Improving the transfection efficiency of post-mitotic neurons.

The transfection of post-mitotic cells, including primary cortical and hippocampal neurons, has proven for the most part to be inefficient. Methods such as DNA/Ca++ phosphate co-precipitation, electroporation, cationic lipids and micro-injection are often toxic to the cell and rarely give a transfection efficiency exceeding 3% of the surviving culture. Virus transfection methods using modified viruses such as adeno and semliki-forest virus have been shown to be more efficient but the procedures are often time consuming and virus infections may interfere with protein processing. In this study, we evaluated the transfection efficiency of cells from E18 rat embryonic cortical and hippocampal tissues using three cationic lipids: LIPOFECTAMINE, LIPOFECTAMINE Plus, and LIPOFECTAMINE 2000. The method of transfection was by a traditional reporter gene beta-galactosidase (pCMV.SPORT-beta-gal). Results show that out of the three cationic lipids tested, LIPOFECTAMINE 2000 allows for a significantly higher transfection efficiency. Transfection efficiency with LIPOFECTAMINE or LIPOFECT-AMINE Plus was <3%. In contrast, transfection efficiency with LIPOFECT-AMINE 2000 was approximately 20-25% with the cortical neurons and 25-30% with the hippocampal neurons.

The total care unit for pediatric hematology and oncology, Princess Margaret Hospital for Children, Perth, Australia.

The total care unit for the treatment of pediatric hematology/oncology in Perth, Australia is so named to embody the philosophy of multidisciplinary care of children and their families. Where possible, patients are treated according to randomized controlled trials of the large cooperative Children's Cancer Group. There is a seamless association of clinical and laboratory research. Hemopoietic stem cell transplantation is managed within the unit, as well as treatment of a range of non-malignant hematological disorders. Long-term follow-up of survivors of childhood cancer is coordinated from the unit.

The prevalence of hepatitis C in survivors of paediatric malignancy.

OBJECTIVE: To assess the prevalence of hepatitis C in 200 patients with paediatric malignancies, surviving in remission more than 5 years from diagnosis, who had received blood product transfusions before 1990 when routine screening of blood products for hepatitis C began. METHOD: The second and third generation Abbott Diagnostics ELISA was used to assess hepatitis C seropositivity. Seropositive patients and those with abnormal liver transaminases were assessed by hepatitis C virus RNA polymerase chain reaction (PCR). RESULTS: A low incidence (4%) of seropositivity for hepatitis C was found in survivors of paediatric malignancy who were transfused prior to routine screening of blood products in this cohort. CONCLUSIONS: All patients identified have evidence of hepatitis and may be at high risk of developing cirrhosis.

Viable cultured neurons in ambient carbon dioxide and hibernation storage for a month.

Neurobasal is a bicarbonate-buffered medium optimized for the growth of embryonic rat hippocampal neurons at pH 7.3 in 5% CO2. Neurons die within hours in this or in other 26 mM bicarbonate buffers when transferred to ambient CO2 (0.2%). Death is associated with a rapid rise in medium pH to 8.1. A new CO2-independent modification of Neurobasal (Hibernate E), when supplemented with B27, can maintain neuron viability for at least 2 days in ambient CO2. This same medium can also be used to store viable brain tissue for up to a month with refrigeration. These advances should facilitate studies of neuron physiology outside the incubator as well as storing and transporting neuronal tissue.

Parents' perceptions of the meaning of quality nursing care.

Quality care is a major issue currently facing the delivery of health care services yet little research has been conducted on what parents of hospitalized children perceive as quality nursing care. A qualitative approach was used to identify the meaning of quality nursing care from parents' perspective and to identify concepts inherent in the process of receiving quality nursing care. Four stages in the process were identified; maneuvering, process of knowing, relationship, and quality care. The concepts suggested a similar experience for all participants, even though the individual stories of the process of receiving quality nursing care were different for each child and parent.

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.

Late leukemic relapse 10 years from diagnosis in a child on recombinant human growth hormone.

A 12 year-old girl developed a late relapse of acute lymphoblastic leukaemia (ALL) 10 years from first presentation. Initial chemotherapy included vincristine, methotrexate, prednisolone, and L-asparaginase with cranial radiotherapy (18 Gy) for central nervous system prophylaxis. Documented growth failure led to recombinant human growth hormone (rhGH) replacement therapy being instituted in May 1989, 6 years from end of therapy and 2 years prior to relapse. Three independent experiments demonstrated no increased cell proliferation in vitro when the patient's thawed cryopreserved fresh leukemic cells were incubated with rhGH. However, a pre-T ALL cell line (PER-255) consistently demonstrated enhanced proliferation when incubated with rhGH (132.1 +/- 13.4%, P < 0.01). Growth hormone has been associated with an increased incidence of leukemia and may be implicated in the late relapse of this child. The use of growth hormone in children with a past history of ALL needs to be examined critically in the light of the potential risk of inducing leukemic relapse.

Segmental durations in the vicinity of prosodic phrase boundaries.

Numerous studies have indicated that prosodic phrase boundaries may be marked by a variety of acoustic phenomena including segmental lengthening. It has not been established, however, whether this lengthening is restricted to the immediate vicinity of the boundary, or if it extends over some larger region. In this study, segmental lengthening in the vicinity of prosodic boundaries is examined and found to be restricted to the rhyme of the syllable preceding the boundary. By using a normalized measure of segmental lengthening, and by compensating for differences in speaking rate, it is also shown that at least four distinct types of boundaries can be distinguished on the basis of this lengthening.

The use of prosody in syntactic disambiguation.

Prosodic structure and syntactic structure are not identical; neither are they unrelated. Knowing when and how the two correspond could yield better quality speech synthesis, could aid in the disambiguation of competing syntactic hypotheses in speech understanding, and could lead to a more comprehensive view of human speech processing. In a set of experiments involving 35 pairs of phonetically similar sentences representing seven types of structural contrasts, the perceptual evidence shows that some, but not all, of the pairs can be disambiguated on the basis of prosodic differences. The phonological evidence relates the disambiguation primarily to boundary phenomena, although prominences sometimes play a role. Finally, phonetic analyses describing the attributes of these phonological markers indicate the importance of both absolute and relative measures.

Late effects of childhood malignancies seen in Western Australia.

Eighty-nine pediatric oncology patients, in remission and off treatment for at least 4 years, were reviewed annually in the Late Effects Clinic of Princess Margaret Hospital for Children in Perth, Western Australia. Interval from time of diagnosis to follow-up ranged from 4 to 23 years (mean 10.8 years). Acute lymphoblastic leukemia (ALL) (40%) and Wilms' tumor (27%) were the most common primary malignancies in this group. Late sequelae included musculoskeletal abnormalities (23 children), growth hormone deficiency (11), second tumors (9), learning difficulties (7), puberty and fertility problems (4), and hypothyroidism (4). These complications were most often related to radiation therapy. The need for prolonged, regular follow-up of survivors of childhood malignancy for early detection of late sequelae and subsequent intervention is stressed.

PER-117: a new human ALL cell line with an immature thymic phenotype.

A new cell line, PER-117, was established from bone marrow cells of an eighteen months old boy with an acute lymphoblastic leukaemia (ALL). The leukaemic origin of cell line PER-117 is indicated by its cytochemical, immunological and cytogenetic similarity to the patient's fresh leukaemic cells. PER-117 carries a marker chromosome which was identified as a translocation between chromosomes 1 and 11. The surface marker analysis revealed that the phenotype of PER-117 is RFB-1+, RFT-1+ (CD5), 3A1+ (CD7), OKT 9+, OKT 10+ and HLA-DR-. Thus, this cell line appears to represent a prothymocyte or stage I thymocyte and preliminary data suggest that it can be induced in vitro to further differentiate.

Perception of temporal differences in speech by "normal-hearing" adults: effects of age and intensity.

Many older people have greater difficulty processing speech at suprathreshold levels than can be explained by standard audiometric configurations. Some of the difficulty may involve the processing of temporal information. Temporal information can signal linguistic distinctions. The voicing distinction, for example, that separates pairs of words such as "rapid" and "rabid" can be signaled by temporal information: longer first vowel and shorter closure characterize "rabid"; shorter vowel and longer closure characterize "rapid." In this study, naturally produced tokens of "rabid" were low-pass filtered at 3500 Hz and edited to create vowel and (silent) closure duration continua. Pure-tone audiograms and speech recognition scores were used to select the ten best-hearing subjects among 50 volunteers over age 55. Randomizations of the stimuli were presented for labeling at intensity levels of 60 and 80 dB HL to this group and to ten normal-hearing volunteers under age 25. Results showed highly significant interactions of age with the temporal factors and with intensity: the older subjects required longer silence durations before reporting "rapid," especially for the shorter vowel durations and for the higher intensity level. These data suggest that age may affect the relative salience of different acoustic cues in speech perception, and that age-related hearing loss may involve deficits in the processing of temporal information, deficits that are not measured by standard audiometry.

Production and immunochemical study of monoclonal antibodies directed against human low density lipoprotein.

The purpose of this work is to produce monoclonal antibodies directed against Human Low Density Lipoprotein (LDL). Because of the importance of an early characterization in isotypes of the monoclonal antibodies we carefully analyzed three different techniques: double gel diffusion, competition ELISA and immunodot isotyping technique. The three methods gave essentially the same results but greatly differed in sensitivity. We are purifying all the IgG monoclonal antibodies by affinity chromatography from mouse ascitic fluid using Protein A-Sepharose. Here we report details of the purification of one of these monoclonal antibodies. Fractions eluted at different pH values displayed multiple bands by isoelectric focusing, which could be stained for proteins and recognized by radiolabelled Protein A with the electroblotting technique.

Simple rapid methods for freezing hybridomas in 96-well microculture plates.

Macroscopic hybridoma colonies were frozen and recovered in a good state of viability in 96-well microculture plates using 2 freezing procedures. These methods offer convenient and rapid means of preserving hybridomas and will permit laboratories developing monoclonal antibodies to distribute workloads to more manageable levels without discarding possibly valuable hybridomas.