Targeting immunoliposomes to EGFR-positive glioblastoma.

BACKGROUND: We assessed the capacity of epidermal growth factor receptor (EGFR)-targeted immunoliposomes to deliver cargo to brain tumor tissue in patients with relapsed glioblastoma harboring an EGFR amplification. We aimed to assess the tolerability and effectiveness of anti-EGFR immunoliposomes loaded with doxorubicin (anti-EGFR ILs-dox) in glioblastoma multiforme patients. PATIENTS AND METHODS: Patients with EGFR-amplified, relapsed glioblastoma were included in this phase I pharmacokinetic trial. Patients received up to four cycles of anti-EGFR ILs-dox. Twenty-four hours later, plasma and cerebrospinal fluid (CSF) samples were obtained. In addition, we also treated three patients with anti-EGFR ILs-dox before resection of their relapsed glioblastoma. Doxorubicin concentrations were measured in plasma, CSF, and tumor tissue. Safety and efficacy parameters were also obtained. RESULTS: There were no or negligible levels of doxorubicin found in the CSF demonstrating that anti-EGFR ILs-dox are not able to cross the blood-brain barrier (BBB). However, significant levels were detected in glioblastoma tissue 24 h after the application, indicating that the disruption of BBB integrity present in high-grade gliomas might enable liposome delivery into tumor tissue. No new safety issues were observed. The median progression-free survival was 1.5 months and the median overall survival was 8 months. One patient undergoing surgery had a very long remission suggesting that neoadjuvant administration may have a positive effect on outcome. CONCLUSIONS: We clearly demonstrate that anti-EGFR-immunoliposomes can be targeted to EGFR-amplified glioblastoma and cargo-in this case doxorubicin-can be delivered, although these immunoliposomes do not cross the intact BBB. (The GBM-LIPO trial was registered as NCT03603379).

Consenso chileno de manejo de fármacos antiepilépticos en algunos síndromes electro-clínicos y otras epilepsias en niños y adolescentes / Chilean consensus on the use of antiepileptic drugs in electro-clinical syndromes

Por iniciativa de tres instituciones: Liga Chilena contra la Epilepsia (LICHE), Sociedad de Epileptología de Chile (SOCEPCHI) y Sociedad de Psiquiatría y Neurología de la Infancia y Adolescencia (SOPNIA) de Chile, se constituye un comité de trabajo que convoca a un consenso de uso de fármacos antiepilépticos (FAEs) en un grupo de 16 Síndromes electro-clínicos y otras Epilepsias en niños y adolescentes. Cuarenta y dos médicos neuropediatras especialistas en Epilepsias de todas las regiones de Chile, participaron en la discusión y realizaron una propuesta de tratamiento farmacológico para cada cuadro. El comité de trabajo realizó un análisis exhaustivo y discusión de los documentos, para finalmente concluir en una recomendación de tratamiento para cada cuadro. Este consenso es una guía práctica de orientación para ayudar a las decisiones de tratamiento en situaciones clínicas concretas. Su objetivo final es ofrecer una mejor calidad de atención a los niños y adolescentes con epilepsias, a través de decisiones fundadas que contribuyan a disminuir la variabilidad de las decisiones terapéuticas.

Committed by three institutions: Liga Chilena contra la Epilepsia (LICHE), Sociedad de Epileptología de Chile (SOCEPCHI) y Sociedad de Psiquiatría y Neurología de la Infancia y Adolescencia (SOPNIA) de Chile, a 6-member working committee called for a meeting of 42 Chilean pediatric epileptologists from all over the country, with the aim of reaching a consensus on the use of antiepileptic drugs in 16 selected children and adolescents electro-clinical syndromes and epilepsies. These treatment proposals were analyzed and fully discussed by the working committee, ending in an antiepileptic drug treatment recommendation guideline for each condition. This consensus is a practical guideline to be used in specific clinical situations, which aims to support treatment decision making. Its main purpose is to offer the best evidence based treatments to our children and adolescents patients with epilepsy, thus contributing to diminish variability in therapeutic decisions.

Diet and cancer.

Large claims have been made for the effectiveness of particular diets in preventing cancer or inhibiting its progression. However, more recent clinical studies have not confirmed this. Instead it seems that rather than specific dietary constituents, total calories influence cancer incidence and progression. In this review article, we summarise and interpret the available evidence for links between diet and cancer.

The potential role of podoplanin in tumour invasion.

Podoplanin is a small mucin-like transmembrane protein, widely expressed in various specialised cell types throughout the body. Here, we revisit the mechanism of podoplanin-mediated tumour invasion. We compare molecular pathways leading to single and collective cell invasion and discuss novel distinct concepts of tumour cell invasion.

The Rho/Rho-kinase and the phosphatidylinositol 3-kinase pathways are essential for spontaneous locomotion of Walker 256 carcinosarcoma cells.

Signal transduction pathways controlling spontaneous locomotion of Walker carcinosarcoma cells are not well understood. We have therefore investigated the role of signalling proteins in development of polarity and locomotion of these cells. Treatment of the cells with 100 ng/ml pertussis toxin had no significant effect on the percentage of polarized cells. In contrast, 2 different phosphatidylinositol 3-kinase inhibitors (wortmannin and LY-294002) markedly reduced the proportion of polarized cells. Spontaneous locomotion of the cells was also significantly inhibited by these two inhibitors. In agreement with these data, we observed localization of the p85alpha subunit of phosphatidylinositol 3-kinase predominantly in the membrane fraction of Walker carcinosarcoma cells, indicating constitutive activation of this enzyme. We also investigated a role of Rho family proteins. Spontaneous development of polarity was almost completely suppressed by electroporation of the cells in the presence of 4 microg/ml C(3) exoenzyme, which specifically ADP-ribosylates and inactivates Rho. Two downstream targets of Rho, the Rho-activated kinases I and II, were also detected predominantly in the particulate membrane fraction, suggesting constitutive activation. A specific Rho-kinase inhibitor (Y-27632) blocked spontaneous polarization and migration in a concentration-dependent manner. Our results indicate that constitutive activation of the Rho/Rho-kinase pathway and of phosphatidylinositol 3-kinase plays an essential part in spontaneous development of polarity and cell locomotion of these cells.

Role of protein kinase C isoforms in locomotion of Walker 256 carcinosarcoma cells.

Treatment with low (nanomolar) concentrations of phorbol-12-myristate-13-acetate (PMA) for 5 to 30 min suppresses locomotion of Walker 256 carcinosarcoma cells, suggesting that activation of protein kinase C (PKC) is a stop signal for tumor cell locomotion. We have compared the effects of PMA on cell shape and motility with down-regulation of specific PKC isoforms. Using specific antibodies, we show that Walker carcinosarcoma cells express PKC isoforms alpha, betaI, betaII, gamma, lambda, mu, eta and zeta. Short-term incubation with PMA induced a marked shift of isoforms alpha, betaI, betaII, gamma and eta to the particulate fraction. Long-term incubation with PMA (0.1 microM, 6 hr) resulted in significant reduction of expression of conventional PKCs alpha, betaI, betaII and gamma and of the novel PKC eta to 10% to 26% of controls. Down-regulation of PKC alpha, betaI and betaII by long-term incubation with PMA was reversible after removal of PMA, whereas that of isoforms gamma and eta was not. The motile properties of cells after down-regulation of PKC isoforms were investigated. Concomitant with down-regulation of PKC isoforms, long-term incubation of cells with PMA resulted in recovery of the polar shape and the ability to migrate. Motility and polarized shape of the down-regulated cells were no longer susceptible to short-term treatment with PMA, showing that active PKC is indeed responsible for the inhibitory effects of PMA. Effects of long-term incubation with PMA on cell shape and motility were reversible. Our findings strongly suggest that PKCs alpha, betaI and betaII activated by PMA are involved in stopping Walker carcinosarcoma cell locomotion.

ECOSYS-87: model calculations of the activity in cattle meat related to deposition time and feeding regimes after a nuclear fallout.

The radioecological model ECOSYS-87 was used to evaluate the effect of countermeasures for reducing the ingestion dose by eating cattle meat after an accidental release of radioactive material. Calculations were performed using a database adapted to Swiss conditions for the case that (1) contaminated grass or hay is replaced by clean fodder; (2) the last 100 days before slaughter, taking place one year after an accident, only uncontaminated fodder is given; and (3) alternative feeding regimes are chosen. Seasonal effects were considered by doing all calculations for a deposition at each month of the year. Feeding uncontaminated forage 100 d before slaughter (case 2) proved to be the most effective countermeasure and reduced the integrated activity in meat by 90% to 99%. The effect of replacing contaminated grass (case 1) was less uniform and depended strongly on the time a deposition occurred. In this case the reduction was between 50% and 100% one year after deposition. The substitution of contaminated hay (case 1) was less effective compared to the substitution of grass. The choice of alternative feeding regimes (case 3) led to a reduction of the integrated activity of up to 40% one year after deposition. The present model calculations clearly reveal the importance of the seasonality and demonstrate the usefulness of such calculations as a basis for generating countermeasures in decision support systems.

[Significance of hepatitis C virus antibodies in asymptomatic blood donors]. / Bedeutung von Hepatitis-C-Virus-Antikörpern bei asymptomatischen Blutspendern.

The aim of the study was to determine the frequency and course of hepatitis C viremia in clinically healthy, anti-HCV positive test subjects, and to ascertain whether the HCV antibodies of the IgM type differed between viremia and immunity. In 21 anti-HCV positive blood donors (test subjects) with normal transaminase activity, two serum samples, taken at an interval of 25 +/- 10 months, have been investigated for HCV-RNA and HCV-IgM antibodies. In a total of 16 test subjects (76%) HCV-RNA was found during the first test and/or the follow-up: 14 of them were positive on both occasions, and one test subject each was HCV-RNA positive exclusively at the first test and the follow-up respectively. At the time of the follow-up the serum transaminase level was elevated in 4 test subjects. 3 of these 4 were HCV-RNA positive also. On the other hand, the results of the HCV-PCR were nonuniform in HCV-IgM antibody negative test subjects. The above results demonstrate that in the majority of clinically healthy, anti-HCV positive test subjects with normal transaminase activity, a viremia exists which persists and the course of which may include inflammatory phases. The proof of HCV-IgM antibodies correlates with a viremia. On the other hand, the lack of HCV-IgM antibodies does not exclude viremia.

The effect of plaque control in subjects with shallow pockets and high prevalence of periodontal pathogens.

In a previous study, it was shown that professional tooth cleaning 3x a week had a significant influence on the subgingival microbiota of shallow pockets. The purpose of this investigation was to study the effect of a single episode of full-mouth supragingival cleaning and oral hygiene instructions in subjects with minimal periodontal disease but high prevalence of putative periodontal pathogens. 10 subjects from Arabic countries, aged between 22 and 48 years, which had previously not been exposed to any dental care other than extractions and fillings, were selected for this trial. DNA probe analysis of subgingival samples, taken in the deepest pocket of each quadrant, showed presence of Porphyromonas gingivalis and Prevotella intermedia in all patients, and presence of Actinobacillus actinomycetemcomitans in 5 individuals. 85% of all samples were P. gingivalis-positive, 83% were positive for P. intermedia and 43% were A. actinomycetemcomitans-positive. 4 weeks after treatment, subgingival microbiological samples were again taken in the same sites. In 8 patients, P. gingivalis could still be detected after treatment. However, the number of P. gingivalis positive samples was reduced from 85% to 38%, and the bacterial counts in positive samples were markedly lower than at baseline. P. intermedia-positive samples were obtained from 7 patients after treatment. 33% of all samples were still positive, but showed markedly reduced bacterial counts. 4 patients still yielded A. actinomycetem comitans-positive samples after treatment. Here, the number of positive samples was reduced to 15%, and the bacterial counts were barely exceeding the detection limit.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis C RNA prevalence in a Western European organ donor pool and virus transmission by organ transplantation.

Liver disease is a common finding after organ transplantation and might in part be due to transmission of hepatitis C virus (HCV). The aim of this study was to determine the prevalence of positive results with different anti-HCV tests and HCV-RNA in a local donor pool and to clarify to what extent HCV was transmitted to organ recipients. Serum samples from 207 consecutive organ donors were analysed retrospectively with anti-HCV ELISA (2nd and 3rd generation), anti-HCV RIBA (2nd generation) and HCV polymerase chain reaction (PCR). Organ recipients at risk were identified and followed up serologically and clinically. Anti-HCV seroprevalance in organ donors was 4.3% for 2nd generation ELISA, 4.8% for 3rd generation ELISA and 1.9% for 2nd generation RIBA. HCV-PCR was positive in 1.4%. Nine organs from four RIBA-positive donors were transplanted into eight recipients of whom four became anti-HCV and PCR positive after transplantation. HCV-PCR became positive several days after transplantation whereas anti-HCV seroconversion took place after 8-9 months. Two recipients developed acute liver disease and another two showed features of mild chronic liver disease but no serious complications due to HCV infection were observed.

Detection of HBs antigen in "anti-HBc alone" positive sera.

The immunoserological finding "anti-HBc alone" is often observed in defined groups of individuals, such as patients with inflammatory hepatopathies, patients on hemodialyses or with organ transplants, i.v. drug users and homosexuals, but it also occurs in up to 1% of Swiss blood-donors. In order to gain further information about whether "anti-HBc alone" reflects late immunity or points to an ongoing or a recently passed hepatitis B virus infection, 153 serum samples were tested for immune-complex-dissociated HBs-antigen, using acid treatment for complex dissociation. Of the samples tested 31% contained complexed HBsAg, the highest rates being found in individuals with hepatopathies (up to 80%), in i.v. drug users (up to 63%) and in hemodialysis patients (40%). The 153 sera were also tested for HBV-DNA by nested PCR. Sixty (39%) probes yielded positive results, comprising 29 (48%) of 60 sera with immune-complexed HBsAg but only 18 (19%) of 93 probes without complexed HBsAg. The results point to the possibility that at least some of the individuals with "anti-HBc alone" still have an ongoing HBV-infection.

[Hepatitis serological finding "anti-HBc alone", circulating viral DNA and interpretation of findings]. / Der Hepatitis-serologische Befund "Anti-HBc allein", zirkulierende virale DNS und Befund-Interpretation.

The immunological finding "anti-HBc alone" (without HBsAg, without anti-HBs) leaves open the question of the state of HBV infection it reflects: a transitory stage of an uncomplicated, eventually prolonged but resolving infection, a chronic or a late state of immunity. A finding of this kind is often observed in immunocompromised individuals (e.g. patients on hemodialysis, drug addicts) but also occurs in up to 1% of the Swiss blood donor population. Of 8800 sera tested for HBV marker in a diagnostic laboratory, 153 individuals showed "anti-HBc alone". They were investigated for circulating hepatitis B desoxyribonucleic acid (HBV-DNA) by polymerase chain reaction (PCR). 60 individuals (39%) showed a positive result. Also taking into consideration anamnestic measurements of conventional HBV markers in 95 individuals and consecutive testing for HBV-DNA in 50 individuals, the following conclusions emerged: 1. A positive finding of HBV-DNS by PCR does not necessarily prove an ongoing HBV infection, hence a negative result does not rule it out. Therefore, the indication to test for this parameter is limited for routine use. 2. The finding of "anti-HBc alone" implies that a HBV-infection is still going on until proven otherwise. This not only might be of consequence for the individual involved, but also raises the question of screening of blood donors and of pregnant women for anti-HBc.

[Polymerase chain reaction in hepatitis C: increased sensitivity via amplification of shorter cDNA fragments]. / Polymerase-Kettenreaktion bei Hepatitis C: höhere Sensitivität durch Amplifikation von kürzeren cDNS-Fragmenten.

The aim of our study was to compare the sensitivity of hepatitis C virus polymerase chain reaction (HCV-PCR) by use of two different primer sets which amplify PCR products of different length. Serum samples of 70 patients with chronic hepatitis C were tested by "nested primer" PCR, using either "NCR primers" that amplify cDNA-fragments of 340 basepairs (bp), or by "PT primers" which amplify fragments of 59 bp only. HCV-RNA was detected in 40 patients (57%) by "NCR primers" and in 69 patients (90%) by "PT primers" (p < 0.001). 23 of 70 patients (33%), which were HCV-RNA negative by "NCR primers", were positive by "PT primers", but no patient negative by "PT primers" was found to be positive by "NCR primers". 20 healthy controls tested by both primer sets were all HCV-RNA negative. We conclude that the sensitivity of HCV-PCR is significantly improved by use of primers that amplify "short" PCR products and recommend the use of "PT primers" for HCV-PCR.

Detection of hepatitis C viraemia in Caucasian patients with hepatocellular carcinoma.

Potential risk factors for the development of hepatocellular carcinoma were analysed in 40 Caucasian patients with this malignancy. A higher proportion (14 of 40; 35%) had evidence of hepatitis C virus (HCV) infection than had evidence of either hepatitis B virus (HBV) carriage (17.5%) or alcohol abuse (30%). In all 14 patients whose sera were reactive by HCV ELISA (Ortho second generation test), the presence of antibodies to HCV were confirmed by recombinant immunoblot assay (Ortho RIBA-2). Furthermore, two independent laboratories detected HCV-RNA in 10 of the 14 (71%) anti-HCV positive sera. Two additional sera were shown to contain HCV-RNA when reanalysed by a modified PCR using oligonucleotide primers designed to amplify a shorter fragment of the 5' noncoding region of the genome. Seven of the anti-HCV positive patients also had evidence of prior HBV infection and 2 admitted to alcohol abuse. HCV infection was the only identifiable risk factor in 6 patients. These data confirm the association between HCV infection and hepatocellular carcinoma and suggest that persistent viral replication accompanies tumour development in the majority of patients whose serum contains anti-HCV.

[Second generation hepatitis-C virus test and polymerase chain reaction in anti-C 100 negative patients with chronic non-A, non-B hepatitis]. / Hepatitis-C-Virus-Test der zweiten Generation und Polymerase-Kettenreaktion bei anti-C 100-negativen Patienten mit chronischer Non-A-Non-B-Hepatitis.

The aim of our study was to evaluate whether a negative HCV test of the first generation (HCV-ELISA 1) using the antigen C100-3 excludes chronic HCV infection, or whether patients exist who are negative for antibodies to C100-3 in spite of chronic hepatitis C. 27 patients with histologically proven chronic non-A, non-B hepatitis, all of whom were HCV-ELISA 1 negative, were tested by the HCV test systems of the second generation (Ortho-HCV-ELISA 2 and Chiron-HCV-RIBA 2) based on the distinct HCV antigens 5-1-1, C100-3, C33c and C22-3. To determine the presence of viremia, serum samples were also tested for HCV-RNA with "nested" PCR. 10 of 27 patients proved to be persistently negative when tested with the second generation assays. One patient showed low grade reactivity by HCV-ELISA 2, but non-reactivity by HCV-RIBA 2. In none of these 11 patients was HCV-RNA detected. 16 (60%) of 27 patients negative with HCV-ELISA 1 were positive with HCV-ELISA 2. HCV-RIBA 2 detected antibodies to the structural core antigen C22-3 in all of these 16 patients and antibodies to the non-structural antigen C33c in 14 of them, while antibodies to 5-1-1 or C100-3 were not found in any of these cases. 10 (63%) of the 16 HCV-ELISA 1 negative, but HCV-ELISA 2 and HCV-RIBA 2 positive patients were positive for HCV-RNA by "nested" PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5' flanking region and chromosomal localization of the gene encoding human platelet membrane glycoprotein Ib alpha.

The human blood platelet membrane glycoprotein Ib (GPIb) functions as a receptor for von Willebrand factor and thrombin. The gene (gpIb alpha) encoding the GPIb alpha-chain was cloned from a genomic cosmid library. The promoter region of this gene was characterized by sequencing two BamHI fragments including 2.8 kb of the 5' flanking region where several Alu repeated elements and purine-rich sequences were found. Possible cis-regulatory elements were identified by comparing the gpIb alpha gene with established consensus sequences known to function as binding sites for transcription factors. To obtain further information on possible megakaryocyte-specific promoter or enhancer sequences, the gpIb alpha promoter region was compared with other genes expressed in platelets that are known so far. The gpIb alpha gene was found to be located on chromosome 17 in region 17p12-ter, by in situ hybridization.