Development and performance of a biomimetic artificial perilymph for in vitro testing of medical devices.

OBJECTIVE: Cochlear implants interface with the fluid in the cochlea called perilymph. The volume of this fluid present in human and animal model cochlea is prohibitively low for isolation for in vitro studies. Thus, there is a need for an artificial perilymph that reflects the complexity of this fluid in terms of competitive protein adsorption. APPROACH: This study established a biomimetic artificial perilymph (BAP) comprising serum albumin, immunoglobulin G, transferrin, inter-alpha-trypsin inhibitor, apolipoprotein A1 and complement C3 to represent the major components of human perilymph. Adsorption of the BAP components to platinum was analysed. MAIN RESULTS: It was established that this six component BAP provided competitive and complex adsorption behaviours consistent with biologically derived complex fluids. Additionally, adsorption of the BAP components to platinum cochlear electrodes resulted in a change in polarisation impedance consistent with that observed for the cochlear device in vivo. SIGNIFICANCE: This study established a BAP fluid suitable for furthering the understanding of the implant environment for electroactive devices that interface with the biological environment.

The structural validity of various thermodynamical models of supercooled water.

The thermodynamic response functions of water exhibit an anomalous increase upon cooling that becomes strongly amplified in the deeply supercooled regime due to structural fluctuations between disordered and tetrahedral local structures. Here, we compare structural data from recent x-ray laser scattering measurements of water at 1 bar and temperatures down to 227 K with structural properties computed for several different water models using molecular dynamics simulations. Based on this comparison, we critically evaluate four different thermodynamic scenarios that have been invoked to explain the unusual behavior of water. The critical point-free model predicts small variations in the tetrahedrality with decreasing temperature, followed by a stepwise change at the liquid-liquid transition around 228 K at ambient pressure. This scenario is not consistent with the experimental data that instead show a smooth and accelerated variation in structure from 320 to 227 K. Both the singularity-free model and ice coarsening hypothesis give trends that indirectly indicate an increase in tetrahedral structure with temperature that is too weak to be consistent with experiment. A model that includes an apparent divergent point (ADP) at high positive pressure, however, predicts structural development consistent with our experimental measurements. The terminology ADP, instead of the commonly used liquid-liquid critical point, is more general in that it focuses on the growing fluctuations, whether or not they result in true criticality. Extrapolating this model beyond the experimental data, we estimate that an ADP in real water may lie around 1500 ± 250 bars and 190 ± 6 K.

Dynamics of nanoconfined water under pressure.

We report a study of the effects of pressure on the diffusivity of water molecules confined in single-wall carbon nanotubes (SWNT) with average mean pore diameter of ~16 Å. The measurements were carried out using high-resolution neutron scattering, over the temperature range 220≤T≤260 K, and at two pressure conditions: ambient and elevated pressure. The high pressure data were collected at constant volume on cooling, with P varying from ~1.92 kbar at temperature T=260 K to ~1.85 kbar at T=220 K. Analysis of the observed dynamic structure factor S(Q,E) reveals the presence of two relaxation processes, a faster diffusion component (FC) associated with the motion of "caged" or restricted molecules, and a slower component arising from the free water molecules diffusing within the SWNT matrix. While the temperature dependence of the slow relaxation time exhibits a Vogel-Fulcher-Tammann law and is non-Arrhenius in nature, the faster component follows an Arrhenius exponential law at both pressure conditions. The application of pressure remarkably slows down the overall molecular dynamics, in agreement with previous observations, but most notably affects the slow relaxation. The faster relaxation shows marginal or no change with pressure within the experimental conditions.

Endothelin-converting enzyme-1 in Alzheimer's disease and vascular dementia.

AIMS: Alzheimer's disease (AD) is believed to be caused by the accumulation of amyloid beta (Aß) peptide within the brain. Endothelin-converting enzyme-1 and 2 (ECE-1 and ECE-2) are expressed in endothelial cells and neurones, respectively, and both cleave 'big endothelin' to produce the vasoconstrictor endothelin-1 (ET-1). ECE-1 and ECE-2 also degrade Aß. AD patients have regionally reduced microvascular blood flow in the brain, with impaired endothelium-dependent relaxation and cerebrovascular autoregulation, and abnormal production of ET-1 has been demonstrated in mice overexpressing amyloid precursor protein. We recently found ECE-2 mRNA and protein to be elevated in the brain in AD. In vitro, expression of ECE-2 was upregulated by Aß. Our aims for this study were to examine expression of ECE-1 (which has 57% homology with ECE-2) in temporal cortex from patients with AD, vascular dementia (VaD) and controls. METHODS: We examined the distribution of ECE-1 with immunohistochemistry, and measured ECE-1 mRNA by real-time polymerase chain reaction (PCR). ECE-1 protein levels were measured by western blot, and results analysed before and after adjustment for factor VIII-related antigen. RESULTS: We showed ECE-1 to be in vascular endothelial cells. We did not find significant differences in ECE-1 mRNA or protein levels (either full-length ECE-1 or the soluble spliced variant, ECE-1sv) in AD or VaD compared with controls. CONCLUSIONS: Our findings suggest that any disease-specific contribution of ECE-1 to the accumulation of Aß or reduction in local microvascular blood flow in AD or VaD is probably small, with abnormal production of ET-1 being more likely to reflect Aß-mediated upregulation of ECE-2.

Determination of hemoglobin expression patterns in erythroid cells of Rana catesbeiana tadpoles.

1. Rana catesbeiana (bullfrog) tadpoles are heterogeneous in the relative amounts of four major tadpole hemoglobins (Hbs), as well as in the relative amounts of two tadpole red blood cell types in the peripheral blood. 2. Previous work has shown that this heterogeneity is present at all stages of larval development and growth. 3. Although some tadpoles lack one of the Hbs in their peripheral blood (i.e. the electrophoretically slowest form, Td-4), the missing Hb can be found in the erythropoietic organ from which it emanates (the kidneys), indicating that the heterogeneity results from quantitative differences in gene expression. 4. We wished to know whether this in vivo regulation is subject to external environmental perturbation and report that tadpoles of known Hb phenotypes regenerate precisely the pre-anemia Hb profile during early as well as late stages of recovery from phenylhydrazine-induced anemia. 5. These and other results indicate that the in vivo mechanism for regulating the pattern of Hb expression has become firmly determined in the erythropoietic system by the earliest larval stage of development.

Hemoglobin switching across vertebrate classes: exchange of developmental signals by cell fusion.

Our aim is to obtain evidence for trans-acting factors that regulate developmental hemoglobin (Hb) switching. Our approach is to fuse erythroid cells that have different developmental programs, allowing the trans-acting factors from the adult cell to have access to the nucleus of the fetal or embryonic cell and vice-versa. After cell fusion, the heteropolykaryons are cultured for six hours, and globin gene expression is assayed at two levels: (1) at the level of mRNAs on dot blots hybridized with globin-specific cDNA probes, and (2) at the level of fully-formed Hb tetramers separated by native polyacrylamide gel electrophoresis (PAGE). Since the donor erythroid cells are from different species, it is easy to determine which globin gene products are from which nucleus. And since there is no nuclear fusion for at least twelve hours, the Hb switching that occurs is due to regulation in trans. Our results show that developmental Hb switching occurs in mouse-frog erythroid cell polykaryons. When DMSO-induced murine erythroleukemia (MEL) cells (which express only adult mouse Hbs) are fused with Rana tadpole RBCs (which express only embryonic and fetal-like Hbs), the resultant heteropolykaryons express adult frog globin mRNA and adult frog Hbs. We conclude that there are developmental stage-specific trans-acting factors for Hb switching, that trans-acting factors from adult mouse erythroid cells can induce expression of adult frog globin genes in a tadpole RBC nucleus, and that Hb switching mechanisms are conserved across vertebrate classes.

In vivo regulation of hemoglobin phenotypes of developing Rana catesbeiana.

We have examined the effects of phenylhydrazine-induced anemia on the in vivo synthesis of specific hemoglobins at larval, metamorphic, and post-metamorphic stages of the bullfrog Rana catesbeiana, and have found that at all stages the animals qualitatively and quantitatively regenerate their pre-anemia hemoglobin profiles, with one exception: Animals approaching or undergoing the metamorphic hemoglobin switch synthesize only adult hemoglobin during recovery from anemia. We conclude that the ontogenetic progression of hemoglobins in R. catesbeiana is regulated at the level of differentiation of distinct erythroid cell lines, each committed to expressing a particular hemoglobin phenotype; this regulation is unperturbed by anemia.

Morbidity and school absence caused by asthma and wheezing illness.

A survey in the London Borough of Croydon was conducted among an entire school cohort, aged about 9 years, to describe the current morbidity from wheezing illness, its relation to social and family factors, and its effects on social and educational development. A postal screening questionnaire was sent to 5100 parents, and 11.1% of the children were reported to have had wheezing illness over the previous 12 months. A sample of 284 parents were subsequently interviewed at home about their child's illness. School absence over the past year caused by wheezing illness was reported by 58%; and in 12% of children this amounted to more than 30 school days. School absence was strongly associated with all other indicators of morbidity-- short and long term. The proportion described as having "asthma' rose from 22% in those with no absence, to 50% in those with more than 30 days absence. Compared with 92 randomly selected controls with no history of wheezing, wheezy children had more atopic conditions, recurrent headaches, and abdominal pains. School absence was associated with parental separation, non-manual occupation of the mother, more than three children in the household, poor maternal mental health, lack of access to a car, and renting of accommodation. The child's illness had substantial effects on the activities of the mother and the rest of the family, but not on the child's social and recreational activities. Children with over 6 weeks' school absence scored appreciably worse on a teacher's assessment of their social, psychological, and educational adjustment.

Medical care of asthma and wheezing illness in children: a community survey.

A survey was carried out to investigate the medical care of asthma and wheezing illness in a school population. Children with current wheezing illness were identified by a screening questionnaire to the parents of 5100 children in one school cohort from all schools in an outer London borough. Of the 89% who responded, 11.1% reported wheezing within the past 12 months. Parents of a sample of 284 wheezy children aged about 9 were interviewed at home about their child's illness and the related use of drugs and services. There was evidence of substantial underuse of services and this was not associated with social, family, or general practice factors. Considerable proportions of children were not having drug treatment, were receiving only non-antiasthmatic drugs, or were using antiasthmatic drugs incorrectly. The most important social and family factor associated with undertreatment was poor maternal mental health, and this factor appeared to explain the observed association of manual social class with undertreatment. Only about half of the most severe group were labelled as having "asthma," and those with this label were more likely to be receiving treatment and using outpatient services. The results show that the potential of modern treatment to prevent disability due to wheezing illness is not being realised despite the existence of a free and accessible health service.

An investigation of 140 deaths associated with volatile substance abuse in the United Kingdom (1971-1981).

1 In the UK, from 1971 to 81, 140 deaths associated with volatile substance abuse (VSA) were identified from press reports and a variety of other sources. Data about each death were obtained from inquest proceedings, post-mortem and toxicological reports, and death certificates. 2 Based on the year 1981, in which 39 deaths occurred, VSA deaths accounted for over 1% of deaths from all causes, and nearly 2% of deaths from injury and poisoning in males aged 10-19 years. 3 The median age was 16.8 years (range 11-63), and 79% were under age 20. The male to female ratio was 13:1. 4 Death rates were highest in conurbations, and in Scotland, Northern Ireland and Northern England. Deaths tended to be more frequent among manual social classes but this association was not marked. 5 The chief substances were butane (28%), solvents in adhesives (23%), other solvents (26%), aerosols (15%) and fire extinguishing agent (5%). Solvents in adhesives were more important with increasing age. 6 Most deaths occurred alone, at home. In about 41% of cases, death appeared to be only indirectly associated with VSA (trauma 8%, plastic bag over head 19%, and inhalation of stomach contents 14%). In 49% death was attributed to the direct toxic effects of the substance; this proportion was highest with aerosols and lowest with solvents in adhesives. 7 The implications of these findings for prevention, monitoring and research are discussed.

Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P815Y.

The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 10(3) tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.

Cell-mediated cytotoxicity against murine cells infected with 6/94 virus, a parainfluenza type 1 isolate from multiple sclerosis brain tissue.

Maximum cytotoxicity to parainfluenza type 1 viral determinants occurs on day 5 post-immunization in mice with and without preexisting anti-6/94 virus antibody. Animals with preexisting anti-6/94 serum antibody, however, exhibited a markedly higher cytotoxic response. Syngeneic, allogeneic, and xenogeneic virus-infected cells induce virus-specific cytotoxicity, but only infected cells histocompatible with the effector cells can serve as susceptible target cells. The effector cell is a T cell, and hence cytotoxicity is thymus dependent. Coincident with the maximum cytotoxic activity on day 5 is the occurrence of pathological lesions, the sites of which are dependent on the route of inoculation.