Quantitative image analysis of HIV-1 infection in lymphoid tissue.

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Regulation of LTP-I secretion from human monocyte-derived macrophages by differentiation and cholesterol accumulation in vitro.

Human macrophages in vitro synthesize and secrete the cholesteryl ester (CE) transfer protein, LTP-I. The effect of differentiation of monocyte-to-macrophage on the synthesis and secretion of LTP-I cholesteryl ester transfer activity was investigated. One marker of macrophage differentiation is expression of the 'scavenger' receptor, which mediates macrophage uptake and degradation of acetylated low-density lipoprotein. Monocytes secreted very little detectable CE transfer activity in the first 24 h following cell isolation. Both CE transfer activity and scavenger receptor activity increased with time in culture. Thus, although circulating monocytes probably do not secrete CE transfer activity, tissue macrophages such as hepatic Kupffer cells may contribute to plasma CE transfer activity. Resident macrophages of the arterial wall are derived from circulating monocytes which enter the vessel wall where they differentiate into macrophages. Such macrophages are the principal source of lipid-laden foam cells of the atherosclerotic plaque. Cholesterol accumulation results when uptake of lipoprotein cholesterol overwhelms the capacity of macrophages to excrete cholesterol. Since LTP-I is postulated to function in reverse cholesterol transport, the effect on LTP-I secretion of loading macrophages with cholesterol was determined after exposure of macrophages to acetylated-LDL or free cholesterol (FC). Cholesterol loading by both these maneuvers resulted in dose-dependent increases in macrophage secretion of CE transfer activity, and there was a significant positive correlation between CE transfer activity secreted and accumulation of CE. Thus, LTP-I may function at the cellular level in maintenance of lipid homeostasis: macrophage LTP-I secretion may be a protective mechanism in response to excess cholesterol accumulation in resident macrophages of the arterial wall.

Subcellular immunolocalization of protein kinase CK2 in normal and carcinoma cells.

CK2 is a messenger-independent protein serine/threonine kinase that has been implicated in cell growth and proliferation. Our recent analysis of squamous cell carcinomas of the head and neck (SCCHN) revealed a significant elevation in CK2 activity in these tumor cells relative to normal mucosa of the upper aerodigestive tract and suggested a correlation with aggressive tumor behavior and poor clinical outcome. In order to further define the distribution of CK2 in these tissues, we have examined the immunohistochemical staining pattern of surgical specimens of both SCCHN tumors and normal upper aerodigestive tract mucosa using a monoclonal antibody directed against the catalytic subunit CK2-alpha of the kinase, and have compared these data with the subcellular distribution of CK2 activity in these same tissues. These measurements showed that CK2 is predominantly localized to the nuclei of the tumor cells, which agreed closely with the immunohistochemical staining pattern of CK2-alpha in tumor cells. The chiefly nuclear distribution of CK2-alpha immunostaining found consistently in SCCHN tumor cells and tumor-infiltrating lymphocytes contrasted with a relatively more predominant cytosolic staining pattern exhibited by various cellular constituents of normal oropharyngeal mucosa. The immunostaining pattern of CK2-alpha revealed that staining was observed in the cells stained for the proliferation-marker Ki-67; however, strong distinct immunostaining for CK2-alpha was also observed in large numbers of other cells in these same tumors, suggesting that CK2 elevation in these tumors is not a reflection of proliferative activity alone, but may also relate to the pathobiological behavior of the tumor.

Secretion of cholesteryl ester transfer protein-lipoprotein complexes by human HepG2 hepatocytes.

We have employed immunoaffinity chromatography to characterize the distribution of cholesteryl ester transfer activity in particles secreted by HepG2 hepatocytes. HepG2-secreted cholesteryl ester transfer activity is associated with apoprotein (apo) A-I (58%) as well as apo A-II (55%), and is not associated with apo B or E. In contrast, our previous studies have shown that most (88%) cholesteryl ester transfer activity in human plasma is associated with apo A-I whereas very little (7%) is associated with apo A-II. Thus, the distribution of cholesteryl ester transfer activity in plasma particles likely reflects active remodeling of nascent particles in the plasma compartment. Further data suggested that HepG2 cells secrete a lipid transfer inhibitor activity which is associated with apo E-containing lipoprotein particles. This inhibitory activity is heat labile.

Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation.

We hypothesized that malignant transformation of normal mucosa of the upper aerodigestive tract to squamous cell carcinoma of the head and neck (SCCHN) might be associated with altered CK2 activity in the chromatin compartment of these tumors. We measured CK2 activity in the cytosol and chromatin of 7 surgical specimens of SCCHN, and 5 specimens of normal oropharyngeal mucosa from non-smokers/non-drinkers. CK2 activity in SCCHN tumors was significantly elevated in both the nuclear chromatin (P < 0.0005) and cytosolic (P <0.04) compartments relative to normal mucosa. These data suggest that activation of dysregulation of the chromatin-associated CK2 signal may play a role in the pathobiology od SCCHN.

Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor.

Overexpression of the epidermal growth factor (EGF) receptor, a hallmark of aerodigestive squamous cell carcinoma of the head and neck (SCCHN), correlates with aggressive tumor behavior. There is evidence that SCCHN cells auto-activate their EGF receptors. The receptor has therefore attracted interest as a potential therapeutic target. We tested the in vitro therapeutic efficacy of PD153035--a potent, specific inhibitor of the tyrosine kinase intrinsic to the EGF receptor--by employing a well-characterized cell line derived from human gingival SCCHN. DNA-synthesis and cell number were assayed for growth-inhibitory effects, phosphorylation of the EGF receptor was quantitated by immunoblot, and cell apoptosis was detected by terminal deoxytransferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) in situ assay. PD153035, at nanomolar concentrations, inhibited autophosphorylation of the EGF receptor induced by EGF stimulation and the inhibition occurred in a dose-dependent manner. Under the same conditions, PD153035 inhibited cell growth, and induced apoptosis of SCCHN cells in vitro. We conclude that selective inhibition of the EGF receptor tyrosine kinase completely abolishes EGF receptor phosphorylation resulting from receptor stimulation, and results in growth inhibition and apoptosis of SCCHN cells in vitro. By inducing cytostasis and apoptosis, this new class of inhibitors may be of therapeutic value against SCCHN.

Chemical warfare agents: estimating oral reference doses.

Health risk assessments for sites contaminated with chemical warfare agents require a comparison of the potential levels of exposure with a characterization of the toxic potency of each chemical. For noncancer health effects, toxic potency is expressed in terms of Reference Doses (RfD). A RfD is a daily exposure level or dose (usually expressed in units of milligrams of chemical per kilogram body weight per day) for the human population, including sensitive subpopulations, that is likely to be without an appreciable risk of deleterious effects. A daily exposure at or below the RfD is not likely to be associated with health risks, but as the amount of chemical that an individual is exposed to increases above the RfD, the probability that an adverse effect will occur also increases. A RfD is derived by first examining the available human or animal toxicity data to identify a dose or exposure that corresponds to a no-observed-adverse-effect level (NOAEL) or a lowest-observed-adverse-effect level (LOAEL). The NOAEL is the exposure level at which there are no statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. Effects may be produced at this level, but they are not considered to be adverse if they do not result in functional impairment or pathological lesions that affect the performance of the whole organism or which reduce an organism's ability to cope with additional challenge. The LOAEL is the lowest exposure level at which there are statistically or biologically significant increases in frequency or severity of adverse effects between the exposed population and its appropriate control. If only a LOAEL is identified by the toxicity data, a NOAEL is estimated by dividing the LOAEL by a factor no greater than 10. This extrapolation factor of 10 or less is termed the LOAEL-to-NOAEL Uncertainty Factor (UFL). The NOAEL is also adjusted by the application of other Uncertainty Factors, including (1) a UFH < or = 10 to ensure that the resulting RfD protects segments of the human population that may be more sensitive to the chemical than the average person; (2) a UFA < or = 10 to extrapolate from the experimental animal species to humans; (3) a UFS < or = 10 to extrapolate from an experimental subchronic exposure study to a potential chronic exposure; and (4) a UFD < or = 10 to ensure that the resulting RfD is protective for all possible adverse effects, particularly those that may not have been adequately evaluated in the available studies. A Modifying Factor (MF), based on a qualitative professional assessment of the data, may also be used to account for other factors (e.g., deficiencies in the critical study) that are not adequately covered by the standard Uncertainty Factors. 1. Agent HD (Sulfur Mustard). RfDe = 7 x 10(-6) mg kg-1 d-1. A LOAEL was identified in a two-generation reproductive toxicity study conducted in rats. A total uncertainty factor of 3000 was applied to account for protection of sensitive subpopulations (10), animal-to-human extrapolation (10), LOAEL-to-NOAEL extrapolation (3), and extrapolation from a subchronic to chronic exposure (10). A LOAEL-to-NOAEL UF of 3, instead of the default value of 10, was used because the critical effect (stomach lesions) was considered to be "mild" in severity and may have been enhanced by the vehicle used (sesame oil in which sulfur mustard is fully soluble) and the route of administration (gavage), which is more likely to result in localized irritant effects. The key study did identify a toxic effect that is consistent with the vesicant properties of sulfur mustard. In none of the other available studies was there any indication of a different effect occurring at a lower exposure level.

A unique case of Bezold's abscess associated with multiple dural sinus thromboses.

OBJECTIVES/HYPOTHESIS: Bezold's abscess and dural sinus thromboses are rare complications of otitis media in the era of antibiotics. Although potentially fatal, they are treatable. We present a unique case report of Bezold's abscess in association with multiple dural sinus thromboses. STUDY DESIGN: Single case report. METHODS: A young female patient's clinical course is presented and discussed. We review the anatomy, incidence, pathogenesis, and treatment of Bezold's abscess and dural sinus thrombosis. RESULTS: After mastoidectomy, neck exploration, broad-spectrum intravenous antibiotics, and anticoagulation therapy, the patient recovered fully and has remained asymptomatic since her discharge from the hospital at 12 months' follow-up. CONCLUSIONS: This is the first reported case of Bezold's abscess associated with a cavernous sinus thrombosis and the third reported case of Bezold's abscess associated with lateral sinus thrombosis. Despite its rarity, Bezold's abscess must be recognized and treated aggressively. Dural sinus thrombosis is relatively more common, and treatment of the underlying cause is essential. The diagnosis and rapid, aggressive treatment of these conditions are essential for an optimal clinical outcome.

Real-time, cine magnetic resonance imaging for evaluation of the pediatric airway.

BACKGROUND: Standard magnetic resonance imaging (MRI) and computed tomographic (CT) modalities are limited in their ability to image dynamic organs. New real-time, dynamic, cine magnetic resonance imaging (CMRI) techniques have the potential to image moving structures. OBJECTIVE: We therefore investigated the feasibility of using CMRI techniques to dynamically image the human airway, to assess laryngeal and tracheal patency and function. METHODS: A cohort of 10 pediatric patients, 10 adult patients, and 10 normal volunteers underwent routine static MRI, as well as CMRI using a Siemens 1.5 T Vision system (Siemens, Erlangen, Germany). Patients also underwent endoscopic evaluation. Cine axial, coronal, and sagittal sequences of the larynx and trachea were obtained during quiet respiration, as well as during a variety of provocative maneuvers. RESULTS: CMRI readily demonstrated normal vocal cord mobility and tracheal stability in normal volunteers. Abnormal vocal mobility was easily appreciated using the CMRI imaging system. Similarly, dynamic effects of tracheomalacia were clearly demonstrated using CMRI. Dynamic extrinsic tracheal compression resulting from mass lesions or anomalous vasculature was also visualized using CMRI. CONCLUSIONS: Cine MRI of the airway has the potential to provide novel data regarding laryngeal and tracheal patency and function. This evolving modality may serve as a valuable adjunct to static MR and CT imaging, as well as endoscopy, in the assessment of the airway.

Results of esophageal biopsies performed during triple endoscopy in the pediatric patient.

BACKGROUND: Endoscopic examination (direct laryngoscopy and bronchoscopy) is the method of choice for diagnosis of respiratory symptoms of unknown cause in children. However, gastroesophageal reflux is being recognized increasingly often as a cause of pediatric respiratory symptoms and is difficult to diagnose on the basis of findings from direct laryngoscopy and bronchoscopy. In cases in which gastroesophageal reflux was included in the differential diagnosis, we additionally performed esophagoscopy with esophageal mucosal biopsies. OBJECTIVES: To determine the feasibility, safety, and efficacy of routinely performing esophageal biopsies during triple endoscopy in children. METHODS: Twenty-four children ranging in age from 2 weeks to 10 years were referred for airway evaluation. Under general anesthesia, children underwent direct laryngoscopy and bronchoscopy and esophagoscopy with mucosal biopsy. RESULTS: Esophageal mucosa biopsy specimens were quickly and safely obtained during endoscopic evaluation. There were no complications. Reflux esophagitis was present in 54% of biopsy specimens, as suggested by basal cell hyperplasia, papillary elongation, and/or inflammatory cell infiltrates. CONCLUSION: Gastroesophageal reflux is often difficult to diagnose in the pediatric population. When direct laryngoscopy and bronchoscopy is performed during examination of the child with airway symptoms, the addition of esophagoscopy with mucosal biopsies will safely and quickly provide data regarding the potential contribution of gastroesophageal reflux.

Nuclear matrix proteins as malignant markers in squamous cell carcinoma of the head and neck.

OBJECTIVE: To test the hypothesis that transformation of normal upper aerodigestive mucosa to squamous cell carcinoma of the head and neck (SCCHN) is associated with specific changes in nuclear matrix (NM) proteins. DESIGN: Retrospective, nonrandomized investigation using a cellular fractionation sequence followed by 2-dimensional gel electrophoresis analysis of NM proteins. SUBJECTS: Nuclear matrix proteins were extracted from a cohort of 12 pathologic SCCHN specimens and 5 normal specimens of oropharyngeal mucosa. RESULTS: All SCCHN specimens examined expressed 11 NM proteins that were not detected in normal mucosa. Conversely, at least 4 NM proteins that were expressed by all specimens of normal mucosa were absent from all SCCHN tumors. Seven NM proteins were common to carcinomas and normal specimens. Spindle cell histological variants of squamous cell carcinoma had distinct NM patterns. CONCLUSIONS: Malignant transformation of normal upper aerodigestive mucosa to SCCHN is associated with specific changes in NM composition. These data suggest that different NM proteins might serve as specific tumor markers.

Noncancer inhalation toxicology of crystalline silica: exposure-response assessment.

Silicosis from inhalation of silica has long been recognized as an occupational hazard. Concern has arisen regarding the potential risk of silicosis from ambient silica (primarily quartz dust). This presentation reviews available data regarding ambient silica levels and estimates of the risk of silicosis at low exposure levels as they relate to the current U.S. Environmental Protection Agency National Ambient Air Quality Standards (NAAQS) for particulate matter. Current data indicate that for individuals not compromised by other respiratory ailments and for ambient environments expected to sustain 10% or less silica fraction in particulate matter with a mean aerodynamic diameter of < or = 10 microns (PM10), maintenance of the 50 micrograms/m3 annual NAAQS for PM10 is adequate to protect against fibrotic effects from ambient silica exposures. Issues such as the large divergence of risk estimates within the occupational setting (particularly at high cumulative exposures) and factors to consider for extrapolating risk in an occupational setting to risk from ambient exposure are discussed.

Child abuse and the otolaryngologist: part I.

Nearly 1 million infants and children are neglected and abused yearly in the United States, with a greater than 1% resulting mortality rate. One half of these children are seen by physicians for abuse-related injuries, and nearly 75% have injuries of the head and neck. Physicians, however, account for reporting only 11% of all cases. As experts trained in diseases and injuries of the head and neck, otolaryngologists are particularly well positioned to recognize abuse in the clinic and in the emergency room and during other consultations. We present an overview of child abuse definitions, risk factors, and legal obligations of the physician. We also review the manifestations of child abuse within the head and neck, with particular attention to the role of the otolaryngologist. We briefly discuss some conditions that may be mistaken for abuse and suggest a practical protocol for management of suspected cases in the clinic.

Outpatient biopsies of the palatine tonsil: access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers.

OBJECTIVES: Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patient's lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN: A pilot cohort study was performed. PATIENTS: We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES: The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS: Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS: Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.

Assessment of congenital bony nasal obstruction by 3-dimensional CT volume rendering.

The nature and extent of congenital bony nasal obstruction is best determined by X-ray computed tomography (CT) imaging. Three-dimensional volume rendering of CT images provides an integrated perspective that can assist in clinical decision making and in operative planning. Clinical cases of choanal atresia and pyriform aperture stenosis are reviewed with their images. The authors propose this modality as an evolving standard for imaging of congenital bony nasal obstruction.

The near complete tracheal ring deformity.

Congenital complete and near-complete tracheal rings are extremely rare tracheal deformities. There are fewer than 100 cases of congenital complete tracheal rings, and only four cases of near-complete described in the literature. Long-segment occurrences of these anomalies are usually incompatible with life. We describe the presentation of the long-segment near complete tracheal ring deformity and long-segment anterior fusion of cartilage rings (cartilage sleeve deformity) in a living infant. The literature is reviewed, and the embryology, presentation, and management of these extremely rare anomalies are discussed.

Securing cochlear implants to the skull: two alternate methods.

In view of the various problems encountered with the traditional methods of securing cochlear implants--including dural tear and suture dissolution following infection--we devised two alternate methods of performing this procedure. We use a titanium mesh or a Gore-Tex patch secured with two 4-mm screws to fix the receiver to the skull. No patient who has undergone either of these procedures at our institution has experienced any of the complications that are associated with the older silk, nylon, and Dacron sutures. Moreover, our two alternate methods are less technically difficult and can be performed in a shorter period of time.