Ultrasonic quantification of geniohyoid cross-sectional area and tissue composition: a preliminary study of age and radiation effects.

BACKGROUND: This study developed and used a new, noninvasive approach to quantify cross-sectional area and tissue composition within the geniohyoid (GH) muscle in normal adults and head and neck cancer patients. METHODS: B-mode ultrasound was used to measure GH cross-sectional area at rest and during four speech gestures and GH tissue composition at rest in normal young adults, patients with SCC head and neck cancer treated with primary radiotherapy, and normal older adults age matched with the patients. RESULTS: Patients exhibited significantly greater GH cross-sectional area than young subjects at rest and in effortful conditions. Significantly greater muscle tissue variability across GH quadrants was observed in patients compared with normal subjects and in older compared with younger subjects. CONCLUSIONS: B-mode ultrasound area analyses and tissue classification techniques can be used to quantify muscle changes, such as those resulting from age, radiotherapy, or rehabilitation for head and neck cancer.

Effects of lingual gestures on blood flow into the tongue: a pilot study.

BACKGROUND: Reduced blood flow has been hypothesized to be a major factor in the formation of postradiation fibrosis. This study examined Doppler ultrasonography as a technique to detect changes in blood flow into the tongue during selected lingual gestures, /t/ and /k/. METHODS: Six normal subjects, three young men (mean age, 26 years) and three older men (mean age, 66 years) were examined in an upright position using Doppler ultrasound imaging of the external carotid artery just below the lingual artery. Measurements were made with a standardized segmentation technique before and after three repetitions of four speech production gestures /t/ and /k/, each with natural and maximal force. RESULTS: Blood flow peak systole increased significantly after the speech gestures (p < .001). Pooled before and after gesture values for older subjects were significantly lower than those for younger subjects (p < or = .05). CONCLUSIONS: Ultrasonography is a clinically useful technique for measuring blood flow during a dynamic gesture and may be useful for measuring effects of tumor treatment and in a lingual exercise program.

Variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion.

The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

Mouse hepatitis virus receptor levels influence virus-induced cytopathology.

We developed human (HeLa) cell lines in which mouse hepatitis virus receptor (MHVR) levels could be regulated by addition of tetracycline. We used these cell lines to determine whether MHVR levels impact the degree of cytopathology induced by infection with the lytic MHV A59 strain. Two cultures were studied; HeLa-MHVRlo (less than 3,000 molecules per cell) and HeLa-MHVRhi (300,000 molecules per cell). Both supported synthesis of infective A59 virus. However, the MHVRlo cells showed no virus-induced cytopathology while the MHVRhi cells uniformly died within 14 hours after infection. This cell death was not related to virus-induced syncytium formation as it occurred even in subconfluent cells overlaid with fusion-blocking antiviral antibodies. MHV A59 spike proteins produced by vaccinia vectors also killed the MHVRhi cells within 12 hours postinfection--MHVRlo cells infected in parallel were intact as judged by trypan blue exclusion. Our current hypothesis is that the accumulation of intracellular complexes composed of spike and MHVR proteins leads to acute single cell lysis.

Treatment research in speech, language and swallowing: lessons from child language disorders.

Three major types of treatment research methodologies are described. Studies on child language intervention are reviewed as examples of trends and methodological issues characterizing treatment research in speech, language, and swallowing within the last 2-3 decades. Principles are drawn from that literature and suggestions for future directions are discussed with particular attention to recent efforts to support clinical trials and treatment outcomes research.

Intracellular complexes of viral spike and cellular receptor accumulate during cytopathic murine coronavirus infections.

Murine hepatitis virus (MHV) infections exhibit remarkable variability in cytopathology, ranging from acutely cytolytic to essentially asymptomatic levels. In this report, we assess the role of the MHV receptor (MHVR) in controlling this variable virus-induced cytopathology. We developed human (HeLa) cell lines in which the MHVR was produced in a regulated fashion by placing MHVR cDNA under the control of an inducible promoter. Depending on the extent of induction, MHVR levels ranged from less than approximately 1,500 molecules per cell (designated R(lo)) to approximately 300,000 molecules per cell (designated R(hi)). Throughout this range, the otherwise MHV-resistant HeLa cells were rendered susceptible to infection. However, infection in the R(lo) cells occurred without any overt evidence of cytopathology, while the corresponding R(hi) cells died within 14 h after infection. When the HeLa-MHVR cells were infected with vaccinia virus recombinants encoding MHV spike (S) proteins, the R(hi) cells succumbed within 12 h postinfection; R(lo) cells infected in parallel were intact, as judged by trypan blue exclusion. This acute cytopathology was not due solely to syncytium formation between the cells producing S and MHVR, because fusion-blocking antiviral antibodies did not prevent it. These findings raised the possibility of an intracellular interaction between S and MHVR in the acute cell death. Indeed, we identified intracellular complexes of S and MHVR via coimmunoprecipitation of endoglycosidase H-sensitive forms of the two proteins. We suggest that MHV infections can become acutely cytopathic once these intracellular complexes rise above a critical threshold level.

A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor.

Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses with novel pathogenic properties.

Identification of a contiguous 6-residue determinant in the MHV receptor that controls the level of virion binding to cells.

Murine carcinoembryonic antigens serve as receptors for the binding and entry of the enveloped coronavirus mouse hepatitis virus (MHV) into cells. Numerous receptor isoforms are now known, and each has extensive differences in its amino terminal immunoglobulin-like domain (NTD) to which MHV binds via its protruding spike proteins. Some of these receptor alterations may affect the ability to bind viral spikes. To identify individual residues controlling virus binding differences, we have used plasmid and vaccinia virus vectors to express two forms of MHV receptor differing only in their NTD. The two receptors, designated biliary glycoproteins (Bgp) 1a and 1bNTD, varied by 29 residues in the 107 amino acid NTD. When expressed from cDNAs in receptor-negative HeLa cells, these two Bgp molecules were displayed on cell surfaces to equivalent levels, as both were equally modified by a membrane-impermeant biotinylation reagent. Infectious center assays revealed that the 1a isoform was 10 to 100 times more effective than 1bNTD in its ability to confer sensitivity to MHV (strain A59) infection. Bgp1a was also more effective than Bgp1bNTD in comparative virus absorption assays, binding 6 times-more MHV (strain A59) and 2.5 times more MHV (strain JHMX). Bgp1a was similarly more effective in promoting the capacity of viral spikes to mediate intercellular membrane fusion as judged by quantitation of syncytia following cocultivation of spike and receptor-bearing cells. To identify residues influencing these differences, we inserted varying numbers of 1b residues into the Bgp1a background via restriction fragment exchange and site-directed mutagenesis. Analysis of the resulting chimeric receptors showed that residues 38 to 43 of the NTD were key determinants of the binding and fusion differences between the two receptors. These residues map to an exposed loop (C-C' loop) in a structural model of the closely related human carcinoembryonic antigen.

Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein.

The envelopes of murine hepatitis virus (MHV) particles are studded with glycoprotein spikes that function both to promote virion binding to its cellular receptor and to mediate virion-cell membrane fusion. In this study, the cysteine-rich spikes were subjected to chemical modification to determine whether such structural alterations impact the virus entry process. Ellman reagent, a membrane-impermeant oxidizing agent which reacts with exposed cysteine residues to effect covalent addition of large thionitrobenzoate moieties, was incubated at 37 degrees C with the JHM strain of MHV. Relative to untreated virus, 1 mM Ellman reagent reduced infectivity by 2 log(10) after 1 h. This level of inhibition was not observed at incubation temperatures below 21 degrees C, suggesting that virion surface proteins undergo thermal transitions that expose cysteine residues to modification by the reagent. Quantitative receptor binding and membrane fusion assays were developed and used to show that Ellman reagent specifically inhibited membrane fusion induced by the MHV JHM spike protein. However, this inhibition was strain specific, because the closely related MHV strain A59 was unaffected. To identify the basis for this strain specificity, spike cDNAs were prepared in which portions encoded either JHM or A59 residues. cDNAs were expressed with vaccinia virus vectors and tested for sensitivity to Ellman reagent in the fusion assays. The results revealed a correlation between the severity of inhibition mediated by Ellman reagent and the presence of a JHM-specific cysteine (Cys-1163). Thus, the presence of this cysteine increases the availability of spikes for a thiol modification that ultimately prevents fusion competence.

Postoperative emesis following otoplasty in children.

Sixty unpremedicated children aged between 3 and 14 years, scheduled for otoplasty, were randomly divided into one of three groups to receive either ondansetron 0.1 mg.kg-1, droperidol 75 micrograms.kg-1, or placebo at induction of anaesthesia. All patients received a standard general anaesthetic using thiopentone, atracurium and halothane. Opioid analgesia was avoided intra-operatively and infiltration with local anaesthetic was used prior to the start of surgery. Children who received ondansetron were less likely to vomit (15%) than those who received either droperidol (40%) or placebo (60%) (p < 0.01). This group also tolerated oral ingestion of fluids and solids earlier than those who received either droperidol or placebo (p < 0.001). There was no difference between the placebo or droperidol group in the incidence of vomiting or time to ingestion of oral fluids and meals. Three patients in the ondansetron group had a self-terminating nodal rhythm which was not associated with any haemodynamic disturbances. Postoperatively there were no untoward incidents in any of the groups and all patients were discharged home the day after surgery.

The effect of a combination of rectal diclofenac and caudal bupivacaine on postoperative analgesia in children.

Both caudal anaesthesia and non-steroidal anti-inflammatory drugs have been used in the management of postoperative pain in children. The aim of the present study was to evaluate the combination of caudal analgesia and rectally administered diclofenac in the treatment of pain following minor surgery in children. Thirty-nine, ASA grade 1 or 2, children undergoing inguinal or penoscrotal surgery were randomly assigned to receive either a caudal block using 0.125% bupivacaine with adrenaline or a similar caudal block in combination with rectally administered diclofenac 1 mg.kg-1. Children given a caudal block alone were more likely to need analgesia in the first 24 h postoperatively. It would appear that the combination of a caudal block and rectal diclofenac in children undergoing minor lower abdominal surgery reduces the need for subsequent analgesia.

Effect of general anaesthesia on prolonged intraoesophageal pH monitoring.

Investigation of gastro-oesophageal reflux often includes endoscopy, usually under general anaesthesia, and pH monitoring. In most cases, the pH probe is passed when the child is awake and is poorly tolerated. The effect of general anaesthesia on pH monitoring is unknown. The aim of the study was to determine if placing the probe in the anaesthetised child gives a representative pH study. Twenty children aged 4 months to 13 years underwent oesophago-gastroduodenoscopy under general anaesthesia. A pH electrode was placed under direct vision in the distal oesophagus. pH monitoring was begun after completion of anaesthesia and continued for 18-24 hours. The study was repeated within 14 days without anaesthetic. The reproducibility of values of percent pH < 4, number of reflux episodes/hour, reflux episodes lasting > 5 min, and longest reflux episode was 85%, 90%, 75%, and 75% respectively. These results are comparable with those in adults and children in whom pH studies were performed on consecutive days (without anaesthetic) keeping all variables constant. Therefore pH data collected in a child within 24 hours of endoscopy under general anaesthesia are representative.

Overexpression of the MHV receptor. Effect on progeny virus secretion.

The intracellular interaction of the coronavirus mouse hepatitis virus (MHV) with its cellular receptor (MHVR) was investigated. Overexpression of MHVR from vaccinia vectors during an ongoing MHV infection resulted in dramatic inhibition of virus production. Infectivity in both cytoplasmic extracts and supernatants was reduced by over three orders of magnitude relative to control cultures in which a truncated MHVR lacking virus binding activity was expressed. Complete MHV virions were not detectable in supernatants of MHVR expressing cells. In the presence of overexpressed MHVR, the coronavirus spike protein was not cleaved into posttranslation products S1 and S2, nor was it fully processed into a form resistant to endoglycosidase H digestion, indicating that intracellular engagement of spike with receptor prevented spike transport and consequent association with virions.

MHVR-independent cell-cell spread of mouse hepatitis virus infection requires neutral pH fusion.

Receptor-specificity is a key determinant of viral tropism. In this report, however, we have demonstrated that cell-associated spread of MHV can bypass the requirement for binding to primary receptors and thereby spread to cells that are resistant to MHV infection. Anti-receptor antibody CC1, which blocks infection by MHV virions, failed to prevent cell-associated spread of MHV to receptor-negative BHK cells or receptor-positive DBT cells. Cell-associated MHV may be utilizing an alternative, low-affinity receptor that is inadequate for functional interaction with MHV virions. Theoretically, dissemination of MHV infection through a receptor-independent, cell-associated mechanism in vivo provides the potential for broader host and tissue range, and for spread of infection despite the presence neutralizing antibodies. Receptor-independent, cell-associated spread of MHV requires neutral pH fusion capability. The low pH-dependent MHV variant OBLV60, which utilizes an endocytic route of entry, does not spread through a receptor-independent mechanism. Additionally, antiviral antibodies that block MHV spike-mediated fusion inhibited cell-associated spread of infection.

Reconstitution of Flock House provirions: a model system for studying structure and assembly.

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Rueckert, J. Virol 6:6728, 1992). We describe here methods for purifying provirions in a state which permitted dissociation and reassembly. Dissociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pH 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of substantial, though subnormal, infectivity. Reconstitution experiments, i.e., remixing components after separating RNA from capsid protein, generated abnormal particles, suggesting the presence in the unfractionated dissociation products of an unidentified "nucleating" component.