Biology of oral mucosa and esophagus.

The mucosal lining of the oral cavity and esophagus functions to protect the underlying tissue from mechanical damage and from the entry of microorganisms and toxic materials that may be present in the oropharynx. In different regions, the mucosa shows adaptation to differing mechanical demands: Masticatory mucosa consists of a stratified squamous keratinized epithelium tightly attached to the underlying tissues by a collagenous connective tissue, whereas lining mucosa comprises a nonkeratinized epithelium supported by a more elastic and flexible connective tissue. The epithelium is constantly replaced by cell division in the deeper layers, and turnover is faster in the lining than in the masticatory regions. Chemotherapeutic agents and radiation limit proliferation of the epithelium so that it becomes thin or ulcerated; this will first occur in the lining regions. The principal patterns of epithelial differentiation are represented by keratinization and nonkeratinization. As keratinocytes enter into differentiation, they become larger and begin to flatten and to accumulate cytokeratin filaments. In addition to the keratins, the differentiating keratinocytes synthesize and retain a number of specific proteins, including profilaggrin, involucrin, and other precursors of the thickening of the cell envelope in the most superficial layers. The concept of epithelial homeostasis implies that cell production in the deeper layers will be balanced by loss of cells from the surface. There is a rapid clearance of surface cells, which acts as a protective mechanism by limiting colonization and invasion of microorganisms adherent to the mucosal surface.

Preinduction activities: a closed malpractice claims perspective.

The American Association of Nurse Anesthetists Foundation conducts an ongoing study of closed malpractice claims that involve nurse anesthetists. A team of 8 CRNA researchers has to date investigated 223 closed claim files from the St Paul Fire and Marine Insurance Company. Research findings have demonstrated that failure to provide appropriate anesthesia care relative to the Scope and Standards for Nurse Anesthesia Practice was significantly associated with adverse anesthetic outcomes. Claims that involved inadequate preinduction activities (n = 22) were analyzed in the context of their compliance with published standards of care. The largest group of claims in this analysis (59%) involved damaging respiratory events, 28% entailed damaging cardiovascular events, and the principal issue in 13% of these claims involved failure to seek available information such as laboratory studies on the medical record. The most prevalent occurrence with damaging respiratory events was undocumented airway assessment in 27% of the claims. In 55% of these claims, the medical history was not completely documented. The surgical procedure categories were general surgical (32%), obstetrical (27%), otolaryngogical (23%), orthopedic (14%), and gynecologic (5%). The involved standards of care are reviewed, and recommendations are made regarding consistent completion of preinduction activities.

Enhancing effect of chitosan on peptide drug delivery across buccal mucosa.

The buccal mucosa represents a potentially important topical route for delivery of peptide or protein drugs with some unique advantages such as the avoidance of hepatic first-pass metabolism and the acidity and protease activity encountered in the gastrointestinal tract. However, the bioavailabilities or relative potencies of intraorally administered peptides are usually quite low, unless permeabilizers are employed. Chitosan, a mucopolysaccharide of marine origin, has been claimed to act both as a bioadhesive and permeabilizer, making it a candidate system for mucosal drug delivery. In this study, the enhancement effect of chitosan in gel form for oral mucosa was investigated with a large bioactive peptide, transforming growth factor-beta (TGF-beta). Chitosan gel was prepared at 2% concentration in dilute lactic acid and TGF-beta was incorporated into the gel. The effect of chitosan as a permeabilizer was determined by measuring the flux of TGF-beta across porcine oral mucosa in an in vitro system. The localization of TGF-beta within the oral mucosa was determined by horizontal sectioning and counting. Chitosan was found to exert a marked permeabilizing effect on buccal mucosa for peptide drug.

Penetration of N-nitrosonornicotine (NNN) across oral mucosa in the presence of ethanol and nicotine.

The effects of ethanol concentrations of 5, 15, 20, 25, 27, 30 and 50% on the penetration of the tobacco-specific carcinogen, nitrosonornicotine (NNN), across porcine oral mucosa were examined using an in vitro perfusion system. Concentrations of ethanol of 25% and above significantly increased the permeability of oral mucosa to NNN, although this increase ceased with 50% ethanol, possibly due to a fixative effect. Nicotine is a consistent component of smoked and smokeless tobacco; the presence of 0.2% nicotine significantly increased the permeability of oral mucosa to NNN and 2% nicotine caused a further increase. Combined use of nicotine and ethanol significantly increased the penetration of NNN across oral mucosa over that of ethanol alone until the concentration of ethanol reached 50%. The results of this study suggest that the synergy between tobacco and alcohol in the etiology of oral cancer may be explained, at least in part, by the local permeabilizing effects of alcohol on the penetration of tobacco-specific (and other) carcinogens across oral mucosa.

Oral mucosal permeability and stability of transforming growth factor beta-3 in vitro.

PURPOSE: To investigate the permeability and localization of topically applied 125I-TGF-beta3 in porcine floor-of-mouth mucosa as a function of concentration and exposure. METHODS: The 125I-TGF-beta3 diluted in three different vehicles was applied to the tissue samples mounted in perfusion cells maintained at 37 degrees C. Flux and Kp values were calculated from the perfusate collected over a 24 hour period. The quantity of 125I-TGF-beta3 present in the tissue was determined by horizontal sectioning and subsequent counting. The stability of 125I-TGF-beta3 in saliva and in the tissue was analyzed by SDS polyacrylamide gradient gel electrophoresis. RESULTS: 125I-TGF-beta3 was relatively stable in saliva and in the epithelium; approximately 50% of the total counts in the deeper epithelium were resident in the 25kDa TGF-beta3 homodimer. A steady-state flux was reached approximately 6 hours post application and Kp value was 4.0+/-0.6 x 10(-6) (mean +/- sem). Penetration of 125I-TGF-beta3 to the basal cell layer was concentration dependent but reached nanomolar concentrations even after extensive surface rinsing, representing over one-thousand fold the IC50 for epithelial cell cycle arrest. CONCLUSIONS: The data suggest that topical application of TGF-beta3 to the oral mucosa in an appropriate vehicle can provide effective therapeutic delivery to the tissue.

Surgery, pain, and immune function.

Research findings have demonstrated that surgery negatively affects immune function. Pain also has deleterious effects on immune function. Most research in this area has been conducted on animals. Psychoneuroimmunology provides the framework for this article. Psychoneurological phenomena, such as stress, depression, and pain, can influence immune system functioning through neuroendocrine pathways. Biological and behavioral processes appear to be closely related and need to be jointly considered when planning and providing anesthesia care. The anesthesia planning process increasingly involves aggressive assessment and treatment of postoperative pain, which may benefit immune function.

AANA Journal course: update for nurse anesthetists--the preoperative pulmonary assessment: is this patient at high risk for surgery?

Postoperative pulmonary complications most often involve atelectasis followed by pneumonia and arterial hypoxemia. The severity of these complications is related to the decreases that occur in vital capacity and functional residual capacity. Astute anesthetists can prospectively identify patients and surgical procedures likely to be associated with pulmonary complications. Upper abdominal surgery carries a 30% to 40% pulmonary complication rate. Vertical laparotomies and lateral thoracotomies are associated with pulmonary risk, as are patients who are obese, emphysemic, asthmatic, or have cardiac disease. Optimal use of clinical data coupled with selective application of diagnostic tests, such as arterial blood gases and pulmonary function tests, help in the development of an appropriate anesthetic management plan that minimizes pulmonary risk, especially in patients with known pulmonary risk factors.

Cations in human rhinoviruses.

All known crystal structures of rhinoviruses have some uninterpreted electron density on their fivefold axes at a distance of about 152 +/- 3 A from the viral center. This density had been assumed to be a Ca2+ ion, based on its shape, height, and the presence of Ca2+ ions in the crystallization solutions. Difference electron density maps between EGTA-soaked crystals of human rhinovirus 14 (HRV14), as well as HRV16, and their corresponding native structures show that this density is an EGTA-chelatable ion. Analysis of the coordination geometry indicates that the ions in HRV3, HRV14, and HRV1A could be Ca2+ and the ion in HRV16 might be Zn2+. These cations may play a role in regulation of rhinovirus stability, although the loss of the ion itself does not seem sufficient to lead to viral disassembly.

Human rhinovirus 3 at 3.0 A resolution.

BACKGROUND: The over 100 serotypes of human rhinoviruses (HRV) are major causative agents of the common cold in humans. These HRVs can be roughly divided into a major and minor group according to their cellular receptors. They can also be divided into two antiviral groups, A and B, based on their sensitivity to different capsid-binding antiviral compounds. The crystal structures of HRV14 and HRV16, major-receptor group rhinoviruses, as well as HRV1A, a minor-receptor group rhinovirus, were determined previously. Sequence comparisons had shown that HRV14 seemed to be an outlier among rhinoviruses. Furthermore, HRV14 was the only virus with no cellular 'pocket factor' in a hydrophobic pocket which is targeted by many capsid-binding antiviral compounds and is thought to regulate viral stability. HRV3, another major-receptor group virus, was chosen for study because it is one of a subset of serotypes that best represents the drug sensitivity of most rhinovirus serotypes. Both HRV3 and HRV14 belong to antiviral group A, while HRV16 and HRV1A belong to antiviral group B. RESULTS: HRV3 was found to be very similar to HRV14 in sequence and structure. Like HRV14, crystallized HRV3 also has no bound pocket factor. The structure of HRV3 complexed with an antiviral compound, WIN56291, was also determined and found to be similar to the same antiviral compound complexed with HRV14. CONCLUSIONS: The amino-acid sequence and structural similarity between HRV3 and HRV14 suggests that rhinoviruses in the same antiviral group have similar amino-acid sequences and structures. The similar amino-acid composition in the pocket region and the viral protein VP1 N termini in all known group B HRV sequences suggests that these viruses may all contain pocket factors and ordered N-terminal amphipathic helices in VP1. Both of these factors contribute to viral stability, which is consistent with the observations that group B rhinoviruses have a higher chance of successful transmission from one host to another and is a possible explanation for the observed higher pathogenicity of these rhinoviruses.

Structural studies on human rhinovirus 14 drug-resistant compensation mutants.

Structures have been determined of three human rhinovirus 14 (HRV14) compensation mutants that have resistance to the antiviral capsid binding compounds WIN 52035 and WIN 52084. In addition, the structure of HRV14 is reported, with a site-directed mutation at residue 1219 in VP1. A spontaneous mutation occurs at the same site in one of the compensation mutants. Some of the mutations are on the viral surface in the canyon and some lie within the hydrophobic binding pocket in VP1 below the ICAM footprint. Those mutant virus strains with mutations on the surface bind better to cells than does wild-type virus. The antiviral compounds bind to the mutant viruses in a manner similar to their binding to wild-type virus. The receptor and WIN compound binding sites overlap, causing competition between receptor attachment and antiviral compound binding. The compensation mutants probably function by shifting the equilibrium in favor of receptor binding. The mutations in the canyon increase the affinity of the virus for the receptor, while the mutations in the pocket probably decrease the affinity of the WIN compounds for the virus by reducing favorable hydrophobic contacts and constricting the pore through which the antiviral compounds are thought to enter the pocket. This is in contrast to the resistant exclusion mutants that block compounds from binding by increasing the bulk of residues within the hydrophobic pocket in VP1.

Human rhinovirus 14 complexed with fragments of active antiviral compounds.

Crystallographic studies of human rhinovirus 14 (HRV14) crystals soaked with fragments of antiviral WIN compounds, at high concentrations (82-200 micrograms/ml), show the compounds bind into the hydrophobic beta-barrel (WIN pocket) of VP1. Two of these short compounds (5-[3,5-dimethyl-4-hydroxyphenyl]-2-methyltetrazole and phenol oxazoline) cause conformational changes in the virus similar to the active, longer WIN compounds. In addition, thermostabilization studies suggest these short WIN compounds provide some stability to the HRV14 capsid. We conclude that the short compounds appear to mimic the cellular cofactors observed in the hydrophobic pocket of VP1 for some picornaviruses. Both cofactors and short WIN compounds bind into the pocket, cause conformational changes in VP1, and provide a small degree of virion stabilization but are unlikely to inhibit attachment. Three specific binding sites for dimethyl sulfoxide (DMSO), used as solvent, were also identified. One of the DMSO molecules binds into the drug binding pocket near the pocket opening, while the other two bind in the canyon near the VP1 protomer-protomer interface.

The structure of human rhinovirus 16.

BACKGROUND: Rhinoviruses and the homologous polioviruses have hydrophobic pockets below their receptor-binding sites, which often contain unidentified electron density ('pocket factors'). Certain antiviral compounds also bind in the pocket, displacing the pocket factor and inhibiting uncoating. However, human rhinovirus (HRV)14, which belongs to the major group of rhinoviruses that use intercellular adhesion molecule-1 (ICAM-1) as a receptor, has an empty pocket. When antiviral compounds bind into the empty pocket of HRV14, the roof of the pocket, which is also the floor of the receptor binding site (the canyon), is deformed, preventing receptor attachment. The role of the pocket in viral infectivity is not known. RESULTS: We have determined the structure of HRV16, another major receptor group rhinovirus serotype, to atomic resolution. Unlike HRV14, the pockets contain electron density resembling a fatty acid, eight or more carbon atoms long. Binding of the antiviral compound WIN 56291 does not cause deformation of the pocket, although it does prevent receptor attachment. CONCLUSIONS: We conjecture that the binding of the receptor to HRV16 can occur only when the pocket is temporarily empty, when it is possible for the canyon floor to be deformed downwards into the pocket. We further propose that the role of the pocket factor is to stabilize virus in transit from one host cell to the next, and that binding of ICAM-1 traps the pocket in the empty state, destabilizing the virus as required for uncoating.

Sedation by infusion: a clinical trial in cardiac surgery patients.

The problem of sedation and analgesia for ventilator-dependent patients was examined in this study. Twenty subjects undergoing elective coronary revascularization surgery at a major medical center were studied. They ranged in age from 49 to 83 years. A randomized, prospective research design was used to place subjects in either an experimental group or a control group. Standard postoperative analgesia with intravenous increments of morphine and midazolam in the control group was compared to treatment with a titratable sufentanil-midazolam infusion in the experimental group. In both groups, hemodynamic variables were measured at selected intervals, sodium nitroprusside consumption was measured, and time to extubation was noted. Data analysis demonstrated no statistically significant differences between the experimental and control groups. The more costly sedative-analgesic infusion appeared to be comparable to conventional treatment with incremental morphine and midazolam based on the results of this study.

A comparison of the anti-rhinoviral drug binding pocket in HRV14 and HRV1A.

The three-dimensional structures of two human rhinovirus serotypes (HRV14 and HRV1A) are compared when complexed with various antiviral agents. Although these agents all bind into the same hydrophobic pocket, the exact viral-drug interactions differ. In the absence of drugs, the pocket is occupied by a fatty acid in HRV1A, but is empty in HRV14 except for two water molecules. The conformation of each drug is dependent upon the shape of the hydrophobic pocket. In HRV14 the major residues determining the shape of the binding site are Y1128, P1174 and M1224, corresponding to I1125, M1169 and I1220 in HRV1A. When there is no cofactor or a drug in the pocket, the entrance to the pocket is open. However, the entrance is closed when the pocket is occupied by a cofactor or a drug. There are relatively small conformational changes when the agents displace the natural cofactor in HRV1A. In contrast, there are much larger conformational changes on binding a drug in HRV14. These differences cause an inhibition of viral attachment in HRV14 but not in HRV1A. Binding of the drugs results in three additional interprotomer hydrogen bonds in HRV14 and one in HRV1A. These hydrogen bonds and a potential loss of flexibility upon efficient packing of the pocket may contribute to the inhibition of uncoating in both serotypes.

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.

Structural analysis of a series of antiviral agents complexed with human rhinovirus 14.

The binding to human rhinovirus 14 of a series of eight antiviral agents that inhibit picornaviral uncoating after entry into host cells has been characterized crystallographically. All of these bind into the same hydrophobic pocket within the viral protein VP1 beta-barrel structure, although the orientation and position of each compound within the pocket was found to differ. The compounds cause the protein shell to be less flexible, thereby inhibiting disassembly. Although the antiviral potency of these compounds varies by 120-fold, they all induce the same conformational changes on the virion. The interactions of these compounds with the viral capsid are consistent with their observed antiviral activities against human rhinovirus 14 drug-resistant mutants and other rhinovirus serotypes. Crystallographic studies of one of these mutants confirm the partial sequencing data and support the finding that this is a single mutation that occurs within the binding pocket.

The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating.

WIN 51711 and WIN 52084 are structurally related, antiviral compounds that inhibit the replication of rhino (common cold) viruses and related picornaviruses. They prevent the pH-mediated uncoating of the viral RNA. The compounds consist of a 3-methylisoxazole group that inserts itself into the hydrophobic interior of the VP1 beta-barrel, a connecting seven-membered aliphatic chain, and a 4-oxazolinylphenoxy group (OP) that covers the entrance to an ion channel in the floor of the "canyon." Viral disassembly may be inhibited by preventing the collapse of the VP1 hydrophobic pocket or by blocking the flow of ions into the virus interior.