[Sleep medicine for the ENT specialist]. / Schlafmedizin für den Hals-Nasen-Ohren-Facharzt.

Sleep-related breathing disorders can be divided into obstructive and central sleep apnea, and hypoventilation syndromes. The diagnosis is made according to an algorithm in which clinical symptoms and a polygraphy or polysomnography usually point the way. After initial diagnosis of the most common obstructive sleep apnea, conservative therapies such as positive airway pressure therapy (PAP therapy), positional therapy, and/or mandibular advancement splint are used in many cases, supplemented by treatment of risk factors. If PAP therapy is not possible, more detailed diagnosis of airway obstruction, often with sleep videoendoscopy, is required. In general, "muscle-sparing" surgical techniques such as tonsillectomy with uvulapalatopharyngoplasty (TE-UPPP) should be considered whenever possible. This is especially important in the surgical treatment of snoring. More surgical therapy alternatives are for example barbed wire pharyngoplasty, tongue pacemaker and bimaxillary advancement. Optimal therapy alternatives should be evaluated in a sleep medicine center.

Cloning and comparative sequence analysis of the gene encoding canine intercellular adhesion molecule-1 (ICAM-1).

Canine intercellular adhesion molecule-1 (ICAM-1) plays a primary role in the adherence of canine neutrophils to endothelial cells and in the cytotoxicity of canine neutrophils for adult cardiac myocytes. We have cloned the canine ICAM-1 gene and have analyzed the conservation of ICAM-1 amino acid (aa) sequences in man, chimpanzee, mouse, rat and dog. Canine ICAM-1 displays 61% identity with human ICAM-1. Cys residues critical to the immunoglobulin (Ig) fold structure and four sites of N-linked glycosylation are absolutely conserved in ICAM-1 from all species. Residues in the cytoplasmic tail associated with cytoskeletal alpha-actinin binding are highly conserved, supporting the hypothesis that intracellular attachment is indeed important for ICAM-1 function. Residues critical for human ICAM-1 binding to the beta 2-integrin leukocyte-function-associated antigen 1 (LFA-1) are highly conserved between all species, whereas those residues demonstrated to play an important role in interaction of human ICAM-1 with macrophage activation complex 1 (Mac-1) are not highly conserved. Residues critical for ICAM-1 binding to rhinovirus and malaria-infected red blood cells (IRBC) are not highly conserved.

Cloning, sequence comparison and in vivo expression of the gene encoding rat P-selectin.

We have cloned the cDNA encoding rat P-selectin (Psel) and have examined the regulation of Psel expression in vivo. Sequence analysis of the complete Psel cDNA demonstrated significant nucleotide and amino-acid identity with human and mouse Psel. Similar to mouse Psel, the rat sequence lacks the equivalent of human complement regulatory protein-like repeat 2 (CR2). Seven potential N-linked glycosylation sites are conserved between the three species, suggesting that carbohydrate modification may play an important role in Psel function. To examine expression of Psel in vivo, levels of Psel mRNA were examined in several different tissues after systemic administration of lipopolysaccharide (LPS). Psel mRNA was undetectable in tissues of vehicle-treated animals. By 3 after LPS administration, Psel mRNA levels were elevated in all tissues examined, the highest levels being seen in the lung. Significant increases in Psel mRNA were also seen in the heart, thymus, spleen and kidney. By 24 h after LPS, mRNA levels for Psel remained elevated in the lung, heart, kidney, thymus and small intestine. Psel mRNA was not detectable in total RNA isolated from purified rat platelets, suggesting that the increased levels of Psel mRNA were the result of upregulation of endothelial gene expression. In addition, only minimal levels of platelet factor 4 mRNA (PF4), used as a platelet-specific marker, were observed in the tissues studied. These data demonstrate that part of the response to acute inflammation in vivo includes the rapid increase in endothelial Psel expression.

Morphologic assessment of leukocyte-endothelial cell interactions in mesenteric venules subjected to ischemia and reperfusion.

Intravital microscopic studies of the mesenteric microcirculation have demonstrated that leukocyte adherence and emigration in postcapillary venules are a characteristic feature of tissues exposed to ischemia-reperfusion. The objectives of this study were to determine whether: (1) neutrophils are the predominant leukocytes that adhere and emigrate in postischemic mesenteric venules, and (2) leukocyte adherence and/or emigration are a prerequisite for reperfusion-induced increases in venular permeability. Leukocyte kinetics in cat mesenteric venules (25-35 microns diameter) were evaluated using both intravital microscopy and quantitative morphometry. The intestine and mesentery were exposed to 60 min of ischemia, followed by 60 min reperfusion. Some animals were pretreated with a monoclonal antibody (MoAb IB4) against the leukocyte adhesion glycoprotein, CD11/CD18. Vessels observed by intravital microscopy and adjacent venules of similar diameter were excised and processed for light (LM) and electron microscopy (EM). Horseradish peroxidase (HRP), administered intravenously, was used to assess vascular permeability by EM. By LM, the control (nonischemic) mesentery is sparsely populated by plasma cells, mast cells, and leukocytes; 30-50% of the resident population is neutrophils. Ischemia-reperfusion led to a significant increase in the number of extravascular cells, with neutrophils accounting for greater than 80% of the total cell population. Control and ischemic venules demonstrated no leakage of HRP into the interstitium. However, venules exposed to ischemia and reperfusion demonstrated HRP leakage between endothelial cells and into the surrounding interstitium; neutrophils were adherent to the luminal surface of the endothelium, transmigrating the vessel wall, and in the surrounding interstitium. Animals pretreated with MoAb IB4 presented the same cell profile as nonischemic controls, with no adherent or transmigrating neutrophils. However, some HRP leakage was noted following reperfusion in venules treated with MoAb IB4. The results of this study indicate that: (1) neutrophils are the predominate leukocytes that adhere and emigrate in postischemic venules, and (2) inhibition of leukocyte adhesion does not completely prevent the venular dysfunction associated with ischemia-reperfusion.

Intracellular variation of rat intestinal mucin granules localized by monoclonal antibodies.

Monoclonal antibodies produced against rat small intestinal mucins were utilized to study variability of stored mucin granules within rat ileal goblet cells. Eleven antibody-secreting hybridoma cultures were produced; six of these uniformly labeled stored mucin granules in virtually all goblet cells, suggesting that some antigenic features are common to all granules. The other five stained goblet cells in the rat small intestinal epithelium nonuniformly. R803, R805, and R807 localized within almost all goblet cells but revealed differential labeling of centrally and peripherally located mucin granules. R804 uniformly labeled the mucin granules of most villous goblet cells; some of the crypt goblet cells were uniformly labeled, but the majority were only partially labeled, resulting in a mottled staining pattern. R808 stained only a small portion of crypt goblet cells; there is, however, an increase in both number of goblet cells labeled and in uniformity of staining of the stored granule mass from the base of the crypt to the surface, resulting in uniform labeling of virtually all goblet cells at the villus tip. This study demonstrates for the first time that rat small intestinal mucin granules are immunologically heterogeneous and nonuniformly distributed within the epithelium. Additionally, staining patterns within the stored granule mass suggest that structurally distinct subpopulations of mucin granules may exist within a single goblet cell.

Cytoskeleton of intestinal goblet cells: role of microtubules in baseline secretion.

To determine the involvement of microtubules (MTs) in granule translocation, autoradiographic analysis of maximal granule movement in the absence or presence of MT inhibitors was performed. Rabbit colonic mucosal explants were pulse-labeled with [3H]glucosamine for 30 min in organ culture, then maintained on nonradioactive medium for 1-6 h. Radio-labeled mucin granules appear in the apical granule mass in 1-2 h, then they migrate to the apical plasma membrane, with a total transit time of 4-6 h. Mucosal explants were treated with either nocodazole or taxol for 30 min, pulse-labeled for 30 min, then maintained in organ culture with the same drug for up to 6 h. Nocodazole binds tubulin, preventing polymerization. In response, granule movement out of the supranuclear region and along the apical granule mass is significantly impeded. Taxol stabilizes MTs, preventing depolymerization. In response, supranuclear MTs are misoriented, but thecal MTs maintain normal orientation. Taxol treatment impedes granule migration out of the supranuclear region of the cell but not migration along the theca. These data suggest that the organization of MTs dictate the spatial organization of the baseline secretory pathway. Microtubules are necessary for granule translocation by providing directed tracks for granule movement, but microtubule dynamics are not the motile mechanism transporting mucin granules to the apical plasma membrane for secretion.

Functional biology of intestinal goblet cells.

Goblet cells reside throughout the length of the small and large intestine and are responsible for the production and maintenance of the protective mucus blanket by synthesizing and secreting high-molecular-weight glycoproteins known as mucins. To elucidate the role of goblet cells in the biology of the intestinal tract, an overview of the physiological implications of the mucus gel is presented, including a concise review of the products secreted by the cell. Because of the unique nature of this highly polarized exocrine cell, the maturational reorganization of the cytoarchitecture and the cellular mechanisms by which goblet cells secrete their products are discussed. This includes elucidation of the baseline secretory pathway, which is dependent on the cytoskeleton for granule movement, and the accelerated secretory pathway, which is independent of the cytoskeleton but requires an extracellular signal to occur. Finally, the involvement of goblet cell mucins in the pathophysiology of intestinal neoplasia and ulcerative colitis are presented.

Cytoskeleton of intestinal goblet cells: role of actin filaments in baseline secretion.

Although microtubules appear necessary to maintain mucin granule transport in intestinal goblet cells, the role of microfilaments in mucus secretion is unknown. To determine the functional significance of microfilaments in goblet cell secretion, fluorescent cytochemistry of microfilaments and autoradiographic studies on granule movement were performed on rabbit intestinal goblet cells, with and without the actin depolymerizing agents, cytochalasin D (cyto D), and dihydro-cytochalasin B (dihydro B). In normal goblet cells, cytochemical localization of F-actin with NBD-phallacidin demonstrated their restriction to the apical surface of the goblet cell. Visualization of the goblet cell apical surface by electron microscopy revealed the presence of a thin layer of cytoplasm overlying the granule mass. Treatment with cyto D and dihydro B eliminated NBD-phallacidin staining of the apical cell surface. Quantitative analysis of baseline granule translocation demonstrated that treatment with cyto D and dihydro B resulted in dramatic acceleration of granule movement through goblet cells. This cellular response results from an increase in baseline secretion and facilitation of secretion of newly synthesized mucins, not stimulation of an accelerated secretory event. These data imply that actin filaments fulfill a barrier function in baseline secretion by hindering granule access to the plasma membrane; once the granule contacts the plasma membrane, exocytosis occurs. Secretion is balanced by the translocation of subjacent granules. In contrast, an accelerated secretory event is not triggered by plasma membrane access alone; this event requires a regulatory signal. We hypothesize that, unlike accelerated secretion, baseline secretion is constitutive, with exocytosis limited solely by the physical constraint of secretory granule access to the apical plasma membrane.

Cytoarchitectural reorganization of rabbit colonic goblet cells during baseline secretion.

Light and electron microscopy were coupled with point counting methods to quantitate shape and volume changes of goblet cells during their migration and maturation from the base of the crypt to the colonic surface epithelium in the rabbit. After differentiation, goblet cells attain a broad pyramidal configuration in the basal third of the crypt. The cells elongate and dramatically decrease in volume as they move into the surface epithelium. The distributions and volume fractions of organelles were found to vary considerably, depending on the location of the goblet cell in the epithelium. Mucin granules are initially synthesized throughout the cytoplasm, but become increasingly concentrated as the cell matures. Organelles involved in synthesis such as the Golgi apparatus and rough endoplasmic reticulum (RER) similarly attain a more concentrated arrangement as the cell moves up in the crypt. The mean cell volume decreases from 1,228.8 microns3 for cells in the basal third of the crypt to 541.3 microns3 for goblet cells on the surface. Most organelles decrease in proportion to this decrease, although a disproportionately large decrease in the RER was measured. When actual subcellular volumes are calculated, a net decrease in several subcellular compartments is detected. This loss of granules and organelles is accomplished by the continual synthesis and secretion of mucin granules. Cytoplasm and organelles become entrapped in the upward movement of granules towards the cell apex, become irretrievably isolated, and are sloughed into the crypt lumen. This process accounts for the decrease in cell volume and contributes to the altered cytoarchitecture of the cell.

Incorporation of chimeric gag protein into retroviral particles.

The product of the Rous sarcoma virus (RSV) gag gene, Pr76gag, is a polyprotein precursor which is cleaved by the viral protease to yield the major structural proteins of the virion during particle assembly in avian host cells. We have recently shown that myristylated forms of the RSV Gag protein can induce particle formation with very high efficiency when expressed in mammalian cells (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). We made use of this mammalian system to examine the abilities of foreign antigens to be incorporated into particles when fused directly to the myristylated Gag protein. Our initial experiments showed that removal of various portions of the viral protease located at the carboxy terminus of the RSV Gag protein did not disrupt particle formation. We therefore chose this region for coupling of iso-1-cytochrome c from Saccharomyces cerevisiae to Gag. This was accomplished by constructing an in-frame fusion of the CYC1 and gag coding sequences at a common restriction endonuclease site. Expression of the chimeric gene resulted in synthesis of the Gag-cytochrome fusion protein and its release into the cell culture medium. The chimeric particles were readily purified by simple centrifugation, and transmission electron microscopy of cells that produced them revealed a morphology similar to that of immature type C retrovirions.