B. Kornhuber

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. Ultra-structural 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.

Modulatory Effects of Human Cytomegalovirus Infection on Malignant Properties of Cancer Cells

Although there is no definitive evidence of the association of human cytomegalovirus (HCMV) infection with human cancers, the oncogenic potential of HCMV has been well established by in vitro studies demonstrating the ability of UV-irradiated or infectious virus to transform a variety of cells. After prolonged passaging the transformed cell type was maintained while HCMV DNA sequences were no more detectable. Three morphological transforming regions (mtr) of HCMV have been identified. The effects of HCMV on cellular functions which may be associated with the malignant phenotype include the expression of oncogenes and transcriptional activation of growth factors and interleukin synthesis. In infected cells, HCMV induces cytoskeletal alterations and changes in expression of cell surface receptors for extracellular matrix proteins which could result in increased motility and dissemination of cancer cells. Several human neuroblastoma cell lines undergo maturation in different neural crest derived cell types upon treatment with oncogenic potential agents, i.e. retinoic acid. The persistent HCMV infection of neuroblastoma cells (> 1 year) is accompanied by the increased expression of oncoproteins (i.e. N-myc) and decreased expression of tyrosine hydroxylase and dopamine-beta-hydroxylase. The activation of the cellular metabolism is due to HCMV binding to cellular receptors (prior to virus gene expression) and to the activity of HCMV immediate early (IE) gene products. IE proteins act directly as transcriptional activators or their activity is mediated by a variety of cellular transcription factors. HCMV infection may result in activation of promoters of cellular genes coding for cytokines, replication enzymes, proto-oncogenes and viral promoters. Recently it has been demonstrated that HCMV IE proteins block apoptosis probably by suppressing the ability of the antioncogene p53 to activate a reporter gene. The interactions of HCMV with tumor suppressor proteins such as p53 or retinoblastoma (pRb) susceptibility protein are reminiscent of those mediated by the oncoproteins of DNA tumor viruses. The acquisition of a fully malignant phenotype by normal cells is thought to require several mutations in a number of cellular genes. In this connection, HCMV may play the role of a nonobligate either direct or indirect cofactor for tumor genesis, e.g. by blocking apoptosis, which may be an essential requirement for tumor progression. Due to the stimulation of growth factors and/or inhibition of antioncogenes by its gene products, HCMV may modulate the malignant potential for tumor cells.

Immune tolerance therapy in paediatric haemophiliacs with factor VIII inhibitors: 14 years follow-up.

We report our clinical experience in the immune tolerance (IT) therapy of 21 paediatric haemophiliacs with FVIII inhibitor: high responders (16HR) received initially FVIII twice daily at a dosage of 50–300 U/kg/day, 11/16 received a concomitant treatment with activated prothrombin complex concentrate (100–200 U/kg/day). Low responders (five LR) received 20–100 FVIII U/kg every second or third day. Inhibitor elimination was achieved in 19/21 patients in a median time of 4 months in HR and 1.5 months in LR. The outcome and length of time needed to induce IT was significantly correlated with FVIII exposure between the first inhibitor detection and onset of IT therapy and to interruption of IT therapy. For a rapid elimination of FVIII inhibitors it is important to start continuous administration of high‐dose FVIII (≥ 100 FVIII U/kg/day) before repeated exposure to FVIII, in order to prevent rebooster effects, prolongation of elimination time, and to reduce expense.

Epidemiology of Inhibitors in Haemophilia A

One of the most serious complications of the treatment of haemophilia A is the development of inhibitors. Former studies mostly considered the prevalence of inhibitor development, thus underestimating its true risk. Prevalences ranged widely (7–18%) probably due to the populations studied and the study design. Recent prospective previously untreated patients (PUP) studies were more comparable because of similar study designs. Eight PUP studies regarding the incidence of factor VIII inhibitors were analyzed: The inhibitor incidences (Independent of severity of haemophilia) ranged from 18.4 to 28%. Evaluating only severe haemophiliacs (factor VIII<2%) significantly higher incidences were found. After 9–36 exposure days (as medians inhibitor development occurred at 0.8‐3.3 years of age (as medians).

Preliminary experiences with triple therapy including nelfinavir and two reverse transcriptase inhibitors in previously untreated Hiv-infected children

OBJECTIVE In an intent-to-treat study increase in CD4 cell count, reduction of viral load, clinical benefit and adverse reactions were examined in HIV-infected previously treatment-naive children taking triple therapy. METHODS sixteen HIV-infected children in category A or B on antiretroviral triple therapy were followed-up for a period of 12 months. In group I eight patients received zidovudine, lamivudine and nelfinavir; in group II eight patients received stavudine, didanosine and nelfinavir. Viral load and CD4 cell count were measured every 4-8 weeks. Plasma nelfinavir levels were assessed once in all patients at baseline and monitored in patients with increasing viral load. RESULTS No significant differences were observed between treatment groups in terms of CD4 cell counts and viral load. A median viral load reduction of 2.8 log10 (range, 1.4-4.2 log10) was achieved over a period of 12 months in both groups. Viral load < 500 copies/ml was found in 69% of patients and viral load < 50 copies/ml in 44% of patients after 12 months. Median CD4 cell count increased from 656 x 10(6) to 850 x 10(6) cells/l after 3 months and was maintained at 813 x 10(6) cells/l after 12 months of treatment. Main side-effects were diarrhoea, rash and hyperlipidaemia. Except for application problems, both regimens were well tolerated. Appropriate formula and individual counselling must be performed during the first weeks of treatment in order to achieve good compliance in paediatric patients. CONCLUSION Triple antiretroviral therapy shows a stronger and more sustained reduction of viral load in HIV-infected children compared with studies combining two nucleoside analogues.

Radiological and orthopedic score in pediatric hemophilic patients with early and late prophylaxis

Abstract In order to evaluate joint alteration, 17 patients with hemophilia A and B were investigated over a period of 4 years (1993–1997). Patients were subdivided into two groups, according to therapy regimens. In group 1 (n=10) prophylactic treatment was initiated until the third year of life. In group 2 (n=7) patients received prophylactic treatment at the age of 5 years and above. To assess alterations in knee, elbow, and ankle joints, the radiological score and the physical examination score of the Orthopedic Advisory Committee of the World Federation of Hemophilia were used. The sum of the scores of these six joints was defined as the patient-dependent score. Patients of group 1 (median age at the end of observation: 10 years) reached a median radiological score of 1.0 (range: 0–13) and an orthopedic score of 0 (range: 0–4), whereas patients of group 2 (median age: 14 years) had a radiological score of 20 (range: 2–47) and an orthopedic score of 8 (range: 0–12), which shows a significant difference (p<0.01). In both treatment groups a manifestation or progression of arthropathic alteration was seen in those children who had repeated joint bleeding (>5) prior to the onset of prophylactic treatment (r=0.90, p>0.01). Altogether, two of 60 joints in group 1 and 12 of 42 joints in group 2 had a radiological score ≥4. Elbow joints were more often affected than knee and ankle joints. In conclusion, the number of joint bleedings before prophylactic treatment was started influenced the progression of arthropathy even in patients with early onset of prophylaxis. The aim of treatment in severe hemophilia should be early prophylaxis before repeated joint bleeding occurs in order to prevent osteoarthropathic alteration.

Long-term productive human cytomegalovirus infection of a human neuroblastoma cell line

Human neuroblastoma cell line UKF‐NB‐4 persistently infected with human cytomegalovirus (HCMV) strain AD169 was established to study the effects of long‐term HCMV infection on virus production and phenotypic characteristics of tumour cells. The cells designated UKF‐NB‐4AD169 were subcultured (80 subcultures) over a period of more than 2 years after initiation of infection. UKF‐NB‐4AD169 cells continued to produce infectious virus in successive passages, with a titre ranging from 9 × 103 to 1 × 105 and from 2 × 101 to 2 × 102 plaque‐forming units per 106 cells and 1 ml culture medium, respectively; 10–20% of the cells produced HCMV‐specific antigens, while 6–13% produced infectious virus progeny. The number of HCMV‐specific DNA copies ranged from 9 × 104 to 9 × 106 per 106 cells. Transmission electron microscopy confirmed the productive nature of HCMV infection. UKF‐NB‐4AD169 cultures proliferated, with population doubling time ranging from 24.5 to 26.6 hr (19.5 to 20.3 hr for UKF‐NB‐4) and cell viability from 79% to 85% (91–96% for UKF‐NB‐4). Significantly lower amounts of tyrosine hydroxylase and decreased activity for dopamine‐β‐hydroxylase than in uninfected cells were observed in UKF‐NB‐4AD169 cells. However, the expression of N‐myc oncoprotein was significantly increased in persistently infected cultures. Our results show that long‐term productive HCMV infection of UKF‐NB‐4 cell line is associated with the modulation of phenotypic properties, which may be related to the biological behaviour of neuroblastoma cells.

In vitro differentiation of human neuroblastoma cells induced by sodium phenylacetate.

Sodium phenylacetate (NaPA) at concentrations ranging from 2 to 6 mM stimulated morphological differentiation of two human neuroblastoma cell lines IMR-32 and UKF-NB-3. These concentrations inhibited growth and DNA synthesis of the cells in a dose dependent manner without significant effect on cell viability. The differentiated cells showed pseudoganglia formation and extension of cellular processes. The morphological differentiation in both cell lines was accompanied by decreased expression of N-myc oncoprotein. These results suggest that NaPA at concentrations, which have been achieved in humans with no significant adverse effects, promotes differentiation of cultured human neuroblastoma cells in association with the reduced expression of the malignant phenotype.

In vitro inhibition of human cytomegalovirus replication by desferrioxamine

Desferrioxamine (DFO) is commonly used in therapy as a chelator of ferric ion in disorders of iron overload. We found that DFO inhibits human cytomegalovirus (HCMV) replication in infected cultures of human foreskin fibroblasts (HFF) at concentrations that have been achieved in humans with no significant adverse effects. The concentrations of DFO required for 50 and 90% reduction in the production of a HCMV-late antigen ranged for several HCMV strains from 3.1 to 4.9 microM and from 14.2 to 17.3 microM, respectively. DFO concentration of 60 microM had no significant effect on the viability of HFF cells. Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of Fe3+.