Formation of antiviral cytoplasmic granules during orthopoxvirus infection.

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.

Information for gene networks in inner ear development: a study centered on the transcription factor gata2.

The search for molecular mechanisms to stimulate sensory regeneration in the mammalian inner ear is commonly based upon developmental studies. This has revealed many genes that regulate the differentiation of sensory cells. A major challenge is to place these genes into the context of functional networks that describe developmental processes more fully and increase the chances of identifying useful therapeutic targets. We used a novel approach to identify genes that are functionally related to the transcription factor gata2. Temporal profiles of gene expression were derived from three conditionally immortal cell lines and clustered to those of gata2 by applying the gamma model for oligonucleotide signals, a statistical method that allows quantitative analysis of oligonucleotide array data. We derived an objective list of 28 genes that clustered with gata2 in all three cell lines. A number of these genes have known functional links with gata2. Genes encoding CCAAT/enhancer binding proteins (C/EBP) and signal transducer and activation of transcription 3 (Stat3) are especially interesting as they are known to bind gata proteins directly. The results provide strong evidence that our experimental approach can reveal functional relationships between genes that regulate fundamental processes in the differentiation of sensory cells in the inner ear.

Vertebral Adaptations to Large Body Size in Theropod Dinosaurs.

Rugose projections on the anterior and posterior aspects of vertebral neural spines appear throughout Amniota and result from the mineralization of the supraspinous and interspinous ligaments via metaplasia, the process of permanent tissue-type transformation. In mammals, this metaplasia is generally pathological or stress induced, but is a normal part of development in some clades of birds. Such structures, though phylogenetically sporadic, appear throughout the fossil record of non-avian theropod dinosaurs, yet their physiological and adaptive significance has remained unexamined. Here we show novel histologic and phylogenetic evidence that neural spine projections were a physiological response to biomechanical stress in large-bodied theropod species. Metaplastic projections also appear to vary between immature and mature individuals of the same species, with immature animals either lacking them or exhibiting smaller projections, supporting the hypothesis that these structures develop through ontogeny as a result of increasing bending stress subjected to the spinal column. Metaplastic mineralization of spinal ligaments would likely affect the flexibility of the spinal column, increasing passive support for body weight. A stiff spinal column would also provide biomechanical support for the primary hip flexors and, therefore, may have played a role in locomotor efficiency and mobility in large-bodied species. This new association of interspinal ligament metaplasia in Theropoda with large body size contributes additional insight to our understanding of the diverse biomechanical coping mechanisms developed throughout Dinosauria, and stresses the significance of phylogenetic methods when testing for biological trends, evolutionary or not.

Differentiation of mammalian vestibular hair cells from conditionally immortal, postnatal supporting cells.

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.

Hair and supporting-cell differentiation during the development of the avian inner ear.

Two monoclonal antibodies and serial section analysis have been used to compare the sites and times at which hair and supporting-cells differentiate in various sensory regions of the chick inner ear during its development. A monoclonal antibody recognising the 275 kD hair-cell antigen, a protein that is specifically associated with the apical surface of hair cells, was used to identify immature hair cells. Another monoclonal antibody, gm-2, which stains the gelatinous membranes of the cochlear duct, sacculus, and utriculus and the epithelial supporting cells of all vestibular structures in the inner ears of early posthatch chicks, was used to detect the onset of supporting-cell differentiation. Although the antigens recognised by the two antibodies are first detected almost simultaneously during the development of each sensory region, their appearance is not always exactly temporally coincident, and their order of appearance, when not coincident, varies between the epithelia but is always the same within any one organ. Also, when the two antigens are first present in any one region, there is not always a very precise overlap in their spatial distribution. These results cannot be explained entirely by a previously proposed model for hair and supporting-cell development in which hair cells differentiate first and prevent undetermined, surrounding cells from becoming hair cells via lateral inhibition. Modified forms of the original model that can accommodate some of these observations are considered and discussed.

Timed markers for the differentiation of the cuticular plate and stereocilia in hair cells from the mouse inner ear.

The differentiation of the cuticular plate and stereocilia in cochleovestibular hair cells from the mouse was traced with monoclonal antibodies raised by in vitro immunization. The cuticular plate is detected first from embryonic days 14-15 (E14-E15), before cell differentiation is apparent, either with scanning electron microscopy or with actin filament labeling. A flat disc of material forms beneath the apical membrane and subsequently expands, forming a fully shaped cuticular plate at postnatal stages 3-5 (P3-P5). A second antibody labels stereocilia from stage E16 to E18. In the cochlea, the label initially appears as a punctate disc on the cell apex and then follows the development of the stereocilia until the adult shape of the bundle forms at P4-P6. Additional antibodies label stereocilia from P4 to P6 and are apparently specific for the inner ear. They do not label the cuticular plate at any stage and do not cross react with tissues of muscle, kidney, eye, tongue, gut, skin, or brain. At stage P12-P14, coinciding with the functional maturity of the ear, they label the apical regions of Deiter's cells. The temporally overlapping sequence of antibody labeling sheds new light on the development of the hair cell apex and allows us to monitor the differentiation of hair cells from their last mitotic division to the initiation of organ function, a period of over 2 weeks.

Auditory hair cell precursors immortalized from the mammalian inner ear.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

Identification of a 53-kDa antigen in the fibrous sheath of avian spermatozoa.

The intracellular fibrous sheath that surrounds the proximal part of the sperm flagellar axoneme in non-passerine birds is structurally different from that of mammals. We raised a monoclonal antibody against the fibrous sheath of cockerel spermatozoa by in vitro immunisation. Indirect immunofluorescence and immunogold labelling showed that the antibody bound specifically to the fibrous sheath. It also labelled the fibrous sheath in quail spermatozoa. In both species the antibody bound an antigen that had a molecular weight of about 53 kDa. In tissue sections from adult cockerel testis the antigen was located in spermatids and spermatozoa with little cross-reactivity with the basal region of the seminiferous epithelium or interstitial tissue. The antibody may prove to be a useful tool in studies of avian spermiogenesis.

Evaluation of the Ca 1 antibody in the diagnosis of invasive breast cancer.

An evaluation of Ca 1 antibody staining was performed on paraffin sections from 136 breast lesions (64 benign and 72 malignant). Although cytoplasmic staining was encountered significantly more often in malignant lesions, the false negative rate was 6.9% and the false positive rate 56.2%. Benign lesions which showed positive staining included gynaecomastia, cystic mastopathy and fibroadenomata. Various other monoclonal antibodies showed staining similar to Ca 1 antibody. Ca 1 antibody was observed to bind to epithelial membrane antigen-coated sepharose beads.

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Purification of mammalian cochlear hair cells using small volume Percoll density gradients.

A micropurification procedure using a discontinuous Percoll density gradient is described for outer hair cells dissociated from the organ of Corti of the guinea pig. Stable, reproducible gradients can be formed in Pyrex fractionation tubes which have a volume of less than 250 microliters. Hair cells can be harvested from gradients in numbers sufficient for analysis on polyacrylamide gels. The procedure is particularly suited to hair cells, and to other situations in which only a small number of cells are available for purification.

Conditional immortalization of hair cells from the inner ear.

The aim of this work was to culture conditionally immortalized cells that possess the potential to differentiate into mechanosensory hair cells. Utricular epithelia at embryonic stage E16 were cultured from the vestibular system of the H2kbtsA58 transgenic mouse (Immortomouse) that carries a conditionally expressed immortalizing gene derived from the simian virus 40. Immunolabelling showed that the immortalizing transgene product, the T antigen (Tag), was expressed in utricular cells under permissive conditions and that it was inactivated under non-permissive conditions. Several morphologically distinct cell types proliferated when Tag was expressed, including those that resembled fibroblasts, nerve cells and epithelial cells. Mixed cultures of cells from the utricle, passaged up to 50 times every 3-4 days over a period of 5 months, were subsequently allowed to differentiate for 10 days by transferring them to non-permissive conditions. Monoclonal antibody markers were used to locate expression of hair cell specific antigens. One antibody that normally labels stereociliary bundles from postnatal stage P4-6 labelled cellular projections from a population of spheroid cells that were distributed across the culture surface. A second antibody that normally labels stereociliary bundles did not label the same structures. We conclude that utricular hair cell progenitors can be derived from the H2kbtsA58 transgenic mouse but that under the experimental conditions used they do not follow the normal pattern of differentiation.

Epithelioid haemangioendothelioma of the liver: objective response to hepatic intra-arterial 5-FU.

A case of an epithelioid haemangioendothelioma of the liver is presented. The tumour was unresectable at laparotomy because of extensive involvement of both lobes of the liver. The histological appearances of the biopsy taken at operation suggested that the lesion was at the more malignant end of the spectrum for these tumours. The patient was treated by cycles of hepatic intra-arterial 5-fluorouracil with relief of symptoms and prolonged survival. It is important to recognize this type of neoplasm which bears resemblance to other liver pathologies, in particular, to sclerosing cholangiocarcinoma.

Visualisation of domains in the avian tectorial and otolithic membranes with monoclonal antibodies.

The staining patterns observed with six monoclonal antibodies (mAbs) raised in vitro against a fraction derived from the utricular macula were examined with cryosections of the auditory and vestibular organs of the avian inner ear. These antibodies revealed several distinct domains within the gelatinous membranes. Three different labelling patterns were observed in the tectorial membrane. Staining was seen either throughout the entire tectorial membrane, restricted to its core, or in a narrow zone lying close to the surface of the basilar papilla. In the maculae, the mAbs stained either the striolar region of the otolithic membrane or the entire structure. One monoclonal which labelled otoconia, stained small otoconia in their entirely, whilst larger otoconia were only labelled around their periphery. Only one of the mAbs stained the cupulae of the semi-circular canal ampullae and this antibody stained neither the tectorial nor the otolithic membranes. These results suggest that there are biochemically distinct regions in the gelatinous membranes of the inner ear and indicate that these matrices are not simply homogeneous extracellular structures.

Rapid KABP survey for evaluation of NGO HIV/AIDS prevention projects.

The Johns Hopkins University HAPA Support Program (HSP) provided technical assistance to Save the Children (SC), a U.S.-based nongovernmental organization, to conduct a survey of knowledge, attitudes, beliefs, and practices (KABP) related to AIDS among rural Zimbabweans. The objectives of the HSP technical assistance were to field test a rapid KABP survey methodology and to assist SC to provide data that would contribute to their final project evaluation. The entire process of planning, implementation, preliminary data analysis, and preparation of a draft report of survey results was completed in a four-week period. A total of 660 respondents, aged 18-45 years, selected by a modified 30-cluster sampling method, were interviewed in two SC project areas. Although knowledge about HIV/AIDS was high, a number of misconceptions about HIV transmission and unfavorable attitudes to people with AIDS were noted. Of five knowledge and attitude variables that could be compared with the baseline survey results, 4 showed favorable changes and 1 showed an unfavorable trend. Comparing responses from those who were educated by SC with those who had other sources of information about HIV/AIDS, higher levels of knowledge were seen in the SC-educated group and, in one area, somewhat greater willingness to care for family members with AIDS. However, there were no differences seen in other attitudes, beliefs, or in practices regarding condom use. The rapid KABP survey approach was successful in providing, with a relatively modest investment of resources, quantitative data useful for project evaluation, and for developing HIV/AIDS-intervention strategies.

Ethical dilemmas in a randomized trial of asthma treatment: can Bayesian statistical analysis explain the results?

OBJECTIVES: The original objective was to determine whether the use of bilevel positive airway pressure (BiPAP) ventilation would reduce the need for endotracheal intubation, the length of hospital stay, and hospital charges in patients with status asthmaticus. The development of physician treatment bias made patient enrollment difficult. The article subsequently describes the use of Bayesian statistics to explain study results when this bias occurs. METHODS: This study was a prospective, randomized controlled clinical trial conducted over a 34.5-month period at an urban university hospital with an emergency department census of 94,000 annual visits. Patients remaining in status asthmaticus after initial standard treatment with inhaled beta-agonists and steroids were randomized to receive BiPAP ventilation plus standard treatment versus standard treatment alone (non-BiPAP), with intubation for either group as needed. Patients with concurrent cardiac or other pulmonary diseases were excluded. The primary outcome measures were endotracheal intubation rate and length of hospital stay. Secondary outcome measures included vital signs (respiratory rate, pulse rate, blood pressure), changes in expiratory peak flow, changes in pulse oximetry values, and hospital charges. Data were analyzed using Fisher's exact test, Mann-Whitney tests, and Bayesian statistics. For patients enrolled in the study more than once, data analysis was performed on the first enrollment only. RESULTS: Nineteen patients were enrolled in the BiPAP group and 16 patients in the non-BiPAP group. Patients were frequently enrolled more than once and the data from the subsequent enrollments were excluded from the analysis. A marked decrease in enrollment, due to physician treatment bias, led to a premature termination of the study. Demographics showed that the groups were similar in age, sex, initial peak flow rate, and arterial blood gas measurements. There was a 7.3% increase (95% CI = -22 to +45) in the intubation rate in the non-BiPAP group (n = 2) compared with that for the BiPAP group (n = 1). No significant difference was seen in length of hospital stay or hospital charges, although there was a favorable trend toward the BiPAP group. Complications encountered in the BiPAP group included one patient with discomfort associated with the nasal BiPAP mask. Bayesian analysis demonstrated that in order for the collected data to be convincing at the 95% confidence level, the prior conviction among treating physicians that BiPAP was a successful treatment modality would have had to be 98.9%. CONCLUSIONS: In this study, BiPAP appeared to have no deleterious effects in patients with status asthmaticus, with a trend toward decreased endotracheal intubation rate, decreased length of hospital stay, and decreased hospital charges. Although further study with more patients is needed to determine the clinical and statistical significance of this intervention, ethical concerns regarding withholding BiPAP treatment from the patients in the control group forced a premature termination of the study in the authors' institution.

Notch signaling and the emergence of auditory hair cells.

OBJECTIVE: Recent insights into the mechanisms that determine a hair cell's fate have emerged from studies on invertebrate sensory organs and the avian inner ear. These mechanisms have important implications for our understanding of the possible therapeutic management of sensorineural deafness. This article reviews the current state of our knowledge regarding mammalian auditory hair cell fate specification. DESIGN: Data were obtained from the MEDLINE database and data presented at the Molecular Biology of Hearing and Deafness Meeting (Bethesda, Md, October 1998). Articles reporting information about cell fate specification and Notch and its ligands were selected. MAIN OUTCOME MEASURES: Data pertaining to cell fate mechanisms, Notch and its ligands, and application to hearing were extracted. RESULTS: The Notch/ligand mechanism is responsible for the specification of the hair cell phenotype. CONCLUSIONS: Major progress has been made in understanding this fundamental process, and its application to hair cell determination is only now being realized. Possible applications could involve the "switching" of supporting cells to hair cells, thus replenishing those hair cells damaged in sensorineural hearing loss.

Departmental audit in histopathology.

Audit is now part of any laboratory service. Histopathology is no exception, and we have set up a system which allows us to review 4% of our specimens. These specimens are identified using a random number generator and reviewed by a consultant pathologist. Both slides and wet specimens are reviewed and graded according to a set scheme. The results from the first year of operation (1990) show a high rate of accuracy with no serious diagnostic disagreements between the auditor and the reporting pathologist. However, some errors which we would wish to prevent were detected and the audit has allowed us to take corrective measures. In our opinion, this form of audit is useful and necessary to maintain good clinical practice. The cost is considerable--histopathology is by its nature labour intensive. Recognition of this fact by health boards is essential if such systems are to continue.

Adaptation of a ciliary basal apparatus to cell shape changes in a contractile epithelium.

Cilia projecting from the surfaces of highly contractile myoepithelia in the sea anemone Metridium senile maintain their basal orientation, and their ability to propel water, at different states of mesentery contraction, despite substantial changes of myoepithelial cell diameter and length. The ciliary basal apparatus in each monociliated myoepithelial cell is structurally well adapted to provide a stable anchorage for the cilium whilst compensating for these shape changes. It is composed of a distal centriole (basal body), a proximal centriole, a striated rootlet 2-3 micron long which is composed of a bundle of 4-6 nm filaments, and an arched rootlet, also striated, which is composed of a relatively loose bundle of 9-11 nm filaments. A single basal foot projects from the side of the distal centriole in the same direction as the path of the cilium during an effective-stroke; its tip is a focus for many microtubules that radiate outward in all directions toward the cell membrane. The arched rootlet forms a single arch in the cell apex, also in the same plane as the path of the cilium during an effective-stroke. The central axis of the basal apparatus, that is through the distal centriole and the striated rootlet, passes through the apex of the arch. The arched rootlet is apparently flexible so that it can increase or decrease its span as the cell increases or decreases in diameter. In pharnyx and siphonoglyph cells from M. senile, which do not undergo great changes in diameter or length, there is no arched rootlet, and the striated rootlet is much longer. The broad structural diversity of the metazoan ciliary basal apparatus must to a large extent be related to the diversity of the structural and mechanical properties of the cells in which it occurs.

Changes in the distribution of filament-containing septate junctions as coelenterate myoepithelial cells change shape.

This paper describes the redistribution of septate junctions during an increase in diameter of myoepithelial cells from mesenteries of the sea anemone Metridium senile (L). Each septum was composed of a filament core, 9.5-10.2 nm in diameter, which had a double row of lateral projections from each side to the adjacent cell membrane. Septa were arranged in patches in which neighbouring septa lay parallel, 28-33 nm apart. When anaesthetized mesenteries were stretched, myoepithelial cell layers decreased from a mean of 32 to 8 micron thick; each cell shortened and its apical diameter increased. The integrity of the septate junctions was, however, maintained. The mean perimeter of septate junctions, corresponding to that of the cells, increased from 20 to 31 micron; mean depth decreased from 3.7 to 2.1 micron. There was no significant change in spacing between septa. Patches of septa, free to move in a fluid matrix of junction cell membranes, may form mobile attachment sites between cells, thus allowing those cells to change shape. Number and distribution density of microvilli decreased when cell diameter increased. This implies that the microvilli contribute membrane to the cell surface as its surface area increases. Gastrodermal cells are compared with epidermal cells that do not undergo dramatic changes in diameter.