Effects of 18-kDa translocator protein knockdown on gene expression of glutamate receptors, transporters, and metabolism, and on cell viability affected by glutamate.

OBJECTIVE: Previously, several important roles for glutamate have been described for the biology of primary brain tumors. For example, glutamate has been suggested to promote glioma cell proliferation by the activation of the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) subtype of glutamate receptors. In the present study, we determined the potential regulatory roles of the 18-kDa translocator protein (TSPO) in the glutamatergic system in relation to cell death of brain tumor cells through knockdown of the TSPO by genetic manipulation. MATERIALS AND METHODS: With microarray analysis and validation of gene expression of particular genes using real-time PCR, we found effects because of small inhibitory RNA knockdown of the TSPO in human U118MG glioblastoma cells on gene expression of glutamate receptors, glutamate transporters, and enzymes for glutamate metabolism. We also applied antisense RNA to silence TSPO in rat C6 glioblastoma cells and assayed the effects on DNA fragmentation, indicative of apoptosis, because of glutamate exposure. RESULTS: In particular, the effects of TSPO silencing in human U118MG cells related to glutamate metabolism indicate a net effect of a reduction in glutamate levels, which may potentially protect the cells in question from cell death. The TSPO knockdown in C6 cells showed that TSPO is required for the induction of apoptosis because of glutamate exposure. CONCLUSION: These findings show that interactions between the TSPO and the glutamatergic system may play a role in tumor development of glioblastoma cells. This may also have implications for our understanding of the involvement of the TSPO in secondary brain damage and neurodegenerative diseases.

The 18 kDa translocator protein influences angiogenesis, as well as aggressiveness, adhesion, migration, and proliferation of glioblastoma cells.

BACKGROUND: It is known that the mitochondrial 18 kDa translocator protein (TSPO) is present in almost all peripheral tissues and also in glial cells in the brain. TSPO levels are typically enhanced in correlation with tumorigenesis of cancer cells including glioblastoma. Relevant for angiogenesis, TSPO is also present in almost all cells of the cardiovascular system. METHODS: We studied the effect of TSPO knockdown by siRNA on various aspects of tumor growth of U118MG glioblastoma cells in two in-vivo models: a nude mouse model with intracerebral implants of U118MG glioblastoma cells and implantation of U118MG glioblastoma cells on the chorionallantoic membrane (CAM) of chicken embryos. In vitro, we further assayed the influence of TSPO on the invasive potential of U118MG cells. RESULTS: TSPO knockdown increased tumor growth in both in-vivo models compared with the scrambled siRNA control. Angiogenesis was also increased by TSPO knockdown as determined by a CAM assay. TSPO knockdown led to a decrease in adhesion to the proteins of the extracellular matrix, including fibronectin, collagen I, collagen IV, laminin I, and fibrinogen. TSPO knockdown also led to an enhancement in the migratory capability of U118MG cells, as determined in a modified Boyden chamber. Application of the TSPO ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) at a concentration of 25 µmol/l in the in-vitro models yielded results similar to those obtained on TSPO knockdown. We found no effects of PK 11195 on TSPO protein expression. Interestingly, at low nmol/l concentrations (around 1 nmol/l), PK 11195 enhanced adhesion to collagen I, suggesting a bimodal concentration effect of PK 11195. CONCLUSION: Intact TSPO appears to be able to counteract the invasive and angiogenic characteristics related to the aggressiveness of U118MG glioblastoma cells in vivo and in vitro.

Antibody levels against tetanus and diphtheria after polychemotherapy for childhood sarcoma: a report from the Late Effects Surveillance System.

BACKGROUND: It is known that antineoplastic treatment may induce secondary immunodeficiency, but studies after childhood sarcoma are rare. Since 1998, the Late Effects Surveillance System (LESS) of the German Society for Paediatric Oncology and Haematology (GPOH) prospectively registers late effects in soft tissue-, osteo- and Ewing's sarcoma patients treated within the therapy trials EICESS-92/EURO-E.W.I.N.G.-99, CWS-96/CWS-2002P, COSS-96 in Austria, Germany and Switzerland. PATIENTS AND METHODS: Antibody levels (AL) against diphtheria and tetanus were used as markers for immunity and classified according to established guidelines for protective AL values. There were 47 eligible relapse-free patients<21 years of age (31 males; 10 osteosarcoma, 12 Ewing's and 25 soft tissue sarcoma patients). Median age at diagnosis was 9.6 (interquartile range: 4.4-14.7) years. RESULTS: A median 7.2 (3.7-12.2) months after end of antineoplastic therapy, in 28% (13/47; 95% CI 16-43%) of patients there were no protective AL (<0.1 IU/ml) against diphtheria and/or tetanus. Diphtheria and tetanus AL were positively correlated (r=0.39; p=0.007). In multivariable analysis, the type of treatment had no effect on AL, similar to tumour type and time of examination after treatment end. Younger patients had significantly lower AL against tetanus (p=0.009) and girls had significantly lower AL against diphtheria than boys (p=0.015). CONCLUSION: Lack of protective AL against tetanus and/or diphtheria is frequent after childhood sarcoma treatment. Prospective surveillance of immunity and, if indicated, re-immunization is warranted in patients treated for childhood cancer.

Tumor localization of an anti-TGF-ß antibody and its effects on gliomas.

Even with current standard-of-care therapies, the prognosis for patients with malignant gliomas is very poor and several new treatment modalities for glioblastomas are currently under investigation. Given the role of TGF-ß in gliomas, anti-TGF-ß strategies against gliomas are currently being investigated. Biodistribution of intravenously injected AF680-labeled 1D11, a pan-neutralizing TGF-ß antibody, was monitored in mice bearing either subcutaneous or orthotopic gliomas using in vivo imaging and fluorescence microscopy. AF680-labeled 1D11 entered both the subcutaneous and intracranial tumors and the antibody was detected within the tumor tissue for several days whereas only low fluorescence was found in organs. The effects of 1D11 on subcutaneous versus orthotopic U87MG and GL261 gliomas in immunocompetent C57BL/6J versus immunodeficient CD1-Foxn1nu mice were observed by direct tumor size measurement, H&E staining and immunohistochemistry. Treatment of immunocompetent mice bearing subcutaneous GL261 tumors with 1D11 resulted in complete remission. In immune deficient mice, the growth of subcutaneous GL261 tumors was increased following treatment with 1D11. Intracranially implanted gliomas in C57Bl/6J mice showed no size reduction after 1D11 treatment but there was reduced invasion of the glioma cells into the adjacent normal brain. Together these data demonstrate that TGF-ß plays different roles in combating the tumor depending on subcutaneous versus orthotopic implantation site.

Monitoring proteins using in vivo near-infrared time-domain optical imaging after 2-O-hexyldiglycerol-mediated transfer to the brain.

PURPOSE: The aim of the present study was to gain insight into the penetration, biodistribution, and fate of globulins in the brain after 2-O-hexyldiglycerol-induced blood-brain barrier opening. PROCEDURES: The spatial distribution of fluorescence probes was investigated after blood-brain barrier opening with intracarotid 2-O-hexyldiglycerol injection. Fluorescence intensity was visualized by microscopy (mice and rats) and by in vivo time-domain optical imaging. RESULTS: There was an increased 2-O-hexyldiglycerol-mediated transfer of fluorescence-labeled globulins into the ipsilateral hemisphere. Sequential in vivo measurements revealed that the increase in protein concentration lasted at least 96 h after administration. Ex vivo detection of tissue fluorescence confirmed the results obtained in vivo. CONCLUSION: Globulins enter the healthy brain in conjunction with 2-O-hexyldiglycerol. Sequential in vivo near-infrared fluorescence measurements enable the visualization of the spatial distribution of antibodies in the brain of living small animals.

Hyperkalemia complicating propranolol treatment of an infantile hemangioma.

Propranolol treatment was recently reported to be successful for the management of severe infantile hemangioma. Known adverse effects of propranolol treatment include transient bradycardia, hypotension, hypoglycemia, and bronchospasm (in patients with underlying spastic respiratory illnesses), which led to a general recommendation to gradually increase propranolol dosage and closely monitor patients' hemodynamics at the onset of therapy. To date, no serious or unexpected adverse effects that required specific intervention have been reported. In this report, we describe the case of a 17-week-old female preterm infant who presented with a large, ulcerated, cutaneous-subcutaneous hemangioma of the right lateral thoracic wall, which we treated successfully with propranolol. A few days into therapy, a potentially life-threatening adverse effect, severe hyperkalemia, was observed and required treatment with loop diuretics, fluids, and nebulized salbutamol to normalize her serum potassium levels. This therapy could be gradually tapered and finally discontinued only after several weeks of propranolol treatment. Our case report indicates that, at least during the initial phase of the propranolol treatment of infantile hemangioma, close monitoring of serum electrolytes, besides the monitoring of hemodynamics and blood glucose, is necessary.

Favourable outcome of patients with childhood acute promyelocytic leukaemia after treatment with reduced cumulative anthracycline doses.

Acute promyelocytic leukaemia (APL) treatment often includes high cumulative doses of anthracyclines, which can cause long-term cardiotoxicity. Here, we report the favourable outcome in 81 paediatric APL patients treated according to the consecutive acute myeloid leukaemia-Berlin/Frankfurt/Muenster (AML-BFM) trials -93/-98/-2004 with an anthracycline-cytarabine regimen in combination with all-trans-retinoid acid (ATRA). Outcomes achieved by treatment with a reduced cumulative anthracycline dose (350 mg/m(2)) were comparable to those reported for studies with higher doses. Five-year overall survival of the total cohort was 89 +/- 4% and event-free survival (pEFS) was 73 +/- 6%. Overall survival was similar when comparing AML-BFM trial periods (trial 93: 88 +/- 8%, 98: 85 +/- 7% and 2004: 94 +/- 8%, P((logrank)) = 0.63). Seventy-five (93%) patients achieved complete remission. Most fatal events occurred during the first 6 weeks of treatment. Long-term cardiotoxicity was observed in one patient. Two patients suffered from secondary haematological malignancies. Salvage treatment was effective in 7/9 patients (78%) with relapsed APL, who now are long-term survivors after second line combination treatment with arsenic trioxide (4/7 patients) and stem cell transplantation (5/7 patients). Our results demonstrate that - combined with ATRA - a lower cumulative anthracycline dose can be used safely to maintain high cure rates and promote the reduction of long-term sequelae, such as cardiotoxicity in APL patients.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Poor outcome for children and adolescents with progressive disease or relapse of lymphoblastic lymphoma: a report from the berlin-frankfurt-muenster group.

PURPOSE: Little is known about the outcome of pediatric patients with lymphoblastic lymphoma (LBL) who suffer from progressive disease or relapse. PATIENTS AND METHODS: We analyzed the pattern of LBL relapses after current non-Hodgkin's lymphoma Berlin-Frankfurt-Muenster (BFM) frontline therapy between April 1990 and March 2003. Relapse therapy was according to acute lymphoblastic leukemia (ALL) -Relapse-BFM protocols or ALL-BFM protocols for high-risk patients. RESULTS: Twenty-eight (11%) of 251 registered patients with precursor T-cell LBL (T-LBL) and six (8%) of 73 patients with precursor B-cell LBL (pB-LBL) suffered from relapse. Of the 28 patients with T-LBL, one died from infection during relapse chemotherapy, 18 failed to achieve stable remission and died from disease progression, and nine reached allogeneic stem-cell transplantation (SCT). Two of these nine patients who underwent SCT died from transplantation-associated toxicity, three died from disease progression, and four are still alive. These four patients are in second remission of their lymphoma for 48, 68, 125, and 131 months, respectively, after allogeneic SCT. One of the four patients developed colon adenocarcinoma 47 months after SCT. Of the six patients with pB-LBL who experienced relapse, one patient died as a result of toxicity of relapse chemotherapy, two died from disease progression after chemotherapy, and three received allogeneic SCT. Of these, two died from subsequent disease progression, and one is still alive 57 months after allogeneic SCT. CONCLUSION: Using modern conventional therapy in the frontline treatment of LBL, 10% of patients suffer from progressive disease or relapse. Because of the extremely poor reinduction success, the salvage rate for these patients is poor, with only a 14% (SE = 6%) overall survival. Long-term survival was only achieved in those few patients who were able to undergo an allogeneic SCT.

Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine.

BACKGROUND: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. METHODS: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. RESULTS: The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. CONCLUSIONS: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.

Blood-brain barrier opening with alkylglycerols: Biodistribution of 1-O-pentylglycerol after intravenous and intracarotid administration in rats.

Short-chain alkylglycerols have been described to increase the penetration of drugs and macromolecules across the blood-brain barrier (BBB) into the central nervous system (CNS) and were considered to be of potential value in the pharmaceutical treatment of CNS disorders. Due to the lack of information on the pharmacological behavior of these compounds in vivo, pharmacokinetics and biodistribution of [14C]- and [3H]-labeled 1-O-pentylglycerol (49 mg/kg, 100 mM) was investigated in normal male Wistar rats after intravenous as well as intracarotid administration. There was a rapid and predominant renal elimination of 1-O-pentylglycerol and more than 70% of administered dose was found in the urine within 270 min. Analysis of the pharmacokinetic parameters after a single i.v. bolus injection of 1-O-pentylglycerol resulted in a peak blood concentration of 0.58+/-0.06 micromol/ml, an initial half life of 23+/-7 min and a terminal half life of 18.8+/-4.1 h. No accumulation of 1-O-pentylglycerol was observed in the brain or other organs while highest concentrations were found in liver and thymus. This was confirmed by autoradiographic studies. Five minutes after intracarotid administration, high radioactivity was found in the ipsilateral brain, whereas after 30 min radioactivity in the brain has dramatically decreased. Autoradiographic images gave evidence of biliary excretion in addition to the renal elimination. There were no signs of cleavage of the O-alkyl bond in vivo as demonstrated by HPLC analysis. In conclusion, 1-O-pentylglycerol is characterized by pharmacological properties appearing very favorable for in vivo use as a permeabilizing drug for increased drug delivery to the brain.

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line.

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.

MAP kinase pathways involved in glioblastoma response to erucylphosphocholine.

Erucylphosphocholine (ErPC) is a promising antineoplastic drug for the treatment of malignant brain tumors. It exerts strong anticancer activity and induces apoptosis even in chemoresistant glioma cells. In the present study, A172 and U373MG glioma cells were treated with ErPC to explore the contribution of MAP kinase family members ERK, JNK and p38 kinase to ErPC-induced cell death. The exposure to ErPC led to activation of JNK and concurrent inhibition of ERK in both cell lines. Using specific MAP kinase inhibitors we confirmed that in U373MG cells ERK was blocked and JNK was activated upon ErPC treatment. Both effects were dependent on caspase activation. In A172 cells, ErPC treatment resulted in an activation of the JNK pathway, whereas the situation with respect to ERK signalling was more complex. We conclude that inhibition of the ERK pathway by ErPC may be related to antiproliferative effects, while activation of the JNK pathway may be responsible for its pro-apoptotic action.

Low occurrence of familial neuroblastomas and ganglioneuromas in five consecutive GPOH neuroblastoma treatment studies.

Familial neuroblastoma is of special interest in view of the oncogenesis of this tumour with its early manifestation in childhood. The inheritance seems to follow an autosomal-dominant mendelian trait with incomplete penetrance. Familial neuroblastomas and ganglioneuromas have not been reported in detail within large treatment studies. A retrospective clinicopathological survey of patients reported to the German neuroblastoma treatment studies over 24 years was performed. Among 2863 patients (2752 neuroblastomas, 111 ganglioneuromas) included in five consecutive trials, only 22 hereditary cases in ten families were observed. Neuroblastomas were found in 18 patients and ganglioneuromas in four, accounting for less than one percent of all cases. Six patients with neuroblastomas had localised disease, seven had stage 4, three had stage 4S, and stage was unknown in two patients. Two families had three affected patients. Contrary to previous reports, age distribution and number of primary tumours in patients with familial data confirm the low prevalence of familial neuroblastoma and may help in counselling the affected families.

Alkylglycerol opening of the blood-brain barrier to small and large fluorescence markers in normal and C6 glioma-bearing rats and isolated rat brain capillaries.

1. The blood-brain barrier (BBB) represents the major impediment to successful delivery of therapeutic agents to target tissue within the central nervous system. Intracarotid alkylglycerols have been shown to increase the transfer of chemotherapeutics across the BBB. 2. We investigated the spatial distribution of intracarotid fluorescein sodium and intravenous lissamine-rhodamine B200 (RB 200)-albumin in the brain of normal and C6 glioma-bearing rats after intracarotid co-administration of 1-O-pentylglycerol (200 mm). To elucidate the mechanisms involved in the alkylglycerol-mediated BBB opening, intraluminal accumulation of fluorescein isothiocyanate (FITC)-dextran 40,000 was studied in freshly isolated rat brain capillaries using confocal microscopy during incubation with different alkylglycerols. Furthermore, 1-O-pentylglycerol-induced increase in delivery of methotrexate (MTX) to the brain was evaluated in nude mice. 3. Microscopic evaluation showed a marked 1-O-pentylglycerol-induced extravasation of fluorescein and RB 200-albumin in the ipsilateral normal brain. In glioma-bearing rats, increased tissue fluorescence was found in both tumor tissue and brain surrounding tumor. Confocal microscopy revealed a time- and concentration-dependent accumulation of FITC-dextran 40,000 within the lumina of isolated rat brain capillaries during incubation with 1-O-pentylglycerol and 2-O-hexyldiglycerol, indicating enhanced paracellular transfer via tight junctions. Intracarotid co-administration of MTX and 1-O-pentylglycerol (200 mm) in nude mice resulted in a significant increase in MTX concentrations in the ipsilateral brain as compared to controls without 1-O-pentylglycerol (P<0.005). 4. In conclusion, 1-O-pentylglycerol increases delivery of small and large compounds to normal brain and brain tumors and this effect is mediated at least in part by enhanced permeability of tight junctions.

Intracarotid administration of short-chain alkylglycerols for increased delivery of methotrexate to the rat brain.

1 The intracarotid administration of alkylglycerols has been reported previously by us to be a novel strategy for increased delivery of various chemotherapeutic drugs to the normal brain and brain tumors in rats. 2 Effectiveness and structure-activity relations of the most promising pentyl- and hexylglycerol derivatives have been elucidated in vivo by analyzing the transfer of methotrexate (MTX) across the blood-brain barrier (BBB) in normal rats. The effects were compared with BBB disruption using hypertonic mannitol or intracarotid infusion of bradykinin. Furthermore, toxicity of the alkylglycerols has been studied in long-term experiments. 3 Apart from 1-O-pentyldiglycerol, all alkylglycerols induced a concentration-dependent increase in MTX delivery to the brain varying from 1.1 to more than 300-fold compared to intra-arterial MTX alone. Enhanced barrier permeability rapidly approached baseline values within 5 and 120 min at the latest. Chemical structure, concentration, time schedule of injections and combination of different alkylglycerols were identified as instruments suited to regulate the MTX accumulation within a wide range. Mannitol 1.4 M resulted in very high MTX levels in the brain as observed using the highest concentrations of alkylglycerols. Intracarotid infusion of bradykinin had only a minor effect on the BBB. Using 1-O-pentylglycerol or 2-O-hexyldiglycerol, both cell culture experiments and long-term in vivo analyses including clinical, laboratory and histopathological evaluations revealed no signs of toxicity. 4 In summary, intracarotid short-chain alkylglycerols constitute a very effective and low toxic strategy for transient opening of the BBB to overcome the limited access of cytotoxic drugs to the brain.

Epidemiological investigation on chronic copper toxicity to children exposed via the public drinking water supply.

Copper in drinking water has been associated with Non-Indian Childhood Cirrhosis (NICC), a form of early childhood liver cirrhosis. This epidemiological study examines the exposition of infants to increased copper concentrations through drinking water from public water supplies in Berlin, Germany, and if this dietary copper intake can cause liver damage in early childhood. In total, water samples from 2944 households with infants were tested for copper. Mean copper concentrations in the two different types of collected composite samples were 0.44 and 0.56 mg/l, respectively. Families having a copper concentration at or above 0.8 mg/l in one or both of the composite samples (29.9% of all sampled households) and a defined minimum ingestion of tap water of their infant were recommended to undergo a paediatric examination. Nearly every of the 541 recommended infants were examined by a local paediatrician and of these 183 received a blood serum analysis, too. None of the infants had clear signs of a liver disease although a few serum parameters lay outside the accompanying reference range and abdominal ultrasound imaging gave slightly unusual results in five cases. Additionally, no signs of a negative health effect could be found in the statistical analysis of the serum parameters GOT, GPT, GGT, total bilirubin, serum copper, or ceruloplasmin in relation to estimated daily and total copper intakes of the infants from tap water. No dose relation of serum parameters and estimated copper intakes could be established. From the results of the study, no confirmed indication of a liver malfunction in infants whose food had been prepared using tap water with an elevated copper concentration could be found and, therefore, no indication of a hazard due to copper pipes connected to public water supplies could be detected.

Impact of conventional chemotherapy on levels of antibodies against vaccine-preventable diseases in children treated for cancer.

Intensive chemotherapy in children with malignancies causes partial immune deficiency, including long-term impairment of humoral immunity. We investigated the levels of antibodies against measles, mumps, polio, rubella, diphtheria, tetanus, and Haemophilus type b (Hib) in 139 children at the time of diagnosis of the malignant disease, during chemotherapy, after cessation of intensive treatment, and after re-vaccination. In general, cytostatic therapy resulted in a significant lowering of antibody levels. A decline of antibodies below the protective level as a consequence of cytostatic treatment was observed in 6% of the children for measles and mumps, in 18%, 12%, and 25% for polio types 1, 2, and 3, and in 21% for diphtheria. By contrast, rubella and tetanus antibodies remained within the protective range in all cases of this study. Re-vaccination 3 to 5 months after cessation of chemotherapy produced antibody levels about as high as those measured prior to therapy. Only 6 out of 83 children with previously positive antigen titres did not respond to re-vaccination. Vaccination or re-vaccination failed in 5 of 13 non-responders for more than 1 antigen, indicating a decreased reactability to vaccinations in some patients.

Increased delivery of erucylphosphocholine to C6 gliomas by chemical opening of the blood-brain barrier using intracarotid pentylglycerol in rats.

BACKGROUND: Erucylphosphocholine (ErPC) has been shown to exert strong antineoplastic effects against various brain tumor cell lines in vitro. Since ErPC only enters the brain after long-term treatment, ineffective drug delivery to the tumor is considered to be the reason for the moderate responses to chemotherapy with ErPC observed in animal brain tumor models. We investigated a recently described method for chemically opening the blood-brain barrier (BBB) using intraarterial administration of alkylglycerols to increase the transfer of ErPC into the brain. METHODS: ErPC (40 mg/kg) was given to C6 glioma-bearing rats either as a single intracarotid bolus injection in the presence or absence of 1- O-pentylglycerol (300 m M) or as an intracarotid infusion in conjunction with bradykinin. Brain tissue concentrations were analyzed and compared to values obtained after intravenous ErPC treatment over 14 and 30 days (cumulative ErPC doses of 210 and 350 mg/kg, respectively). RESULTS: Pentylglycerol-induced BBB opening resulted in a significant increase in ErPC delivery to the tumor (17-fold) and, to a lesser extent, to the surrounding ipsilateral brain (7-fold) compared to intraarterial ErPC administration without alkylglycerol ( P<0.05). Furthermore, the resulting ErPC concentrations in the brain tumor exceeded those obtained in tumor and tumor-free brain after long-term intravenous ErPC administration. In contrast to this, intracarotid bradykinin did not increase the transfer of ErPC to the tumor or tumor-free brain. CONCLUSIONS: The intracarotid administration of pentylglycerol represents a novel and nontoxic method of overcoming the limited access of ErPC to both brain tumors and brain tissue adjacent to tumors. The present results provide further evidence that chemical opening of the BBB by intraarterial alkylglycerols is a promising new concept for improving delivery of chemotherapeutic agents to brain tumors.

Erucylphosphocholine-induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation.

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.