regression. Two weeks after the end of treatment no tumor cells were microscopically detectable. Animals were free of tumor for more than 90 days. Histologic examination of inner organs and bone marrow did not reveal any apparent toxic effects of AphiG. These data strongly indicate that AphiG deserves further evaluation as a specific treatment for neuroblastoma.

Azidothymidine resistance of H9 human T-cell lymphoma cells is associated with decreased sensitivity to antitumor agents and inhibition of apoptosis.

Biology of HIV-1 associated neoplasias is modulated by viral and host factors. In addition the development of tumors and their response to therapy may be further influenced by long-term treatment of HIV-1 patients with nucleoside analogs such as AZT (3'-azido-3'deoxythymidine), ddI (2',3'-dideoxyinosine), ddC (2',3'-dideoxycytidine), d4T (2',3'-didehydro-2'3'-dideoxythymidine), and 3TC [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] alone or in combination. As these compounds can trigger mechanisms involved in chemoresistance, we tested whether prolonged in vitro treatment of H9 cells (T-cell lymphoma) with AZT alters sensitivity of lymphoma cells to antitumor agents used for AIDS-associated malignancies. H9 cells grown for more than two years in medium containing 250 microM AZT developed resistance to the toxic effects of AZT while retaining sensitivity for other nucleoside analogs including ddC or cytosine arabinoside (ARA-C). These cells designated H9rAZT250 were 2 to 10-fold less sensitive to the toxic effects of antitumor agents, including cisplatin (CDDP), vincristine (VCR), doxorubicin (DOX) and etoposide (VP-16), when compared with parental H9 cells. The resistance of H9rAZT250 cells to antitumor agents was associated with inhibition of apoptosis as demonstrated by ultrastructural investigations and DNA-fragmentation assay (ELISA). The expression of the antiapoptotic gene bcl-2 was increased in H9rAZT250 cells while expression of other genes involved in the regulation of apoptosis such as c-myc, p53 and Fas was not changed. These results demonstrate that prolonged in vitro treatment of H9 lymphoma cells with AZT results in the development of resistance to antitumor agents in association with inhibition of apoptosis and increased expression of bcl-2. Therefore AZT long-term treatment of some HIV-1 patients with malignancies may have affected behavior of tumor cells including response to therapy.

Survival of very young children with medulloblastoma (primitive neuroectodermal tumor of the posterior fossa) treated with craniospinal irradiation.

PURPOSE: Very young children with medulloblastoma are considered to have a worse prognosis than older children. As radiotherapy remains an important part of the treatment, the adverse prognosis could be due to inadequate radiation treatment rather than biological factors. We analyzed the published literature to examine the impact of radiotherapy on survival in this group. METHODS AND MATERIALS: A Medline search was performed and we reviewed studies of treatment of medulloblastoma where radiotherapy was delivered using megavoltage equipment and the minimum follow-up allowed the calculation of 5-year survival rates. RESULTS: Thirty-nine studies were published between 1979 and 1996 with a treatment including craniospinal irradiation and boost to the posterior fossa. Eleven studies comprising 1366 patients analyzed survival by age at diagnosis. Eight of 11 studies showed a worse 5-year survival for the younger patient group which reached statistical significance in two. There is also a suggestion of a higher proportion of children with metastatic disease at presentation in the very young age group. The usual policy in younger children was to give a lower dose of radiotherapy to the craniospinal axis (CSA) and posterior fossa (PF) with reduction of dose in the range of 15 to 25% compared to standard treatment. As dose reduction to the posterior fossa is associated with worse survival and local recurrence is the predominant site of failure, the major determinant of worse survival in very young children with medulloblastoma may be suboptimal radiotherapy. Protocols including postoperative chemotherapy with delayed, omitted, or only local tumor irradiation do not reach survival rates of protocols with standard radiotherapy, also suggesting a continued importance for irradiation. CONCLUSION: Very young children with medulloblastoma have a worse prognosis than older children. Inadequate radiation dose and technique to the primary tumor region may be a major contributing factor. Current chemotherapeutic regimes alone are not sufficient to compensate for reduced radiation doses and volumes.

Persistent human cytomegalovirus infection induces drug resistance and alteration of programmed cell death in human neuroblastoma cells.

Infection with human cytomegalovirus (HCMV) is a common and generally asymptomatic affection in childhood. Its role in neuroblastoma (NB) patients has not yet been elucidated. As evidence grows that HCMV interacts with apoptotic signaling due to the interaction of HCMV gene products with cellular proteins of apoptotic pathways, we used human NB cell line UKF-NB-2 persistently infected with HCMV strain AD169 to study the effects of long-term HCMV infection on programmed cell death of neuroectodermal tumor cells. The cells designated UKF-NB-2AD169 continued to produce infectious virus in successive subcultures over a period of more than 1 year. Up to 20% of cells expressed viral genes or produced infectious virus after initiation of infection. UKF-NB-2AD169 cells were significantly less sensitive to the cytotoxic agents cisplatinum and etoposide than parental (noninfected) UKF-NB-2 cells. These effects were associated with decreased ability of UKF-NB-2AD169 cells to undergo apoptosis and continuous viral replication. UKF-NB-2AD169 cells showed increased levels of antiapoptosis Bcl-2 protein (up to 12-fold), whereas expression of p53 and c-myc was not changed. Treatment of UKF-NB-2AD169 cells with ganciclovir, abolishing virus production, reestablished sensitivity to chemotherapy, lowered Bcl-2 expression, and facilitated inducibility of apoptosis to the level of the parental cell line. The results demonstrate that persistent HCMV infection confers resistance to cytotoxic agents on neuroectodermal tumor cells and protects from apoptosis, probably due to increased levels of Bcl-2 protein. Hence, it is conceivable that HCMV infection before or during tumorigenesis may contribute in some NB patients to failure of therapy.

Sodium valproate inhibits in vivo growth of human neuroblastoma cells.

Sodium valproate (VPA) belongs to the group of simple branched-chain fatty acids and due its anticonvulsive activity is broadly applied in the treatment of epilepsy. We previously showed that VPA is able to induce cellular differentiation, to enhance immunogenicity and to inhibit proliferation of human neuroblastoma (NB) cells in vitro. Furthermore, we demonstrated that VPA inhibits proliferation, enhances neural cell adhesion molecule expression and decreases CD44 expression of human and rat glioma cells in vitro. In the present study we investigated the antitumoral effects of VPA on established human NB xenografts from UKF-NB-3 human NB cells in athymic (nude) mice. When the animals developed s.c. tumors of about 100 mm3 volume they were treated with 400 or 200 mg/kg/day VPA i.p. At the end of the treatment period (40 days) tumor volumes in animals treated with 400 and 200 mg/kg VPA were about 4- (p < 0.0001) and 2-fold (p < 0.0005) smaller than in the saline-treated control group, respectively. Histological examination of the remnant tumors of treated animals revealed induction of differentiation by induction of stroma-rich tumors and nodules that contained elongated NB cells. Pyknotic nuclei and apoptotic bodies indicated induction of apoptosis. We conclude that VPA is able to abrogate NB growth in vivo and may therefore be useful in the treatment of NB patients.

Antitumor activity of sodium valproate in cultures of human neuroblastoma cells.

Valproic acid (VPA) is a simple branched-chain fatty acid that has anticonvulsant activity and is widely used in the treatment of epilepsy. VPA was found to effect growth and differentiation of human neuroblastoma (NB) cells in vitro at concentrations that have been achieved in humans with no significant adverse effects. Treatment of UKF-NB-2 and UKF-NB-3 NB cell lines with VPA at concentrations ranging from 0.5 to 2 mM resulted in neuronal morphological differentiation characterized by extension of cellular processes without significant effects on cell viability. Ultrastructural features of VPA-treated cells were consistent with the neuronal type of differentiation. VPA treatment of NB cells was associated with decreased expression of N-myc oncoprotein and increased expression of neural cell adhesion molecule in their membrane. Treatment of NB cells with 0.5 mM VPA increased their sensitivity to lymphokine-activated killer lysis. The results indicate that VPA, at non-toxic pharmacological concentrations, arrests the growth, induces differentiation and increases immunogenicity of NB cells through non-toxic mechanisms.

Aphidicolin induces myogenic differentiation in the human rhabdomyosarcoma cell line KFR.

The effects of aphidicolin, a specific inhibitor of DNA polymerase alpha, on cell growth, DNA synthesis and myogenic differentiation in the human alveolar rhabdomyosarcoma cell line KFR were studied. The treatment with aphidicolin at 5 x 10(-6) M concentration, which completely inhibited DNA synthesis and cell growth, induced morphological differentiation of small mononuclear cells to elongated, multinucleated (myotube-like) structures. The morphological differentiation was accompanied by the expression of skeletal muscle myosin; about 30% myosin-positive cells were observed after 14 days of treatment, compared to 2.3% in untreated cultures. The results showed that aphidicolin induces differentiation of human rhabdomyosarcoma cells and that multinucleated myotube-like elements may develop simply by cell fusion without cell division and DNA synthesis.

A rapid method for the determination of human CEA/mouse anti-CEA immune complexes in patients undergoing immunoscintigraphy.

We report here on a new and quick procedure to isolate human (hu) CEA mouse anti CEA immune complexes of sera from patients admitted for immunoscintigraphy with radiolabeled monoclonal anti CEA antibody. This method employs rabbit anti mouse IgG immune affinity chromatography together with a commercial CEA-IRMA kit for the specific isolation of immune complexes. By applying this procedure we were able to show that immune complex formation increased in parallel to CEA serum concentration. The formation of immune complexes did not significantly affect tumor detection by immunoscintigraphy.

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase.

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.

Reduced methyl group acceptance of 1-beta-D-arabinofuranosylcytosine-containing DNA polymers.

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.